WO2013026123A1 - Use of a protein nanopore for the detection, identification, quantification and real-time monitoring of microcystins in aqueous systems - Google Patents
Use of a protein nanopore for the detection, identification, quantification and real-time monitoring of microcystins in aqueous systems Download PDFInfo
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- 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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- 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).
Abstract
According to the present invention, there are several methods for detecting microcystins, which have a limited capacity to adequately identify and monitor in real time structural variants of said toxins. The present inventive method and inventive device enable the detection, identification, quantification and real-time monitoring of microcystins in aqueous media. The inventive method is based on the fact that the influx and interaction of microcystins in the aqueous opening of a protein nanopore, formed by alpha-toxin incorporated into a resistive matrix, induces discretized events in the ion current that flows through the nanopore. The temporal series of said events is representative of and correlated with the microcystin structural variant present in the solution contained in one of the conductive reservoirs. The segmental statistical analysis of the temporal series, combining the method of least squares and genetic optimization algorithm, allows real-time discrimination of the microcystin variants. The inventive device consists basically of a nanopore incorporated into a lipid bilayer that separates two conductive reservoirs, wherein the microcystins are placed in one of the reservoirs.
Description
"UTILIZAÇÃO DE UM NANOPORO PROTEICO PARA DETECÇÃO, IDENTIFICAÇÃO, QUANTIFICAÇÃO E MONITORAMENTO EM TEMPO REAL DE MICROCISTINAS EM SISTEMAS AQUOSOS". "USE OF A PROTEIN NANOPOR FOR DETECTION, IDENTIFICATION, QUANTIFICATION AND REAL-TIME MONITORING OF MICROCYSTINS IN WATER SYSTEMS".
A presente invenção refere-se a um processo baseado na utilização de um nanoporo protéico para detecção, identificação, quantificação e monitoramento em tempo real de toxinas de baixo peso molecular, especificamente, as microcistinas em meios aquosos, bem como a um dispositivo para realização do processo. 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.
Sabe-se que dois principais processos são atualmente empregados para detecção e identificação de microcistinas e outras cianotoxinas em meios aquosos, baseado principalmente em ensaios biológicos, bioquímicos e em suas propriedades físico- químicas. Estes processos diferem em seus princípios de detecção, identificação, complexidade de execução e custos operacionais. Dentre os processos bioquímicos normalmente realiza-se o ensaio imunoenzimático (ELISA) e o ensaio de inibição da proteína fosfatase. O ELISA apesar de apresentar sensibilidade adequada, seu principal empecilho de ordem técnica, consiste nas reações cruzadas que podem levar a subestimação da concentração das cianotoxinas analisadas. Outrossim, o ensaio de inibição da proteína fosfatase é bastante sensível, porém, requer proteínas altamente purificadas de difícil disponibilização no mercado e ainda necessitam da utilização de fosfato radioativo, o que onera consideravelmente sua operacionalização, uma vez que exige o cumprimento das normas de segurança para manipulação de material radioativo. Os processos físico-químicos por sua vez são analíticos, e consideram as propriedades físico-químicas das microcistinas, tais como, a presença de grupos cromóforos sensíveis à radiação ultravioleta (UV) presentes na estrutura molecular destas cianotoxinas, cuja reatividade deve-se a grupos funcionais específicos, e também em sua estrutura ou peso molecular. Dentre os processos físico-químicos utilizam-se: cromatografia líquida de alta performance (HPLC), eletroforese capilar (EC), ressonância magnética nuclear (NMR) e a espectroscopia de massa (MS). A principal desvantagem da HPLC consiste na impossibilidade de identificação de variantes estruturais presentes em amostras de microcistinas, além da ausência de padrões específicos que podem inviabilizar sua ampla utilização, uma vez que, a identificação da amostra se dá pelo tempo de retenção. A EC apresenta baixa sensibilidade e também se faz necessário o pré-processamento da amostra no sentido de produzir produtos fluorescentes capazes de serem sensibilizados por um laser de comprimento de onda específico. Por outro lado a espectroscopia de massa e a ressonância magnética nuclear apesar de serem métodos eficientes para a determinação da estrutura molecular são bastante onerosas; e enquanto que o primeiro é destrutivo (impossibilitando o reaproveitamento da amostra analisada, para realização de contraprovas), no segundo é indispensável uma quantidade razoável (normalmente miligramas) de material de elevado grau de pureza. It is known that two main processes are currently employed for detection and identification of microcystins and other cyanotoxins in aqueous media, based mainly on biological, biochemical and physicochemical properties. These processes differ in their principles of detection, identification, complexity of execution and operating costs. Among the biochemical processes, the enzyme-linked immunosorbent assay (ELISA) and the protein phosphatase inhibition assay are usually performed. Although ELISA presents adequate sensitivity, its main technical impediment is the cross-reactions that may lead to underestimation of the concentration of cyanotoxins analyzed. Moreover, 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). The main disadvantage of HPLC is the impossibility of identifying structural variants present in microcystin samples, besides the absence of specific patterns that can make its wide use unfeasible, since the identification of the sample is due to the retention time. EC has low sensitivity and sample pre-processing is also required to produce fluorescent products capable of being sensitized by a specific wavelength laser. On the other hand, mass spectroscopy and nuclear magnetic resonance, although efficient methods for determining molecular structure, are quite costly; and while the former is destructive (making it impossible to reuse the analyzed sample for counterproofing), the latter requires a reasonable amount (usually milligrams) of high purity material.
Todos estes processos físico-químicos requerem pessoal altamente especializado e são muito onerosos para serem empregados de forma rotineira e em grande escala.
Adicionalmente todos os processos supracitados não permitem o momtoramento em tempo real de microcistinas, uma vez que, se faz necessário a coleta da amostra, na maioria das vezes, seu pré-processamento e finalmente a análise propriamente dita. All of these physicochemical processes require highly skilled personnel and are too costly to employ on a routine and large scale. In addition, all the above processes do not allow real-time microcystin monitoring, since it is necessary to collect the sample, most of the time, its preprocessing and finally the analysis itself.
Nesta invenção utilizasse os conhecimentos da biomimética e se desenvolveu um processo para detecção, identificação, quantificação e monitoramento em tempo real de microcistinas em meio aquoso. O elemento de reconhecimento molecular é uma a nanoestrutura, o nanoporo protéico formado pela alfatoxina nativa, depositada e referenciada no Protein Data Bank sob código - PDB ID 7AHL. In this invention the biomimetic knowledge was used and a process for detection, identification, quantification and real time monitoring of microcystins in aqueous medium was developed. 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.
Sabe-se, por exemplo, da patente US2010122907-A1 da utilização do nanoporo da alfatoxina para determinação da massa molecular de polímeros neutros, especificamente o polietilenoglicol, porém, desconhecesse o uso deste nanoporo como elemento de detecção, identificação, quantificação e monitoramento de variantes estruturais de microcistinas. For example, 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.
O nanoporo protéico da alfatoxina (aHL) é uma nanoestrutura formada por sete subunidades monoméricas, que se auto-inserem em uma bicamada lipídica, criando uma via aquosa para a passagem de diferentes partículas, desde, que apresentem diâmetro menor que a região mais estreita do nanoporo. A estrutura cristalina tridimensional e a estequiometria deste nanoporo são conhecidas. A geometria do poro aquoso e seu posicionamento assimétrico em relação ao plano da membrana lipídica, também já foram elucidados em condições dinâmicas empregando substâncias não eletrolíticas. Existem duas regiões do poro, uma com diâmetro de 4,6 nm que fica praticamente fora da membrana, e outra, que fica totalmente inserido na membrana e tem diâmetro de ~2 nm, sendo composto por 14 folhas β-barril. Estes dois domínios estão separados por uma constrição de -1 ,4 nm de diâmetro. Adicionalmente o nanoporo formado pela alfatoxina apresenta elevada estabilidade e condutância iônica, facilidade de incorporação em membranas naturais e bicamadas lipídicas planas sintéticas. Alfatoxin protein nanopore (aHL) 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. There are two regions of the pore, one with a diameter of 4.6 nm that is practically outside the membrane, and another that is fully inserted in the membrane and has a diameter of ~ 2 nm, consisting of 14 β-barrel leaves. These two domains are separated by a constriction of -1.4 nm in diameter. Additionally, the alpha-toxin nanopore presents high stability and ionic conductance, easy incorporation into natural membranes and synthetic flat lipid bilayers.
Na figura 1, (A), esta representado um esquema do nanoporo protéico (1) inserido na membrana lipídica (2) construída em uma barreira resistiva (3) que separa dois reservatórios condutivos (I e II), e, na (B), o registro da corrente iônica captada por um dos eletrodos (4). O mecanismo de detecção de microcistinas (5) pelo nanoporo da alfatoxina se dá pela alteração transitória discretizada, denominada de agora em diante de evento de bloqueio (figura 1B), na corrente iônica que flui através do lume aquoso do nanoporo, quando da permeação de uma molécula de microcistina por uma das entradas do nanoporo. Na figura 1 B é demonstrado o tempo interbloqueio (1), que corresponde ao nanoporo DESOCUPADO, e, o evento de bloqueio que é caracterizado por amplitude (2) e tempo de duração (3). A amplitude depende do volume relativo ocupado pela microcistina quando presente no nanoporo, e, corresponde a diminuição na condutância do nanoporo, comparativamente a situação em que a microcistina não o ocupa; enquanto que o tempo de duração do evento corresponde ao tempo de residência de uma molécula de microcistina no lume aquoso, ou seja, o tempo em que o nanoporo fica OCUPADO.
A figura 2 representa que a série temporal dos eventos de bloqueios juntamente com os tempos interbloqueios correlacionasse com a variante estrutural da microcistina, portanto, é uma espécie de sua "IMPRESSÃO DIGITAL" (A), e que a análise da série temporal dos eventos de bloqueio relativos a cada valor médio de condutância, é operacionalizada traçando-se um histograma de todos os tempos, gerando uma distribuição de tempos característicos de cada variante da microcistina (B). Igualmente a frequência dos eventos de bloqueio, ou seja, o intervalo de tempo interbloqueios depende da concentração das variantes estruturais da microcistina presente na solução contida no reservatório de onde ela advém. In Figure 1, (A), 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). Likewise, 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.
Na figura 3 representa-se de forma esquemática o diagrama modular do método de análise, e, a diferença entre os valores médios da condutância do nanoporo na ausência e na presença das variantes da microcistina no lume aquoso do nanoporo, de agora em diante denominada, condutância residual, que permite, através de um gráfico bidimensional, a identificação em tempo real das variantes estruturais de microcistinas. 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.
Na figura 4 representasse a análise dos tempos interbloqueios, que representam a ausência de microcistina no interior do nanoporo, denominado de agora em diante, tempo característico de não-ocupação, representado por τοη· A forma inversa, 1/τοη, denominado de agora em diante como taxa de transição, é proporcional a concentração da microcistina, o que permite determinar a concentração de microcistina na solução. 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 τ οη · The inverse form, 1 / τ οη , 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.
A figura 5 ilustra a montagem mecânica do aparato, ou seja, a câmara experimental (1) empregada para realização do processo. Os dois reservatórios (I) e (II) onde se colocam soluções iónicas, são separados por uma barreira resistiva (2) composta por uma película não condutora que apresenta em sua região central, um pequeno orifício circular de 50μΜ de diâmetro. Neste orifício usando lipídeo sintético, constrói-se uma bicamada lipídica (3). Cada um dos reservatórios contendo solução eletrolítica é acoplado eletricamente ao amplificador de alta impedância configurado como conversor corrente-voltagem (não representado na figura), através de eletrodo prata-cloreto de prata (Ag-AgCl) (4) mantidos em pontes salinas do tipo agarose 2% em cloreto de potássio 3M, acomodado em ponteiras plásticas com volume de 200 ί, não esquematizada na figura. A corrente que flui através do nanoporo é condicionada por meio de filtro passa-baixa do tipo Butterworth, posteriormente digitalizada por um sistema de registro (5) formado por uma placa conversora analógico-digital, e finalmente armazenada diretamente na memória de um microcomputador (não representado na figura).
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 50μΜ diameter. In this hole using synthetic lipid, 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 ί, 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).
Claims
1. Processo para detecção de toxinas de baixo peso molecular, especificamente microcistinas em meio aquoso, caracterizado pelo fato de que a interação de moléculas de microcistinas através do lume aquoso do nanoporo protéico formado pela alfatoxina nativa, gera alterações discretizadas típicas no fluxo da corrente iônica que o permeia e, que estas alterações são dependentes desta classe de toxinas. 1. A process for the detection of low molecular weight toxins, specifically microcystins in aqueous medium, characterized by the fact that the interaction of microcystin molecules through the aqueous lumen of protein formed by native alfatoxin generates typical discretized changes in ionic current flow. that permeates it and that these changes are dependent on this class of toxins.
2. Processo para identificação de variantes estruturais de microcistinas em meio aquoso, caracterizado pelo fato de que a interação de cada variante estrutural da microcistina através do lume aquoso do nanoporo protéico formado pela alfatoxina nativa, provoca alterações discretizadas específicas no fluxo de corrente iônica que o permeia, e, que estas alterações são dependentes das propriedades estruturais de cada variante da microcistina. 2. A process for the identification of structural variants of microcystins in aqueous medium, characterized by the fact that the interaction of each structural variant of microcystin through the aqueous lumen of protein formed by native alfatoxin causes specific discretized changes in the ionic current flow that and that these changes are dependent on the structural properties of each microcystin variant.
3. Processo para quantificação de microcistinas em meio aquoso, caracterizado pelo fato de que a interação de cada variante estrutural da microcistina através do lume aquoso do nanoporo protéico formado pela alfatoxina nativa, provoca alterações discretizadas específicas no fluxo de corrente iônica que o permeia, e, que a distribuição dos intervalos de tempo entre os eventos sucessivos de bloqueio de corrente iônica provocados por interação de cada variante estrutural da microcistina com o lume aquoso do nanoporo protéico, são dependentes da concentração ou quantidade destas moléculas no reservatório de onde elas advêm. A process for quantification of microcystins in aqueous medium, characterized by the fact that the interaction of each structural variant of microcystin through the aqueous lumen of the protein nanopore formed by native alfatoxin causes specific discretized changes in the ionic current flow that permeates it, and , that the distribution of time intervals between successive ionic current blocking events caused by the interaction of each structural variant of microcystin with the aqueous nanopore protein fire, are dependent on the concentration or quantity of these molecules in the reservoir from which they originate.
4. Processo para o monitoramento em tempo real de microcistinas em meio aquoso, caracterizado pelo fato de que a interação de cada variante estrutural da microcistina através do lume aquoso do nanoporo protéico formado pela alfatoxina nativa, provoca alterações discretizadas específicas para cada microcistina e que sua comparação com um banco de dados permite a identificação instantânea de cada molécula de microcistina que interagir com o nanoporo. 4. A process for real-time monitoring of microcystins in aqueous medium, characterized by the fact that the interaction of each structural variant of microcystin through the aqueous lumen of protein formed by native alpha-toxin causes specific discretized changes for each microcystin and its Comparison with a database allows instant identification of each microcystin molecule that interacts with the nanopore.
5. Dispositivo de acordo com as reivindicações 1 , 2, 3 e 4 caracterizado por uma câmara experimental composta por material isolante e inerte, dividida em dois reservatórios condutores separados por uma barreira resistiva, no qual há uma única via de condução elétrica. Device according to claims 1, 2, 3 and 4, characterized by an experimental chamber composed of insulating and inert material, divided into two conductive reservoirs separated by a resistive barrier, in which there is a single electric conduction path.
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| BRMU9102088U BRMU9102088U2 (en) | 2011-08-25 | 2011-08-25 | use of a protein nanopore for detection, identification, quantification and real time monitoring of microcystins in aqueous systems |
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| CN103830280B (en) * | 2014-03-11 | 2017-03-22 | 丽江广润生物科技有限公司 | Preparation method of spirulina extract |
| CN111323469A (en) * | 2020-02-14 | 2020-06-23 | 中国科学院重庆绿色智能技术研究院 | Immunoglobulin M detection method based on nanopore hydrolysis reaction |
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| 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/en not_active Application Discontinuation
-
2012
- 2012-08-29 WO PCT/BR2012/000322 patent/WO2013026123A1/en active Application Filing
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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 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103512934A (en) * | 2013-10-14 | 2014-01-15 | 无锡艾科瑞思产品设计与研究有限公司 | Method and device for monitoring algal toxin-LR in water body on line |
| CN103830280B (en) * | 2014-03-11 | 2017-03-22 | 丽江广润生物科技有限公司 | Preparation method of spirulina extract |
| CN111323469A (en) * | 2020-02-14 | 2020-06-23 | 中国科学院重庆绿色智能技术研究院 | Immunoglobulin M detection method based on nanopore hydrolysis reaction |
Also Published As
| Publication number | Publication date |
|---|---|
| BRMU9102088U2 (en) | 2016-04-26 |
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