CN1712940A - Production of fluorescent sensor based on soluble conjugated polymer - Google Patents
Production of fluorescent sensor based on soluble conjugated polymer Download PDFInfo
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- CN1712940A CN1712940A CN 200510019207 CN200510019207A CN1712940A CN 1712940 A CN1712940 A CN 1712940A CN 200510019207 CN200510019207 CN 200510019207 CN 200510019207 A CN200510019207 A CN 200510019207A CN 1712940 A CN1712940 A CN 1712940A
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Abstract
A method for preparing fluorescent transducer based on water soluble and conjugated polymer includes preparing water soluble anion fluorescent conjugated polymer of poly {5 - methoxyl - 2-3 (3 - sulfonylation) oxypropyl group - 1, 4 - p - benzene frame ethane} and dissolving it in water to obtain reaction liquid A; dissolving biological macromolecule as probe in water to obtain reaction liquid B; mixing liquid A and B with water to obtain mixed liquid C; filling liquid C in fluorescent chromometer for measuring its fluorescent intensity ; adding sample to be tested in liquid C for measuring its fluorescent intensity .
Description
Technical field
The present invention relates to the interdisciplinary field of subjects such as biology, medical science, pharmacy, material, chemistry, virology.The preparation method who more specifically relates to a kind of fluorescent optical sensor based on soluble conjugated polymer.
Background technology
Environmental toxin, important pathogenic microorganisms and virus are harm humans life security and healthy formidable enemy, the bioterrorism shade increases, new virus constantly occurs, this controlled disease also may break out again, give human existence, social stability and economic development bring serious threat and challenge.At aspects such as disease early diagnosis, environmental monitoring and protection, virus and bacterial monitorings, people press for has real-time, quick, sensitive and specific detection means.People recognize that progressively the public arena is more vulnerable to the biochemical attack of terrorism, and the sensitivity and the selectivity of detection technique proposed more and more higher requirement, and these technology also can be widely used in medical diagnosis and biomedical research.Chemical/biological sensors technology based on water soluble fluorescent conjugated polymer is a kind of quick, the high-sensitive chemical/biological sensing technology that development in recent years is got up.This type of sensor has become an important directions of chemistry and biology cross discipline research field.The application of water soluble fluorescent conjugated polymer in biology sensor will realize to biological sample (big molecule or micromolecule) in real time, fast, homogeneous phase, sensitivity and selectivity detect.
The method of identification biomolecule (big molecule or micromolecule), for example enzyme-linked immunosorbent assay (ELISA) and immune competition law (micromolecule detection), selectivity is good, but operation steps is many, wastes time and energy, the cost height.Other has optionally ligand-receptor detection method such as polymerase chain reaction (PCR) method, usually need be with biomolecule fixedly (as DNA chip and protein-chip) on matrix or the film.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of the fluorescent optical sensor based on soluble conjugated polymer.The employing water soluble fluorescent conjugated polymer is that the sensor properties of sensitive material is stable, and highly sensitive to biology sample detection, selectivity is good, highly versatile.
To achieve these goals, the present invention adopts following technical measures:
Its step is as follows:
(1) with 4-metoxyphenol, 3-propylene chlorohydrin, thiophane, sodium metal, absolute ethyl alcohol, ether, pyridine, methylsufonyl chloride, concentrated hydrochloric acid, anhydrous sodium sulfate, chloroform, acetone, sodium iodide, sodium chloride, the concentrated sulphuric acid, anhydrous magnesium sulfate (MgSO
4), sodium sulphite (Na
2SO
3); NaOH (NaOH); N; N dimethyl formamide (DMF); thionyl chloride; formaldehyde; dioxane, benzene, absolute methanol are raw material; by refluxing; purify with Zeo-karb; vacuum drying; decompression distillation; rotary evaporation or freeze drying crystallization; technology such as recrystallization [Huang Tao chief editor; the Zhang Zhimin associate editor, Experiment of Organic Chemistry (second edition), Beijing; Higher Education Publishing House; 1998] polymer poly (5-methoxyl-2-(3-sulfonylation) propoxyl group-1,4-phenylenevinylene) of preparation good water solubility (MPS-PPV) or poly-(5-methoxyl-2-(4-sulfonylation lithium butoxy)-1,4-phenylenevinylene) (MBL-PPV); it is dissolved in water or alcohol (methyl alcohol; or ethanol; or propyl alcohol; or butanols; or tetrahydrofuran) in, getting concentration is 10
-10Mol/L～10
-5Mol/L reactant liquor A (MPS-PPV or MBL-PPV); Conjugated polymer is water-soluble or pure dissolubility; Conjugated polymer is negative ion or kation poly-phenylene vinylene (ppv) derivant or polyparaphenylene's acetylene ene derivative of water-soluble or pure dissolubility.
(2) biomacromolecule or biotinylated metal complex are dissolved in the solvent as probe, getting concentration is 10
-10Mol/L～10
-5Mol/L reactant liquor B (antiviral antibody solution or Re-Biotin solution or Ru-Biotin solution or Ir-Biotin solution); Described biomacromolecule is antibody or antigen.
Described biotinylated metal complex is biotinylation rhenium (I) complex, biotinylation ruthenium (II) complex, biotinylation iridium (III) complex, or biotinylated other metal complex; Or the conjugate of metal complex and antigen/antibody, DNA/RNA, ligand/receptor; Described solvent is water or methyl alcohol or ethanol or propyl alcohol or butanols or tetrahydrofuran (any one);
(3) extract reaction solution A, reactant liquor B and water or 0.01molL
-1Phosphate buffered solution (pH is 7.4) or 0.1molL
-1Hartshorn salt buffer solution (pH is 8.9) is pressed amount of substance and was mixed than 10: 10: 1～1: 1: 10, gets mixed solution C;
(4) getting mixed solution C 500～1000 μ L in micro-fluorescence cuvette, with certain excitation wave. long 280～450nm excites, and measures its fluorescence intensity at suitable emission wavelength 350～600nm place.
(5) add sample such as viral antigen or avidin/streptavidin in aforementioned mixed solution C, excite, measure its fluorescence intensity in same transmitted wave strong point with identical excitation wavelength.
This sensor is used for the detection of hemorrhagic fever viruse antibody/antigen or Avidin or streptavidin, can finish at 1-15 minute.
The present invention compared with prior art has the following advantages and effect:
Compare with Enzyme Linked Immunoadsorbent Assay and chip technology, this law is highly sensitive, easy fast, good, the highly versatile of selectivity.It is applied to antigen-antibody reaction or DNA: DNA, and (or DNA: RNA) reaction detection will make existing standard detection technology generation fundamental change.If a kind of biomolecule such as antibody or antigen both can be used as reinforcing agent or quencher, again can by with the fluorescent characteristic of the specificity interaction change system of corresponding antigen or antibody, just can set up a kind of general immune sensing technology.
Based on the preparation of the fluorescent optical sensor of soluble conjugated polymer will realize biological sample (big molecule) micromolecular in real time as SARS virus, avian influenza virus, anthrax virus, AIDS virus, hemorrhagic fever viruse antibody/antigen and other, fast, homogeneous phase, sensitivity and selectivity detect.
Response fast (1～200 second), good hydrophilic property, highly sensitive, selectivity is good, highly versatile.
Description of drawings
Fig. 1 changes MPS-PPV (1.0 * 10 for pH value of solution
-5MolL
-1)-antibody L133 (4.0 * 10
-8MolL
-1) influence of system fluorescence intensity
PH4.0～9.0:Britton-Robinson buffer solution (orthophosphoric acid, acetate, boric acid three sour mixed solutions and NaOH solution composition)
Fig. 2 is response characteristic and the reversibility of sensor to hemorrhagic fever viruse antibody-L133/ antigen-EHF
Fig. 2 A fluorescent spectrum curve (a) 1.0 * 10
-5MolL
-1MPS-PPV, (b) a+1.6 * 10
-7MolL
-1Antibody-L133, (c) b+2.9 * 10
-10MolL
-1Antigen-EHF
Fig. 2 B fluorescent spectrum curve (a) 1.0 * 10
-5MolL
-1MPS-PPV, (b) a+2.9 * 10
-9MolL
-1Antigen-EHF, (c) b+1.6 * 10
-7MolL
-1Antibody-L133
Fig. 3 excites and emission spectrum for sensitive material MPS-PPV's
Excitation wavelength 390nm, emission wavelength 497nm
Fig. 4 is the influence of antibody-L133 concentration change to MPS-PPV aqueous solution fluorescence intensity
1.0 * 10
-5MolL
-1Inject the antibody-L133 of variable concentrations in the MPS-PPV aqueous solution
Fig. 5 is a hemorrhagic fever viruse immunosensor synoptic diagram (acting between MPS-PPV and antibody-L133 and the antigen-EHF)
Polymkeric substance MPS-PPV is subjected to antibody-L133 perturbation strengthens fluorescence, behind adding antigen-EHF, because the specificity between antibody-antigen interacts, makes fluorescent weakening
Fig. 6 is the atomic force micrograph between polymkeric substance MPS-PPV, MPS-PPV and antibody-L133 or antigen-EHF and the three
(a)MPS-PPV，(b)MPS-PPV+L133，(c)MPS-PPV+EHF，(d)MPS-PPV+L133+EHF
Image size: (a, b, c) 3.0 * 3.0 μ m
2, (d) 1.5 * 1.5 μ m
2
Fig. 7 is transmission electron microscopy figure: (a) MPS-PPV, (b) MPS-PPV+L133+EHF
Fig. 8 is streptavidin (SA) sensor synoptic diagram (being recovered by fluorescence behind the MPS-PPV fluorescence of the Re-Biotin quencher adding streptavidin)
Embodiment
Embodiment 1:
A kind of preparation method of the fluorescent optical sensor based on soluble conjugated polymer the steps include:
(1) MPS-PPV or the MBL-PPV with preparation is dissolved in water or methyl alcohol or ethanol or propyl alcohol or the butanols, and getting concentration is 10
-10Mol/L～10
-5Mol/L reactant liquor A;
(2) with biotinylated metal complex such as biotinylation rhenium (I) complex---hexafluorophosphoric acidization-N-4-pyridine-methylamino biotin-three carbonyl-2,9-dimethyl-4,7-diphenyl-1, the 10-phenanthrolene closes rhenium (I) (Re-Biotin) as probe in water-soluble or methyl alcohol or ethanol or propyl alcohol or butanols or the tetrahydrofuran, concentration be 10
-10Mol/L～10
-5Mol/L reactant liquor B;
(3) extract reaction solution A 10～100 μ L, reactant liquor B 10～100 μ L and water or 0.01molL
-1Phosphate buffered solution (pH=7.4) or 0.1molL
-1Hartshorn salt buffer solution (pH=8.9) 800～1000 μ L are mixed, get mixed solution C;
(4) get mixed solution C 500～1000 μ L in micro-fluorescence cuvette, excite, measure its fluorescence intensity at suitable emission wavelength 350～600nm place with certain excitation wavelength 280～450nm.
(5) add sample Avidin or streptavidin in aforementioned mixed solution C, excite, measure its fluorescence intensity (see figure 8) in same transmitted wave strong point with identical excitation wavelength.
Embodiment 2:
A kind of preparation method of the fluorescent optical sensor based on soluble conjugated polymer the steps include:
(1) MPS-PPV or the MBL-PPV with preparation is dissolved in water or methyl alcohol or ethanol or propyl alcohol or the butanols, and getting concentration is 10
-10Mol/L～10
-5Mol/L reactant liquor A (see figure 3);
(2) with biomacromolecule such as hemorrhagic fever viruse antibody as probe in water-soluble or methyl alcohol or ethanol or propyl alcohol or butanols or the tetrahydrofuran, concentration be 10
-10Mol/L～10
-5Mol/L reactant liquor B;
(3) extract reaction solution A 10～100 μ L, reactant liquor B 10～100 μ L and water or 0.01molL
-1Phosphate buffered solution (pH=7.4) or 0.1molL
-1Hartshorn salt buffer solution (pH=8.9) 800～1000 μ L are mixed, get mixed solution C;
(4) get mixed solution C 500～1000 μ L in micro-fluorescence cuvette, excite, measure its fluorescence intensity (seeing Fig. 2, Fig. 4) at suitable emission wavelength 350～600nm place with certain excitation wavelength 280～450nm.
(5) add sample such as hemorrhagic fever viruse antigen in aforementioned mixed solution C, excite, measure its fluorescence intensity (see figure 2) in same transmitted wave strong point with identical excitation wavelength.
Claims (5)
1, a kind of preparation method of the fluorescent optical sensor based on soluble conjugated polymer comprises the following steps:
A, be raw material with the absolute methanol, by refluxing, the polymkeric substance for preparing good water solubility with Zeo-karb purification, vacuum drying, decompression distillation, rotary evaporation or freeze drying crystallization, recrystallization, it is dissolved in water or methyl alcohol or ethanol or propyl alcohol or butanols or the tetrahydrofuran, and getting concentration is 10-10mol/L～10-5mol/L reactant liquor A;
B, biomacromolecule or biotinylated metal complex are dissolved in aqueous solvent or alcohol or the tetrahydrofuran as probe, concentration be 10
-10Mol/L～10
-5Mol/L reactant liquor B;
C, extract reaction solution A, reactant liquor B and water or 0.01molL
-1Phosphate buffered solution pH is 7.4 or 0.1molL
-1Hartshorn salt buffer solution pH is 8.9, presses amount of substance and mixes than 10: 10: 1～1: 1: 10, gets mixed solution C;
D, get mixed solution C 500～1000 μ L in micro-fluorescence cuvette, wavelength 280～450nm excites, and measures the fluorescence intensity at its emission wavelength 350～600nm place;
E, adding sample or avidin/streptavidin excite with identical excitation wavelength in aforementioned mixed solution C, measure its fluorescence intensity in same transmitted wave strong point.
2, the preparation method of a kind of fluorescent optical sensor based on soluble conjugated polymer according to claim 1, it is characterized in that: conjugated polymer is water-soluble or pure dissolubility.
3, the preparation method of a kind of fluorescent optical sensor based on soluble conjugated polymer according to claim 1, it is characterized in that: conjugated polymer is negative ion or kation poly-phenylene vinylene (ppv) derivant or polyparaphenylene's acetylene-derivative of water-soluble or pure dissolubility.
4, the preparation method of a kind of fluorescent optical sensor based on soluble conjugated polymer according to claim 1 is characterized in that: solvent be water or methyl alcohol or ethanol or propyl alcohol or butanols,
5, the preparation method of a kind of fluorescent optical sensor based on soluble conjugated polymer according to claim 1, it is characterized in that described biotinylated metal complex is biotinylation rhenium (I) complex, biotinylation ruthenium (II) complex, biotinylation iridium (III) complex, or the conjugate of metal complex and antigen/antibody, DNA/RNA, ligand/receptor.
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| CN1712940A true CN1712940A (en) | 2005-12-28 |
| CN100458417C CN100458417C (en) | 2009-02-04 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864294A (en) * | 2010-06-09 | 2010-10-20 | 中国科学院上海微***与信息技术研究所 | Composite sensing material of fluorescent conjugated polymer and organic metal complex and application |
| CN1987466B (en) * | 2006-12-22 | 2011-08-31 | 武汉大学 | In-situ preparing method for micro porous plate pH sensor |
| CN102516990A (en) * | 2011-11-30 | 2012-06-27 | 东南大学 | Application of polyphenylene vinylene (PPV) polymer serving as fluorescent indicator in oxygen sensor based on fluorescence quenching |
| CN103674913A (en) * | 2013-12-04 | 2014-03-26 | 南京邮电大学 | Fluorescence method and kit for testing lymphocyte homing receptor |
| CN105115945A (en) * | 2015-06-26 | 2015-12-02 | 安徽师范大学 | gamma-globulin detection method |
| CN114431560A (en) * | 2021-12-30 | 2022-05-06 | 广州市赛特检测有限公司 | Mask for rapidly detecting new coronavirus and biological probe modification method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4422544B2 (en) * | 2003-05-23 | 2010-02-24 | 関東化学株式会社 | Side chain conductive polymer |
| CN1594314A (en) * | 2004-06-24 | 2005-03-16 | 复旦大学 | Fluorene based water soluble conjugated polymer and process for preparing same |
-
2005
- 2005-08-02 CN CNB2005100192074A patent/CN100458417C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1987466B (en) * | 2006-12-22 | 2011-08-31 | 武汉大学 | In-situ preparing method for micro porous plate pH sensor |
| CN101864294A (en) * | 2010-06-09 | 2010-10-20 | 中国科学院上海微***与信息技术研究所 | Composite sensing material of fluorescent conjugated polymer and organic metal complex and application |
| CN102516990A (en) * | 2011-11-30 | 2012-06-27 | 东南大学 | Application of polyphenylene vinylene (PPV) polymer serving as fluorescent indicator in oxygen sensor based on fluorescence quenching |
| CN102516990B (en) * | 2011-11-30 | 2013-12-18 | 东南大学 | Application of polyphenylene vinylene (PPV) polymer serving as fluorescent indicator in oxygen sensor based on fluorescence quenching |
| CN103674913A (en) * | 2013-12-04 | 2014-03-26 | 南京邮电大学 | Fluorescence method and kit for testing lymphocyte homing receptor |
| CN103674913B (en) * | 2013-12-04 | 2015-10-21 | 南京邮电大学 | A kind of fluorescent method and kit thereof detecting lymphocyte homing receptor |
| CN105115945A (en) * | 2015-06-26 | 2015-12-02 | 安徽师范大学 | gamma-globulin detection method |
| CN105115945B (en) * | 2015-06-26 | 2018-01-19 | 安徽师范大学 | The detection method of gamma Globulin |
| CN114431560A (en) * | 2021-12-30 | 2022-05-06 | 广州市赛特检测有限公司 | Mask for rapidly detecting new coronavirus and biological probe modification method |
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| Publication number | Publication date |
|---|---|
| CN100458417C (en) | 2009-02-04 |
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