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 PDF

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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
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WO
WIPO (PCT)
Prior art keywords
microcystin
microcystins
nanopore
aqueous
interaction
Prior art date
Application number
PCT/BR2012/000322
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English (en)
French (fr)
Portuguese (pt)
Inventor
Cláudio Gabriel RODRIGUES
Sergio Fernandovith CHEVTCHENKO
Oleg Vladimirovich KRASILNIKOV
Dijanah Cota MACHADO
Juliana PEREIRA DE AGUIAR
Janilson José da SILVA JUNIOR
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Universidade Federal De Pernambuco - Ufpe
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Application filed by Universidade Federal De Pernambuco - Ufpe filed Critical Universidade Federal De Pernambuco - Ufpe
Publication of WO2013026123A1 publication Critical patent/WO2013026123A1/pt

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material
    • G01N33/48707Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721Investigating 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)
PCT/BR2012/000322 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 WO2013026123A1 (pt)

Applications Claiming Priority (2)

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BRMU9102088-3 2011-08-25
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

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Cited By (3)

* Cited by examiner, † Cited by third party
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)

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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

Patent Citations (6)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

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