WO2008116244A1 - Dispositif, notamment biopuce, d'identification de micro-organismes - Google Patents

Dispositif, notamment biopuce, d'identification de micro-organismes Download PDF

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Publication number
WO2008116244A1
WO2008116244A1 PCT/AT2008/000111 AT2008000111W WO2008116244A1 WO 2008116244 A1 WO2008116244 A1 WO 2008116244A1 AT 2008000111 W AT2008000111 W AT 2008000111W WO 2008116244 A1 WO2008116244 A1 WO 2008116244A1
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WO
WIPO (PCT)
Prior art keywords
micro
organisms
sensor
dielectric
measuring
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PCT/AT2008/000111
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German (de)
English (en)
Inventor
Peter Ertl
Rudolf Heer
Michael Kast
Christoph Stepper
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Austrian Research Centers Gmbh - Arc
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Publication of WO2008116244A1 publication Critical patent/WO2008116244A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance

Definitions

  • Device in particular biochip, for the identification of microorganisms
  • Modern microbial identification methods can be classified either as genotypic or as phenotypic methods.
  • Genotypic methods include all forms of DNA and RNA analyzes that examine the genetic material of microorganisms for specific sequences to determine the presence of certain enzymes, stress factors,
  • Phenotypic identification methods assess the proportion of the genome of a cell culture that is currently expressed. These methods include the traditional ways of studying the growth of cell culture under different conditions, as well as performing biochemical screening to determine metabolic capabilities. There is already a large number of inventions in microtiter plate format (Lit .: 6-31).
  • phenotypic identification methods already described, measure the cell wall potential with the help of fluorescent dyes in the presence of bioactive chemicals (Lit .: 32). These include database-based or fingerprinting methods using pattern recognition analyzes that detect signal patterns and assign them to known organisms.
  • the best known method for phenotypic characterization of cells is flow cytometry.
  • counting, testing and sorting of suspended cells take place in a fluid stream.
  • This method allows the determination of the properties of single cells flowing through a measuring chamber.
  • Coulter Counter Another established method for cell characterization comes in the so-called Coulter Counter for use.
  • cells located in a weak electrolyte solution solution are passed through a small opening between two electrodes and analyzed by means of impedance spectroscopy.
  • the Coulter principle is based on particles or cells passing through the measurement zone, displacing the electrolyte. By analyzing the pulses it is then possible to deduce the size distribution of the cells.
  • V dielectric gauges for the study of suspended cells, such as in fermentors, by means of alternating electric fields have been developed in recent years (V).
  • An important feature of dielectric spectroscopy is the ability to obtain quantitative signals over a wide frequency spectrum at low amplitudes. This provides the opportunity to study biological systems almost without interference. Dielectric spectroscopy has therefore already been used to study cell sedimentation, aggregation, cell division and growth in a liquid environment (VI).
  • the present invention uses interdigital electrode structures ( ⁇ lDES) as micro-dielectric sensors. These sensors show increased sensitivity compared to standard two-electrode systems (V, VII). Photolithographically generated biochip integrated interdigital electrode structures offer the advantage of accurate Dimensioning of the electric field distribution and thus ultimately the sensitivity (VIII).
  • a fluorescent marker is introduced into the cell culture, which may adversely affect the natural behavior of the cells.
  • the size of the marker molecules which is the size of the substance to be analyzed, e.g. in the antigen-antibody reaction, and thus exerts a significant influence on the kinetics of the system, crucial.
  • Many of the markers are not long-term stable, which leads to bleaching of the fluorescence markers on repeated excitation. For this reason, long-term tests with such systems are hardly feasible.
  • WO 01/79529 A1 relates to a device and a method for the detection of pathogenic substances or microorganisms, which can occur especially in food infections.
  • the identification of the substances takes place after their binding to antibodies which are immobilized on metallic electrodes, indirectly by influencing the Farraday currents forming between the metal electrodes.
  • the metabolism of - now also immobilized - microorganisms changes the electrolytic composition of the medium and thus influences the expression of Farraday's currents.
  • electrolytic buffer solutions with low conductivity and special markers are used.
  • steps of sample preparation and enrichment are implemented.
  • WO 01/51921 A1 is an application of organic co-polymer coatings with defined surface charges to prevent the
  • the protective coating is the
  • Electrodes have been optimized for repulsion of substances of any kind.
  • the invention relates to a device, in particular a biochip, according to the
  • the present invention enables the non-contact, real-time and continuous, as well as non-invasive, acquisition of dielectric data of an adherent culture. It is able to provide information about the identity, vitality, morphology, mobility and dielectric properties of the organisms.
  • the optical or spectroscopic data obtained can therefore be used both for the rapid identification of organisms and for the determination of relative changes in the phenotypic cell behavior over longer periods of time. A reduction made possible by the new facility
  • Determination of cellular doubling rates can be used.
  • the small sensor dimensions allow an effective measurement of 100 to 1000 cells, which are introduced into the measuring chambers or applied to the sensor surface by gravimetric methods.
  • Cell duplication rates can be measured by the present invention continuously and over short periods of time.
  • a unique feature of the subject invention is also the combination of dielectric measurement spectra for the high-resolution rapid determination of morphological differences of living cell assemblies based on embedded in fluidic microchips, contactless micro-dielectric sensors. Further description of the invention
  • Microchip technology in biology follows the trend of miniaturization of analytical measurement methods using MEMS (micro-electromechanical systems) technology, as it greatly reduces the overall cost of complex analysis and opens up new possibilities for analytical systems (IX).
  • MEMS micro-electromechanical systems
  • IX analytical systems
  • the invention described herein is based on the measurement of relative deflections versus a reference branch in an alternating electric field over a wide frequency range.
  • the biological systems to be studied are grown in a weak alternating electric field, e.g. with ⁇ 15 mV, polarized and any morphological, biochemical or biological change that affects the dielectric properties of the plasma membrane, the cytoplasm, nucleus and other cell components detected rapidly and with high resolution.
  • the method not only offers the ability to distinguish between living and dead cells, to track cell growth and to distinguish structural features, but also to identify different organisms.
  • the measuring device such as e.g. The MultiChannel potentiostat VMP3, Princeton Applied Research, is commercially available, well evaluated and well characterized, while the microfluidic biochip and micro-dielectric sensors are fabricated using standard semiconductor technologies.
  • the new device according to the invention differs fundamentally from the device and method according to WO 01/79529 A1 due to the complete suppression of Farraday currents.
  • the detection is not performed according to the invention indirectly via metabolic products. There is no sample preparation or concentration. In addition, neither markers nor antibodies are used in the device according to the invention.
  • the device according to the invention is based on the application of thin bioaffinity protective layers to interdigital electrode structures.
  • the bioaffinity according to the invention is in complete contrast to the properties of the protective coating in the abovementioned WO-A1 and in the first place makes it possible to attach the microorganisms to the sensor surface protected against Faraday currents.
  • the invention specifically selected dielectric properties of the protective layer according to the invention allow the use of low electrical alternating potentials (with about 15 mV amplitude) and thus enable linear dielectric spectroscopy.
  • the thickness of the shielding layer which is advantageously to be adhered to is more clearly indicated by the intrinsic phragm 3, the thickness of the individual layer layers forming the same being related to the intrinsic layer 4.
  • the inferior total thicknesses of the shielding layer plus the same adhering organism layer are to be inferred from the effect in the sense of the effectiveness of the field strengths used.
  • the A n s p r u c h 6 called in the invention preferably used
  • Metals or metal alloys for the micro-sensor are Metals or metal alloys for the micro-sensor.
  • Claim 7 relates to the fluidic structure underlying the new bio-chip.
  • a construction structure of the biochip to be used which is advantageously suitable for the described documents is disclosed in claim 8.
  • Claims 11, 12 and 23 relate to the concentration of the organisms in the suspension which is favorable for the analysis, and advantageously to the covering of the measuring surface of the measuring chamber with the same.
  • the claim 14 contains information regarding the preferred
  • claims 15 to 19 favorably indicate ways of using the device according to the invention for various investigations on organisms or microorganisms. The invention will be explained in more detail with reference to FIGS.
  • FIG. 1 shows the basic design of a biochip 100 according to the invention, which consists of a glass substrate 1, which includes the micro-sensors 4 and a polymer part 2, the microfluidics, substantially comprising the inflows A and C with the reference numeral 6 and 9, the microchannels 7, the injector 10 and the actual measuring chamber 5 and the reference measuring chamber 5 ', accommodated exists.
  • the Biochip 100 incorporates several sensors and duct systems connected to reservoirs and pumping and heating systems through appropriate connections, including the. Referring to Fig. 1A (b) in detail. The sectional view of Fig.
  • 1A) b) clearly shows the glass substrate 1, the interdigital comb structure of the micro-sensor 4 with the "comb teeth” 42 and their insulating shielding layer 40 with the thickness ds, e.g. with a thickness of up to 500 nm, in which the micro-sensor 4 is embedded.
  • This multi-layered layer 40 is adjoined at the top by the measuring chamber 5 incorporated or etched into the polymer 2, the measuring surface 50 of which is preferably opaque, coated or covered with the (micro) organisms (8) to be examined and which has a thickness do, wherein it is important that the two thicknesses ds plus do do not exceed 5.1 or 5.51 microns.
  • FIG. 1B) a) shows - with otherwise identical reference numerals meanings - a close-up of the measuring chamber 5 with fluid channels 9 and 7, while FIG. 1B) b) shows a section of the interdigital comb structure 42 of the mic in the form of a metallization 11 Sensor 4 shows.
  • FIGS. 1A and 1B shows the integration of the microchip or biochip shown in FIGS. 1A and 1B into the overall system of analysis with heating system 101 for heating or cooling of chip 100, of at least one pump 102 for the delivery of nutrient media, Test media and rinsing media, an injection system 103 for the introduction of the organism material to be examined in the measuring chamber 5 of the biochip 100 or for the loading or occupancy of the electrically isolated sensors with the organisms 8, all components of the fluidic system.
  • the integrated reference arm of the biochip 100 shown in FIG. 1A) a) with the reference measuring chamber 5 1 offers the possibility for background and interference-free measurements.
  • the interdigital electrode structures ( ⁇ lDES) 4 are embedded beneath a multilayer system forming the shielding layer 40 for isolation.
  • Several hard or soft passivation materials such as silicon nitrate, silica, glass (SOG) and / or epoxy polymers are suitable. They are applied over a large area by means of lithographic methods on the chip 100.
  • the essential physical separation of the ⁇ lDES structure from the liquid environment eliminates the direct interaction of the electroactive substances and ions contained therein with the sensor electrode surface. Signal influences that could be caused by the electrode polarization or air bubble adhesion are prevented in this way.
  • Saline buffer drift analyzes have given a relative standard deviation of 1.5% over a 20 h period. It is important to ensure that the micro-dielectric sensors are completely shielded without losing sensitivity.
  • FIG. 2A shows, on the basis of a diagram: current in amps to voltage in volts, the results of an electrochemical analysis of the passivation quality of the insulating layers used according to the invention versus non-isolated sensors in the presence of a highly electroactive substance, in this case 10 mM potassium hexacyanoferrate, curve 1. It however, no detectable amount of faradaic current was measured using a 550 nm thick insulation or shielding layer, curve 2. Further, calculations using the "conformal mapping technique" show that a loss of sensitivity below 8% is due to insulating layer thicknesses less than 500 nm hold is.
  • a highly electroactive substance in this case 10 mM potassium hexacyanoferrate
  • the non-contact sensor of the ⁇ LDES of the micro-sensor has, for example, 200 fingers spaced 5 ⁇ m apart, which are 5 ⁇ m wide and 1000 ⁇ m long exhibit. This means that biological components that are close to the sensor surface have the greatest influence on the measurement signal, since 95% of the electric field lines and thus of the effective signal component lie below 5.51 ⁇ m, which can be seen in FIG. 2B, which shows the dependence of the signal component on the layer thickness is to be referred. For this reason, the new micro-dielectric sensors behave differently from the previously known or existing dielectric sensors, since in these the biological systems must be in direct or close contact with their surface.
  • micro-dielectric sensors shielded according to the invention show the greatest distractions as a result of different cell composition.
  • FIG. 3 shows, on the basis of a diagram: impedance ( ⁇ IZI / ohms) to log concentration (cfu / ⁇ L), clearly that the yeast cells, in many cases larger Pichia pastoris, generate lower signals than the bacteria Staphylococcus xylosus and Bacillus subtilis after sensor saturation.
  • Gram-positive bacteria such as S. xylosus and B. subtilis
  • Gram-negative bacteria such as E. coli and Serratia forticola with similar cell sizes
  • the micro-dielectric sensors isolated according to the invention are particularly sensitive to differences in cell morphology.
  • the distinction between Gram-positive and Gram-negative bacteria is of great importance in clinical medicine, since certain antibiotics may only be used in Gram-positive or even Gram-negative pathogens.
  • Fig. 3B with the diagram impedance (ohms) to log conc. (Cfu / ⁇ L).
  • Figures 4A / 4B show graphs of impedance (ohms) to various microorganisms and IZI (ohms) to frequency (Hz) results of live and dead yeast cells, in the specific case of Candida albicans. Growth tests carried out on agar plates with yeast cells exposed for 2 h to an ultrasound bath confirmed that complete destruction of the yeast culture was ensured in this way.
  • the ability of the new device to distinguish cell morphological features by means of dielectric spectroscopy of surface-living biological systems can also be used to identify them.
  • the prerequisite is that the measuring range or the measuring surface is saturated with cells is. Cell counts of greater than 10 7 cfu / mL are generally sufficient for completely loading the described sensor geometry with biological material.
  • Figures 5A and B show pattern recognition plots of cells and culture media using chemometric data analysis. Cultivation of yeast and bacterial strains took place externally in shake flasks under standardized growth conditions.
  • the culture medium was initially removed from the cells and measured separately.
  • FIGS. 5A and 5B show within the framework of a scheme: factor 1 / factor 2 or PC1 / PC2 pattern recognition plots of organisms and their growth media. Since the groupings were obtained only with the cells and not from the different growth media, it is to be assumed that changes of the medium by the investigating and to be determined organism do not significantly influence the identification.
  • the principal component analysis (PCA, factor analysis) used in the subject system uses the impedance and phase data of individual microorganism dielectric spectra to generate a data template.
  • each column of the template comprises 501 values and each experiment is given its own row, eg 501 x 17 in the case of Figure 5B.
  • the template is converted to a Lotus file and loaded into MATLAB version 7.01.
  • the factor analysis is performed using the Chemometric Toolbox for MATLAB, Version 3.02, and includes the generation of reduced eigenvectors, the study of the normal distribution and elimination of the noise component, and the calculation of the different factors, namely Factor 1, Factor 2 or Principle Components (PC) 1 and 2 for each data set.
  • PC Principle Components
  • a further field of application of the new dielectric identification method is the rapid recognition of morphological changes in a cell line which is already known. Cells growing on defined surfaces are exposed to an external stimulus and the phenotypic response of the cell population is continuously measured. External environmental influences include a wide range of possibilities, such as temperature fluctuations, variations in flow rates, different media compositions or material additions, such as Nanoparticles available.
  • FIGS 6A and 6B show by diagrams: IZI (ohms) at time (h) the behavior of a yeast culture in the proliferation chamber of the bio-chip with variation of the flow rate.
  • Figures 6A and 6B demonstrate that the sensor of the present invention is sensitive to changes in cell composition, unlike existing technologies that primarily allow only the measurement of increases or decreases in cell numbers. That is, even if the organisms do not die and their total cell count thus remains constant, the effects can be traced at the cellular level, optionally detecting directly subcellular structures, e.g. the cell wall od. Like., Within an entire cell population is possible.
  • amphotericin B accumulates particularly in the plasma membrane of fungi and thus inhibits the growth of fungi.
  • the inhibitory effect can also be seen from FIG. 7B, since the cell population increases only slightly over a period of 10 h, which also showed growth tests under standard conditions.
  • the present invention is particularly suitable for rapid analyzes, such as for the phenotypic discrimination and identification of different primary and standard cell types, and in particular for the differentiation of Gram-positive and Gram-negative bacteria.
  • the cells to be examined organisms u. Like. Must be on defined surfaces, below which are shielded by the insulating shielding micro-dielectric sensors.
  • the method according to the invention thus relates to a non-invasive and marker-free method for identifying organisms, in particular microorganisms, and comprises the following steps: After obtaining a pure culture of an unknown microorganism, a certain amount of sample is applied to the passivated and isolated from the test solution through the shielding layer Sensor surface applied to load the measuring area or the measuring surface of the measuring chamber entirely with cells. A single cell layer consisting of cells is sufficient in this case, since the sensors have only a small measurement depth, namely 95% signal component at layer depths of less than 5.1 microns.
  • the dielectric properties of the biological sample are measured by means of cellular dielectric spectroscopy.
  • the over a wide Frequency range obtained impedance signals are specific to the cell types contained in the sample and are therefore suitable directly for their identification.
  • respective changes in the dielectric properties of an adherent cell culture can be dynamically measured and thus show relative changes in cell populations in real time.
  • Fig. 1 A) graphic representation of the biochip layout, B) image extracts of
  • Fig. 3 A) Comparison of the sensor signals (amount of impedance at 50 kHz) with
  • Fig. 4 A) Block diagram of live, treated with Amphoterizine B and ultrasound
  • Fig. 5 Pattern recognition plot of impedance data of A) cell-free medium extract of three micro-organisms. E. coli K12 (0), B.subtilis (T) and P. pastoris ( ⁇ ) were cultured under standard conditions, harvested (exponential phase), centrifuged and 1 ⁇ l each of the supernatant injected into the biochip and allowed to settle for 30 minutes every measurement.
  • B Pattem recognition plot of phase values of ß. subtilis (T) 1 S. xylosis (o), E. coli K12 (0), P. pastoris ( ⁇ ), and S. fonticola ( ⁇ ) pure cultures.
  • Fig. 6 Influence of flow rates and shear forces on the growth course of A) Pichia pastoris and B) C. albicans yeast cultures
  • Fig. 7 (A) Dynamic behavior of a Candida biofilm after addition of 0.5 ⁇ g / mL amphotericin B (arrow) where (a) impedance signals and (b) phase values for 50 kHz are plotted against the culture time. (B) Photos (before and after the addition of amphotericin B) from the growth chamber in the C. albicans

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  • Organic Chemistry (AREA)
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  • Engineering & Computer Science (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

L'invention concerne un dispositif, notamment une biopuce, destinée à la recherche de phénotypes, à l'identification et à la caractérisation de micro-organismes par spectroscopie diélectrique sans apport de marqueur, au moyen d'un microcapteur doté d'une structure en peigne métallique, d'une chambre de mesure située au-dessus de cette structure et recevant les micro-organismes, et d'une unité servant à fournir des champs alternatifs de haute fréquence et à saisir des variations d'impédance. L'invention est caractérisée en ce que la chambre de mesure (5) à alimenter en organismes (8) ou bien sa surface de mesure (50) à doter des organismes (8) ainsi que les organismes (8) eux-mêmes sont séparés par une couche de protection (40), de préférence à plusieurs strates, perméable seulement aux champs alternatifs électriques émis par le microcapteur (4), cette couche étant composée d'un matériau n'étant normalement pas perméable, électriquement non conducteur, ni magnétique, ni diélectrique, la séparation étant faite par rapport à la métallisation (41) de la structure en peigne (42) du microcapteur (4), appliquée sur un support de verre (1) et orientée vers la chambre de mesure (5) ou vers sa surface de mesure (50).
PCT/AT2008/000111 2007-03-27 2008-03-27 Dispositif, notamment biopuce, d'identification de micro-organismes WO2008116244A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AT4802007A AT505106A1 (de) 2007-03-27 2007-03-27 Einrichtung, insbesondere bio-chip, zur identifizierung von mikro-organismen
ATA480/2007 2007-03-27

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10073074B1 (en) 2014-04-25 2018-09-11 Iowa State University Research Foundation, Inc. Low RF-band impedance spectroscopy based sensor for in-situ, wireless soil sensing
CN109107619A (zh) * 2017-06-23 2019-01-01 南京理工大学 复合介质层数字微流控芯片及其制备方法
CN110177882A (zh) * 2016-10-12 2019-08-27 克拉利斯公司 使用单细胞分析进行疾病诊断的组合物和方法
CN114858888A (zh) * 2022-04-02 2022-08-05 中山大学 一种测定海洋微生物附着的方法

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WO2001079529A1 (fr) * 2000-04-17 2001-10-25 Purdue Research Foundation Biocapteur et procede associe
WO2002046357A1 (fr) * 2000-10-26 2002-06-13 The Trustees Of Princeton University, Princeton University Procede et appareil de spectroscopie dielectrique de solutions biologiques
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10073074B1 (en) 2014-04-25 2018-09-11 Iowa State University Research Foundation, Inc. Low RF-band impedance spectroscopy based sensor for in-situ, wireless soil sensing
CN110177882A (zh) * 2016-10-12 2019-08-27 克拉利斯公司 使用单细胞分析进行疾病诊断的组合物和方法
CN109107619A (zh) * 2017-06-23 2019-01-01 南京理工大学 复合介质层数字微流控芯片及其制备方法
CN114858888A (zh) * 2022-04-02 2022-08-05 中山大学 一种测定海洋微生物附着的方法

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