WO2013026123A1 - Utilização de um nanoporo proteico para detecção, identificação, quantificação e monitoramento em tempo real de microcistinas em sistemas aquosos - Google Patents
Utilização de um nanoporo proteico para detecção, identificação, quantificação e monitoramento em tempo real de microcistinas em sistemas aquosos Download PDFInfo
- Publication number
- WO2013026123A1 WO2013026123A1 PCT/BR2012/000322 BR2012000322W WO2013026123A1 WO 2013026123 A1 WO2013026123 A1 WO 2013026123A1 BR 2012000322 W BR2012000322 W BR 2012000322W WO 2013026123 A1 WO2013026123 A1 WO 2013026123A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- microcystin
- microcystins
- nanopore
- aqueous
- interaction
- Prior art date
Links
- 108010049746 Microcystins Proteins 0.000 title claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 13
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 13
- 238000001514 detection method Methods 0.000 title claims abstract description 10
- 238000012544 monitoring process Methods 0.000 title claims abstract description 8
- 238000011002 quantification Methods 0.000 title claims abstract description 7
- SRUWWOSWHXIIIA-UKPGNTDSSA-N Cyanoginosin Chemical compound N1C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](C)[C@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C(=C)N(C)C(=O)CC[C@H](C(O)=O)N(C)C(=O)[C@@H](C)[C@@H]1\C=C\C(\C)=C\[C@H](C)[C@@H](O)CC1=CC=CC=C1 SRUWWOSWHXIIIA-UKPGNTDSSA-N 0.000 claims abstract description 23
- 108010067094 microcystin Proteins 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000012736 aqueous medium Substances 0.000 claims abstract description 8
- 101710092462 Alpha-hemolysin Proteins 0.000 claims abstract description 5
- 101710197219 Alpha-toxin Proteins 0.000 claims abstract description 5
- 101710124951 Phospholipase C Proteins 0.000 claims abstract description 5
- 239000002776 alpha toxin Substances 0.000 claims abstract description 5
- 239000003053 toxin Substances 0.000 claims abstract description 4
- 231100000765 toxin Toxicity 0.000 claims abstract description 4
- 108700012359 toxins Proteins 0.000 claims abstract description 4
- 230000003993 interaction Effects 0.000 claims abstract 6
- 230000008569 process Effects 0.000 claims description 13
- 230000000903 blocking effect Effects 0.000 claims description 6
- 230000004888 barrier function Effects 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 2
- 230000001419 dependent effect Effects 0.000 claims 3
- 239000012466 permeate Substances 0.000 claims 2
- 239000000232 Lipid Bilayer Substances 0.000 abstract description 4
- 230000002596 correlated effect Effects 0.000 abstract description 2
- 230000002123 temporal effect Effects 0.000 abstract 2
- 230000002068 genetic effect Effects 0.000 abstract 1
- 230000004941 influx Effects 0.000 abstract 1
- 239000011159 matrix material Substances 0.000 abstract 1
- 238000005457 optimization Methods 0.000 abstract 1
- 238000007619 statistical method Methods 0.000 abstract 1
- 239000012528 membrane Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 2
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000007781 pre-processing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- GTKRFUAGOKINCA-UHFFFAOYSA-M chlorosilver;silver Chemical compound [Ag].[Ag]Cl GTKRFUAGOKINCA-UHFFFAOYSA-M 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
Definitions
- the present invention relates to a process based on the use of a protein nanopore for detection, identification, quantification and real-time monitoring of low molecular weight toxins, specifically microcystins in aqueous media, as well as a device for performing the assay. process.
- the protein phosphatase inhibition assay is very sensitive, but it requires highly purified proteins that are difficult to make available on the market and still require the use of radioactive phosphate, which considerably increases its operation, as it requires compliance with safety standards. for handling radioactive material.
- the physicochemical processes are analytical, and consider the physicochemical properties of microcystins, such as the presence of chromophor groups sensitive to ultraviolet radiation (UV) present in the molecular structure of these cyanotoxins, whose reactivity is due to specific functional factors, and also in their structure or molecular weight.
- Physical-chemical processes include: high performance liquid chromatography (HPLC), capillary electrophoresis (EC), nuclear magnetic resonance (NMR) and mass spectroscopy (MS).
- HPLC high purity liquid crystal
- the molecular recognition element is a nanostructure, the protein nanopore formed by native alfatoxin, deposited and referenced in the Protein Data Bank under code - PDB ID 7AHL.
- US2010122907-A1 is known for the use of alpha-toxin nanopore to determine the molecular mass of neutral polymers, specifically polyethylene glycol, but the use of this nanopore for detection, identification, quantification and monitoring of variants is unknown. microcystins.
- Alfatoxin protein nanopore is a nanostructure formed by seven monomeric subunits, which self-insert into a lipid bilayer, creating an aqueous pathway for the passage of different particles, provided they have a diameter smaller than the narrowest region of the Nanopore.
- the three-dimensional crystal structure and stoichiometry of this nanopore are known.
- the aqueous pore geometry and its asymmetric positioning in relation to the lipid membrane plane have also been elucidated under dynamic conditions using non-electrolytic substances.
- the alpha-toxin nanopore presents high stability and ionic conductance, easy incorporation into natural membranes and synthetic flat lipid bilayers.
- FIG 1 there is a schematic of the protein nanopore (1) inserted in the lipid membrane (2) constructed in a resistive barrier (3) that separates two conductive reservoirs (I and II), and in (B) , the registration of the ionic current captured by one of the electrodes (4).
- the mechanism of detection of microcystins (5) by the alpha-toxin nanopore occurs by the discretized transient change, now called the blocking event ( Figure 1B), in the ionic current that flows through the aqueous nanopore lumen upon permeation of a microcystin molecule through one of the nanopore inlets.
- Figure 1B shows the interlock time (1), which corresponds to the UNOCOCATED nanopore, and the blocking event that is characterized by amplitude (2) and duration time (3).
- the amplitude depends on the relative volume occupied by microcystin when present in the nanopore, and corresponds to the decrease in nanopore conductance, compared to the situation in which microcystin does not occupy it; whereas the duration of the event corresponds to the residence time of a microcystin molecule in the aqueous flame, ie the time when the nanopore is BUSY.
- Figure 2 represents that the time series of blocking events along with interlocking times correlated with the structural variant of microcystin, therefore, is a kind of its "DIGITAL IMPRESSION" (A), and that the analysis of the time series of blocking events relative to each average conductance value is operationalized by plotting a histogram of all times, generating a characteristic time distribution of each microcystin variant (B).
- the frequency of blocking events that is, the interlock time interval depends on the concentration of the structural variants of the microcystin present in the solution contained in the reservoir from which it comes.
- Figure 3 is a schematic representation of the modular diagram of the method of analysis and the difference between the mean values of nanopore conductance in the absence and presence of microcystin variants in the aqueous nanopore flame, hereafter referred to as residual conductance, which allows, through a two-dimensional graph, real-time identification of structural variants of microcystins.
- Figure 4 represents the analysis of interlocking times, which represent the absence of microcystin inside the nanopore, called henceforth, characteristic non-occupation time, represented by ⁇ ⁇ ⁇
- the inverse form, 1 / ⁇ ⁇ called Now, as a transition rate, it is proportional to the concentration of microcystin, which allows to determine the concentration of microcystin in the solution.
- Figure 5 illustrates the mechanical assembly of the apparatus, that is, the experimental chamber (1) employed to perform the process.
- the two reservoirs (I) and (II) in which ionic solutions are placed are separated by a resistive barrier (2) composed of a nonconductive film which has in its central region a small circular hole of 50 ⁇ diameter.
- a lipid bilayer (3) is constructed.
- Each reservoir containing electrolyte solution is electrically coupled to the high impedance amplifier configured as a current-voltage converter (not shown in the figure) by silver-chloride-silver electrode (Ag-AgCl) (4) maintained on saline bridges of the type 2% agarose in 3M potassium chloride, housed in plastic tips with a volume of 200 ⁇ , not shown in the figure.
- the current flowing through the nanopore is conditioned by a Butterworth low-pass filter, then digitalized by a recording system (5) formed by an analog-to-digital converter board, and finally stored directly in a microcomputer's memory. represented in the figure).
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- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Nanotechnology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| BRMU9102088U BRMU9102088U2 (pt) | 2011-08-25 | 2011-08-25 | utilização de um nanoporo protéico para detecção, identificação, quantificação e monitoramento em tempo real de microcistinas em sistemas aquosos |
| BRMU9102088-3 | 2011-08-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2013026123A1 true WO2013026123A1 (pt) | 2013-02-28 |
Family
ID=47745792
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/BR2012/000322 WO2013026123A1 (pt) | 2011-08-25 | 2012-08-29 | Utilização de um nanoporo proteico para detecção, identificação, quantificação e monitoramento em tempo real de microcistinas em sistemas aquosos |
Country Status (2)
| Country | Link |
|---|---|
| BR (1) | BRMU9102088U2 (it) |
| WO (1) | WO2013026123A1 (it) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103512934A (zh) * | 2013-10-14 | 2014-01-15 | 无锡艾科瑞思产品设计与研究有限公司 | 一种在线监测水体中藻毒素-lr的方法及装置 |
| CN103830280B (zh) * | 2014-03-11 | 2017-03-22 | 丽江广润生物科技有限公司 | 一种螺旋藻提取物的制备方法 |
| CN111323469A (zh) * | 2020-02-14 | 2020-06-23 | 中国科学院重庆绿色智能技术研究院 | 一种基于纳米孔水解反应的免疫球蛋白m检测方法 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001059453A2 (en) * | 2000-02-11 | 2001-08-16 | The Texas A & M University System | Biosensor compositions and methods of use |
| WO2003095669A1 (en) * | 2002-05-10 | 2003-11-20 | The Texas A & M University System | Stochastic sensing through covalent interactions |
| WO2007084103A2 (en) * | 2004-12-21 | 2007-07-26 | The Texas A & M University System | High temperature ion channels and pores |
| WO2009077734A2 (en) * | 2007-12-19 | 2009-06-25 | Oxford Nanopore Technologies Limited | Formation of layers of amphiphilic molecules |
| US20100072080A1 (en) * | 2008-05-05 | 2010-03-25 | The Regents Of The University Of California | Functionalized Nanopipette Biosensor |
| US20100122907A1 (en) * | 2008-05-06 | 2010-05-20 | Government of the United States of America, | Single molecule mass or size spectrometry in solution using a solitary nanopore |
-
2011
- 2011-08-25 BR BRMU9102088U patent/BRMU9102088U2/pt not_active Application Discontinuation
-
2012
- 2012-08-29 WO PCT/BR2012/000322 patent/WO2013026123A1/pt active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001059453A2 (en) * | 2000-02-11 | 2001-08-16 | The Texas A & M University System | Biosensor compositions and methods of use |
| WO2003095669A1 (en) * | 2002-05-10 | 2003-11-20 | The Texas A & M University System | Stochastic sensing through covalent interactions |
| WO2007084103A2 (en) * | 2004-12-21 | 2007-07-26 | The Texas A & M University System | High temperature ion channels and pores |
| WO2009077734A2 (en) * | 2007-12-19 | 2009-06-25 | Oxford Nanopore Technologies Limited | Formation of layers of amphiphilic molecules |
| US20100072080A1 (en) * | 2008-05-05 | 2010-03-25 | The Regents Of The University Of California | Functionalized Nanopipette Biosensor |
| US20100122907A1 (en) * | 2008-05-06 | 2010-05-20 | Government of the United States of America, | Single molecule mass or size spectrometry in solution using a solitary nanopore |
Non-Patent Citations (2)
| Title |
|---|
| JANILSON JOSE DA SILVA JUNIOR: "Utilizaçao de Nanoporo protéicos no desenvolvimento de Sensores", TESE DE MESTRADO UNIVERSIDADE FEDERAL DE PERNANBUCO, vol. 1, 1 August 2011 (2011-08-01), pages 85 * |
| SILVA JUNIOR ET AL.: "NANOPORO PROTEICO FORMADO PELA ALFATOXINA COMO BIOSSENSORES DE MOLECULAS EM SISTEMAS AQUOSOS.", 1° ENCONTRO BRASILEIRO PARA INOVAÇÃO TERAPÊUTICA, 2009 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103512934A (zh) * | 2013-10-14 | 2014-01-15 | 无锡艾科瑞思产品设计与研究有限公司 | 一种在线监测水体中藻毒素-lr的方法及装置 |
| CN103830280B (zh) * | 2014-03-11 | 2017-03-22 | 丽江广润生物科技有限公司 | 一种螺旋藻提取物的制备方法 |
| CN111323469A (zh) * | 2020-02-14 | 2020-06-23 | 中国科学院重庆绿色智能技术研究院 | 一种基于纳米孔水解反应的免疫球蛋白m检测方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| BRMU9102088U2 (pt) | 2016-04-26 |
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