ES2319018B1 - METHOD FOR SEPARATION AND DETECTION OF PROTOZOARS. - Google Patents
METHOD FOR SEPARATION AND DETECTION OF PROTOZOARS. Download PDFInfo
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- 230000019469 detection of protozoan Effects 0.000 claims abstract description 7
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- 210000004369 blood Anatomy 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
Método para separación y detección de protozoarios.Method for separation and detection of protozoa
Un método que permite la separación y detección de protozoarios, con potencial para extenderse a especies de fisiología similar, a través de la ingestión por los mismos de material magnético biocompatible. El método tiene las siguientes etapas: realizar un cultivo de la especie con ferrofluido biocompatible consistente en nanopartículas de magnetita de tamaños en el rango aproximado de 20 < d < 150 nm recubiertas con dextrano, en concentraciones de 10 a 80 mg/ml, separar la población de parásitos que han incorporado efectivamente las partículas magnéticas, a través de centrifugación en gradiente discontinuo de densidades; y detectar la población de parásitos magnetizados a través de un magnetómetro o susceptómetro, que permite medir la imantación del cultivo; y comparar la imanación del cultivo con la del ferrofluido puro como referencia.A method that allows separation and detection of protozoa, with the potential to spread to species of similar physiology, through ingestion by them of biocompatible magnetic material. The method has the following stages: carry out a culture of the species with ferrofluid biocompatible consisting of magnetite nanoparticles of sizes in the approximate range of 20 <d <150 nm coated with dextran, in concentrations of 10 to 80 mg / ml, separate population of parasites that have effectively incorporated the particles magnetic, through discontinuous gradient centrifugation of densities; and detect the population of magnetized parasites to through a magnetometer or susceptometer, which allows you to measure the crop magnetization; and compare the magnetization of the crop with the of pure ferrofluid as a reference.
Description
Método para separación y detección de protozoarios.Method for separation and detection of protozoa
El objeto de la presente invención es un método que permite la separación y detección de protozoarios a través de la ingestión por los mismos de material magnético biocompatible, con potencial para extenderse a otros organismos unicelulares eucariotas (reino Protista) de fisiología relacionada tales como algas, bacterias, y hongos.The object of the present invention is a method which allows the separation and detection of protozoa through their ingestion of biocompatible magnetic material, with potential to spread to other single-celled organisms eukaryotes (Protist kingdom) of related physiology such as Algae, bacteria, and fungi.
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La propagación de diversas enfermedades infecciosas a través de especies de protozoarios en animales y humanos es uno de los principales problemas de la salud pública en diversos países en desarrollo. En un considerable número de estas enfermedades (por ej. Tripanosomiasis Africana o Americana, o Leishmaniasis), no se dispone hasta hoy de vacunas para prevenirlas ni medicamentos eficientes para su tratamiento. Las alternativas terapéuticas actualmente disponibles presentan problemas tales como poca eficiencia terapéutica, efectos colaterales severos y el surgimiento de cepas resistentes [ver referencia SIL05].The spread of various diseases infectious through protozoan species in animals and humans is one of the main public health problems in Various developing countries. In a considerable number of these diseases (eg African or American trypanosomiasis, or Leishmaniasis), vaccines to prevent them are not available until today or efficient medications for treatment. The alternatives currently available therapies present problems such as poor therapeutic efficiency, severe side effects and emergence of resistant strains [see reference SIL05].
La tripanosomiasis que presenta la mayor diversidad de complicaciones clínicas es la americana, siendo caracterizada por una fase inicial (aguda), con una parasitemia evidente, seguida de una fase indeterminada, asintomática, que evoluciona a una fase crónica, con sintomatologías características. Dependiendo de cada fase, los exámenes recomendados para la detección en laboratorio incluyen: observación directa de sangre (frotis y gota gruesa), técnicas de concentración de sangre (Strout, microstrout), hemocultivo e incluso métodos indirectos serológicos (hemaglutinación directa o indirecta, inmunofluorescencia indirecta y ELISA) [ver referencia WHO06]. Estos métodos son complejos y costosos. Por otro lado, métodos tales como la reacción en cadena de la polimerasa (PCR por sus siglas Polymerase Chain Reaction en inglés) todavía no forma parte de la rutina laboratorial diagnóstica, por lo tanto hasta ahora ésta técnica sólo está disponible en laboratorios de investigación.Trypanosomiasis presenting the greatest diversity of clinical complications is the American, being characterized by an initial (acute) phase, with a parasitemia evident, followed by an indeterminate, asymptomatic phase, which It evolves into a chronic phase, with characteristic symptoms. Depending on each phase, the recommended exams for Laboratory detection include: direct blood observation (smear and thick drop), blood concentration techniques (Strout, microstrout), blood culture and even indirect methods serological (direct or indirect hemagglutination, indirect immunofluorescence and ELISA) [see reference WHO06]. These Methods are complex and expensive. On the other hand, methods such as polymerase chain reaction (PCR) Polymerase Chain Reaction in English) is not yet part of the diagnostic laboratory routine, so far this one Technique is only available in research laboratories.
Frente a estas dificultades para establecer exámenes fiables en clínicas y laboratorios, la posibilidad de detección de parásitos a través de técnicas magnéticas conlleva la evidente ventaja de ser un método no invasivo, rápido y de bajo costo.Facing these difficulties to establish reliable examinations in clinics and laboratories, the possibility of Detection of parasites through magnetic techniques involves the obvious advantage of being a non-invasive, fast and low method cost.
Los usos previamente reportados de nanopartículas magnéticas en bacterias o unidades biológicas menores están relacionados a la aplicación de gradientes de campo magnético para separación y fijación [GU05,BUC03]. Sin embargo, la presente propuesta esta basada en la inclusión (fagocitosis) de las nanopartículas magnéticas por los protozoarios, y posterior análisis y detección por técnicas magnetométricas estándar.The previously reported uses of magnetic nanoparticles in bacteria or biological units minors are related to the application of field gradients magnetic for separation and fixing [GU05, BUC03]. However, the This proposal is based on the inclusion (phagocytosis) of the magnetic nanoparticles by the protozoa, and subsequent analysis and detection by standard magnetometric techniques.
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La presente invención se refiere a un método para la detección y cuantificación de la concentración de parásitos presentes en una muestra de sangre. La técnica propuesta utiliza como elemento básico una suspensión coloidal biocompatible de magnetita o hierro en base acuosa, de concentración variable. El material magnético consiste en una distribución de nanopartículas magnéticas, compuestas de núcleos magnéticos de magnetita (Fe_{3}O_{4}) de <d> = 20 nm y coberturas de polisacáridos (por ej. Dextrano) o péptidos.The present invention relates to a method for the detection and quantification of parasite concentration present in a blood sample. The proposed technique uses as a basic element a biocompatible colloidal suspension of magnetite or water-based iron, of variable concentration. He magnetic material consists of a distribution of nanoparticles magnetic, composed of magnetite magnetic cores (Fe_ {O} {4}) of <d> = 20 nm and coverage of polysaccharides (eg Dextran) or peptides.
Este método permite la separación y detección de protozoarios (ya verificada en dos especies, y con potencial para extenderse a especies de fisiología similar) a través de la ingestión de material magnético biocompatible. La detección de parásitos en concentraciones de 10^{4}-10^{5} individuos/ml similares a las concentraciones en sangre.This method allows the separation and detection of protozoa (already verified in two species, and with potential for extend to species of similar physiology) through the ingestion of biocompatible magnetic material. The detection of parasites in concentrations of 10 4 -10 5 individuals / ml similar to blood concentrations.
El método de la invención es potencialmente adaptable a la detección in vivo, dependiendo de la respuesta (fisiología) de cada protozoario en los diferentes estadios de su ciclo de vida.The method of the invention is potentially adaptable to in vivo detection, depending on the response (physiology) of each protozoan at different stages of its life cycle.
El proceso de separación propuesto garantiza que la señal magnética está vinculada a los parásitos asociados a parte del material magnético.The proposed separation process guarantees that the magnetic signal is linked to the parasites associated with part of the magnetic material.
La técnica de detección de los parásitos magnetizados posee, respecto a las actuales técnicas de laboratorio, las ventajas de ser rápido (2-10 seg en la detección), de bajo coste (infraestructura usual de un Lab. de análisis) y sencilla implementación (utilización de técnicas de separación por centrifugación en un gradiente discontinuo de densidades comúnmente empleadas).The parasite detection technique magnetized, with respect to the current techniques of laboratory, the advantages of being fast (2-10 sec in detection), low cost (usual infrastructure of a Lab. of analysis) and simple implementation (using techniques of centrifugal separation in a discontinuous gradient of densities commonly used).
El proceso consiste en tres etapas principales:The process consists of three stages Main:
1. Cultivo de la especie con ferrofluido
biocompatible consistente en nanopartículas de magnetita de tamaños
en el rango aproximado de 20 < d < 150 nm recubiertas con
polisacáridos/péptidos, en concentraciones de 10 hasta
80
mg/ml.1. Cultivation of the species with biocompatible ferrofluid consisting of magnetite nanoparticles of sizes in the approximate range of 20 <d <150 nm coated with polysaccharides / peptides, in concentrations of 10 to
80 mg / ml
2. Separación de la población de parásitos que han incorporado efectivamente las partículas magnéticas, a través de centrifugación en gradiente discontinuo de densidades.2. Separation of the population of parasites that have effectively incorporated magnetic particles, through of discontinuous gradient density centrifugation.
3. Detección de la población de parásitos magnetizados se realiza a través de un magnetómetro o susceptómetro, que permite medir la imantación del cultivo y compararlo con el ferrofluido puro como referencia.3. Detection of the parasite population magnetized is done through a magnetometer or susceptometer, which allows measuring the magnetization of the crop and compare it with pure ferrofluid as a reference.
Las etapas del proceso se describirán ahora en detalle en una descripción de experimentos específicos realizados.The stages of the process will now be described in detail in a description of specific experiments made.
En diversos experimentos realizados, las etapas del método se llevaron a la práctica de la forma siguiente:In various experiments performed, the stages of the method were implemented as follows:
Los epimastigotes de T. cruzi cepa CL clon 14 son crecidos en medio LIT suplementado con 10% de SFB, mantenidos en fase exponencial por repique cada 24/48 horas. Se hizo un segundo experimento idéntico al primero, con T cruzi, con diferente proceso de resuspensión, llevando a los mismos resultados.The epimastigotes of T. cruzi strain CL clone 14 are grown in LIT medium supplemented with 10% SFB, maintained in exponential phase by ringing every 24/48 hours. It became a second experiment identical to the first, with T cruzi, with different resuspension process, leading to the same results.
Dos ferrofluidos (coloides) : #1 (350 nm) y #4 (20 nm). Incubación durante la noche (aprox. 16 hs de incubación 10^{9} células en 4 ml de medio de cultivo).Two ferrofluids (colloids): # 1 (350 nm) and # 4 (20 nm). Incubation overnight (approx. 16 hours of incubation 10 9 cells in 4 ml of culture medium).
Usando sacarosa para formar un gradiente discontinuo de densidad, se separa la mezcla de parásitos asociados a partículas de las partículas que sobran (partículas libres). En detalle:Using sucrose to form a gradient discontinuous density, the mixture of associated parasites is separated to particles of the remaining particles (free particles). In detail:
1. Resuspender la muestra agitando.1. Resuspend the sample by shaking.
2. En un tubo Falcon de 15 ml pipetear 2 ml de sacarosa 2 M en PBS (colchón de sacarosa). Sobre este colchón, pipetear el cultivo. Centrifugación (3000 x g) 10 minutos en colchón de Sacarosa 2 M.2. In a 15 ml Falcon tube pipette 2 ml of 2 M sucrose in PBS (sucrose mattress). On this mattress, Pipette the culture. Centrifugation (3000 x g) 10 minutes in mattress of Sacarosa 2 M.
Un experimento idéntico, utilizando el protozoário Crithidia deanei mostró comportamiento similar, excepto que no hubo formación de pellet. Se cultivaron en medio de Warren suplementado con 10% SFB.An identical experiment, using the Protozoan Crithidia deanei showed similar behavior, except that there was no pellet formation. They were grown in the middle of Warren supplemented with 10% SFB.
Los parásitos fueron detectados a través de la observación de la temperatura de bloqueo en las curvas M(T) en modo zero-field-cooling. También fue observada la señal magnética en los ciclos de imanación M(H) a temperatura ambiente.The parasites were detected through the observation of the blocking temperature in the M (T) curves in zero-field-cooling mode. Too the magnetic signal was observed in the magnetization cycles M (H) at room temperature.
Para todas las muestras, medimosFor all samples, we measure
a. M(T) a 100 G en modo ZFC para ver el bloqueo de las partículas, yto. M (T) at 100 G in ZFC mode to see the particle blockage, and
b. los ciclos M(H) a 10 K y 240 K.b. the M (H) cycles at 10 K and 240 K.
Los protozoos que han fagocitado las partículas magnéticas poseen imanación suficiente para ser atraídos por un imán, con lo que puede implementarse una etapa previa de concentración vía separación magnética, tanto en el tubo de ensayo cuanto en un portaobjetos de microscopio. Se ve a simple vista la aglomeración en la pared del tubo.The protozoa that have phagocyted the particles Magnets have enough magnetization to be attracted to a magnet, so that a previous stage of concentration via magnetic separation, both in the test tube how much on a microscope slide. You can see the naked eye agglomeration in the tube wall.
Los limites de detección varían según el instrumento utilizado, pero su limite inferior puede estimarse a partir de la sensibilidad de magnetómetros SQUID de uso corriente [ver referencia SQU97]. Estos magnetómetros pueden detectar señales del orden de 10^{-6} emu, [ver referencia SQU00] lo que correspondería a 10^{6}-10^{8} partículas de Fe_{3}O_{4} con diámetros d = 150 y 20 nm, respectivamente. Para transformar estos valores de 10^{6} - 10^{8} partículas/ml en concentración de protozoarios, debe corregirse por un factor que depende del numero medio de partículas fagocitadas (10 partículas de 150 nm, o 50 partículas de 20 nm por protozoario), lo que implica un limite de detección menor que 10^{5} protozoarios/ml, muy por debajo de las concentraciones usuales en sangre de portadores infectados en el estadio inicial.Detection limits vary by instrument used, but its lower limit can be estimated at from the sensitivity of current use SQUID magnetometers [see reference SQU97]. These magnetometers can detect signals of the order of 10 - 6 emu, [see reference SQU00] what would correspond to 10 6 -10 8 particles of Fe 3 O 4 with diameters d = 150 and 20 nm, respectively. To transform these values of 10 6 - 10 8 particles / ml in concentration of protozoa, it must be corrected by a factor that depends on the average number of phagocytized particles (10 particles 150 nm, or 50 particles of 20 nm per protozoan), which implies a detection limit of less than 10 5 protozoa / ml, well below the usual blood concentrations of infected carriers in the initial stage.
Una vez descritas suficientemente las características de la invención, sólo debe añadirse que pueden realizarse modificaciones de detalle siempre que no afecten a las características esenciales de la misma, tal como se reivindica en las reivindicaciones siguientes.Once sufficiently described characteristics of the invention, it should only be added that they can make detailed modifications as long as they do not affect the essential characteristics thereof, as claimed in the following claims.
[SIL05] Silber et al. (2005) Curr. Drug Targets - Infect. Disorders, 5, 1, 53-64.[SIL05] Silber et al . ( 2005 ) Curr. Drug Targets - Infect. Disorders , 5, 1, 53-64.
[WHO06] WHO Statistical Information System Website, at http://www.who.int/ctd/chagas/disease.htm. [WHO06] WHO Statistical Information System Website, at http://www.who.int/ctd/chagas/disease.htm.
[BUC03] BUCAK S, "Protein separations using colloidal magnetic nanoparticles" BIOTECHNOLOGY PROGRESS 19 : 477 2003 [BUC03] BUCAK S, "Protein separations using colloidal magnetic nanoparticles" BIOTECHNOLOGY PROGRESS 19: 477 2003
[GU05] GU H.W., Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 125 : 15702 2003 [GU05] GU HW, Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 125: 15702 2003
[SQU97] R. Kötitz, T. Bunte, W. Weitchies, L. Trahms, J. Appl. Phys. 81 (1997) 4317.[SQU97] R. Kötitz , T. Bunte , W. Weitchies , L. Trahms , J. Appl. Phys. 81 ( 1997 ) 4317.
[SQU00] H. L. Grossman, Y. R. Chemla, Y. Poon, R. Stevens, J. Clarke and M. D. Alper, Eurosensors XIV (2000) 27.[SQU00] HL Grossman , YR Chemla , Y. Poon , R. Stevens , J. Clarke and MD Alper , Eurosensors XIV ( 2000 ) 27.
Claims (2)
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- realizar un cultivo de la especie con ferrofluido biocompatible consistente en nanopartículas de magnetita de tamaños en el rango aproximado de 20 < d < 150 nm recubiertas con polisacáridos o péptidos, en concentraciones en el rango de 10-80 mg/ml;perform a crop of the species with biocompatible ferrofluid consisting of nanoparticles of magnetite sizes in the approximate range of 20 <d <150 nm coated with polysaccharides or peptides, in concentrations in the range of 10-80 mg / ml;
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- separar la población de de parásitos que han incorporado efectivamente las partículas magnéticas, a través de centrifugación en gradiente discontinuo de densidades;separate the population of parasites that have effectively incorporated magnetic particles, through of centrifugation in discontinuous gradient of densities;
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- detectar la población de parásitos magnetizados a través de un magnetómetro o susceptómetro, que permite medir la imantación del cultivo; y comparar la imanación del cultivo con la del ferrofluido puro como referencia.detect the population of parasites magnetized through a magnetometer or susceptometer, which allows to measure the magnetization of the crop; and compare the magnetization of the culture with that of pure ferrofluid as a reference.
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| SE8401125L (en) * | 1984-03-01 | 1985-09-02 | Ulf Schroder | SET UP DIAGNOSTIZATION IN VITRO AND DIAGNOSTIC MEDICINE |
| US5279936A (en) * | 1989-12-22 | 1994-01-18 | Syntex (U.S.A.) Inc. | Method of separation employing magnetic particles and second medium |
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| (IVO SAFARIK, MIRKA SAFARIKOVA) 05.02.1999, "{}Use of magnetic techniques for the isolation of cells"{} Journal of Chromatography B Biomedical Sciences and Applications (1999), Vol. 722, N$^{o}$. 1-2, páginas 33-53; ISSN 0378-4347. Todo el documento. * |
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