WO2008116244A1 - Device, particularly a biochip, for identifying microorganisms - Google Patents

Device, particularly a biochip, for identifying microorganisms 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
measuring
dielectric
Prior art date
Application number
PCT/AT2008/000111
Other languages
German (de)
French (fr)
Inventor
Peter Ertl
Rudolf Heer
Michael Kast
Christoph Stepper
Original Assignee
Austrian Research Centers Gmbh - Arc
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Publication date
Priority to AT4802007A priority Critical patent/AT505106A1/en
Priority to ATA480/2007 priority
Application filed by Austrian Research Centers Gmbh - Arc filed Critical Austrian Research Centers Gmbh - Arc
Publication of WO2008116244A1 publication Critical patent/WO2008116244A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means
    • G01N27/02Investigating or analysing materials by the use of electric, electro-chemical, or magnetic means by investigating the impedance of the material

Abstract

The invention relates to a device, particularly a bio-chip, for the phenotypical analysis and for the identification and characterization of microorganisms, by means of dielectric spectroscopy without the addition of markers by using a microsensor with a metal comb structure and a measuring chamber, which is arranged above the microsensor and receives the microorganism, and a unit for feeding high-frequency alternating fields and for impedance change detection, characterized in that the measurement chamber (5) to receive the organisms (8), or the measuring surface (50) thereof to be provided with the organisms (8), and the organisms (8) per se are separated by a preferably multilayer shielding layer (40), which is permeable only to the electric alternating fields emitted by the microsensor (4) and made of otherwise non-permeable, electrically non-conductive, non-magnetic and non-dielectric material from the plating (41) of the comb structure (42) of the micro sensor (4) applied to a glass substrate (1) and facing the measuring chamber (5) or the measuring surface (50) thereof.

Description

 Device, in particular biochip, for the identification of microorganisms

Background and state of the art

The identification of phenotypic traits and the rapid detection of morphological changes under certain conditions is an integral part of many cell biological characterizations. Traditionally used methods for the identification of microorganisms usually consist of

Enrichment steps followed by a series of studies, such as morphological appearance, biochemical screening, growth characterization or serotypic and serological confirmations.

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,

Antibiotic resistance mechanisms od. Like. To detect. Many genotypic microbial

Identification methods are described in the literature. (Lit.: 1-5)

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).

Further 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.

Various measuring methods have already been proposed and used for this purpose, in particular pyrolysis mass spectrometry, magnetic resonance data which generate a specific pattern, or the binding of microorganisms to modified surfaces are described in the literature (references: 33-35).

The best known method for phenotypic characterization of cells is flow cytometry. Here, 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.

Another established method for cell characterization comes in the so-called Coulter Counter for use. In this case, 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.

A variety of bioimpedance methods are also described in the literature. In this case, the cell characterization is carried out by means of electrodes which are in contact with the solution. In most cases, the change in solution conductance is measured, and in this way information about the cell count as well as about the metabolic activity of cell populations is obtained.

Furthermore, impedance methods for determining membrane receptors, such as ion channels, ion pumping, chemical influences, or binding of specific antibody-antigen ligands to receptors have been described (refs. 37-39). For example, integrated micro-dielectric sensors have already been used for the

Detection of DNA molecules (Lit .: 36) or different organic and inorganic substances used after separation by capillary electrophoresis (I). Another application of dielectric sensors in flow cytometry has been to determine the cell cycle of individual cells, as this can demonstrate an increase in the cellular DNA content of living cells (II).

Fast measurements are of particular importance for the phenotypic characterization of cells and above all for the observation of the dynamic behavior of living organisms (III). As a method for label-free identification of living cells, dielectric spectroscopy is used in the present invention. This measurement method is ideal for the non-invasive characterization of biological systems (IV).

A number of 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 (μlDES) 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).

The previously mentioned known standard methods are usually tedious and labor intensive. They generally take periods of several days to several weeks. Often, a variety of indirect tests must be performed. Most phenotypic rapid tests are based on the detection of certain surface molecules, which can be measured optically, electrically or by other measuring methods.

In almost all cases, a fluorescent marker is introduced into the cell culture, which may adversely affect the natural behavior of the cells. Often, 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.

Furthermore, currently only a limited number of markers that can display only specific events are available. The experimental procedure is thus strongly determined by the selected marker. In many cases, only an endpoint detection is possible because many processes, such as hybridization processes, od only under defined process conditions, ie at a certain temperature, in CO 2 atmosphere. Like. Run in incubators.

The analysis is only possible at certain points during the process or after its completion. In particular, the kinetics of the process via a continuous measurement, in particular online monitoring, not accessible. A major disadvantage of commercially available cell analysis systems, therefore, is the inability to dynamically observe phenotypic changes in cells, for example, during cell aging, introduction of external stimuli, and other influences. In addition, the following state of the art should be considered:

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. To increase the sensitivity, electrolytic buffer solutions with low conductivity and special markers are used. In addition, steps of sample preparation and enrichment are implemented.

Furthermore, WO 01/51921 A1 is an application of organic co-polymer coatings with defined surface charges to prevent the

Contamination of electrode surfaces with biological substances.

By means of high electrical alternating potentials (with about 1 V amplitude) at different frequencies, non-linear dielectric spectra are recorded on the samples. This method known from W0-A1 requires a special four-electrode geometry. For example, yeast cells, but also

Enzymes in cell membranes are detected. 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

O b e r b e g r i f f of the A n s p r e c h e s 1, which has the m e rk m e l e mentioned in the e n e ce of this claim.

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

Test cycles in the development of new therapeutics as well as in the quantification of their properties under physiologically relevant conditions represents a tremendous enrichment for the data quality, for example in investigations of toxicity. In addition, the inventive device for rapid and simple

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). , The production of biochips is based on photolithographic processes that were originally developed for microelectronics.

The biggest advantage of the chip technology lies in the actual miniaturization of the measuring method. Here, additional physical properties that are not possible in large volumes, such as in the diffusion-assisted mixture, can be used. Furthermore, the successful establishment of genomics has shown that miniaturized systems are more efficient, faster and more accurate analytical methods (X). These properties are particularly important in cell analysis, since a high sensitivity is a prerequisite of or for real-time measurements (Xl). The establishment of cell cultures on chips has led to new and more efficient methods for the study of single cells and cell assemblies over extended periods of time (XII). A large number of cell chips have already been described in the literature for the controlled on-chip manipulation of cells (XIII, XIV). 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.

For this reason, 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.

In particular, the two mentioned as prior art, both WO-A1, and to the differences between the objects disclosed therein and the invention, the following should be noted: 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 specially shaped sensor geometry and the use of thin, bioaffinity, insulating layers, which separate the metal structures of the 'physiological media that allow noncontact analysis and identification of microorganisms based on their dielectric properties. 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.

In contrast to WQ 01/51921 A1, 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. In addition, 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. By the two aforementioned WO-A1-fonts, the subject invention subject matter is not taken individually. A combination of the two WO-A1-fonts can not lead to the present invention, since the objects of WO 01/79529 A1 are based on completely different principles and WO 01/51921 A1 describes surface coatings which are diametrically opposed to the measuring principle provided according to the invention.

It has been shown in practice that it is particularly favorable to form the shielding layer with a plurality of special layer layers, as can be seen from the A n s p r u c h 2.

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.

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.

Another essential subject of the invention is the investigation method according to. The preamble of claim 9, which the to the characterizing part of this claim - entnehmenden

Has process features.

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

Voltage values for the supply of the comb structure of the micro-sensor.

Finally, 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.

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.

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.

1C 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. It also shows the electrical system responsible for the measurement and output of the measured data, with the potentiostat 104 generating the high-frequency fields and the same supplying the micro-sensors of the biochip 100 and impedance, and the personal computer 105 for recording, analyzing and comparing the impedance Data with databases and for the classification and quantification of the studied (micro) organisms 8.

Furthermore, 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.

In order to obtain signals which are as stable as possible, the interdigital electrode structures (μlDES) 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 μlDES 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.

The non-contact sensor of the μLDES of the micro-sensor has, for example, 200 fingers spaced 5 μm apart, which are 5 μm wide and 1000 μm 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 μm, 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.

While commercially available systems are particularly sensitive to cell size and cell count, the 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 (ΔIZI / ohms) to log concentration (cfu / μL), 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.

The fact that the Gram-positive bacteria, such as S. xylosus and B. subtilis, which have a different membrane composition compared to Gram-negative bacteria, such as E. coli and Serratia forticola with similar cell sizes, further indicates that 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. Reference is made to Fig. 3B, with the diagram impedance (ohms) to log conc. (Cfu / μL).

Further evidence for the existence of a morphology-dependent measurement is the detection and differentiation of living and dead cells.

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.

Consequently, 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. However, 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.

Subsequently, a minimum of four samples from each cell culture were removed and injected into the bio-chip and characterized by dielectric spectroscopy. Each point in the graph therefore represents the entire data set of a measurement sample. The generation of abstract vectors (scores) by means of Principle Component Analysis (PCR) is a widespread method of analysis for certain patterns, such as groupings or

Determine relationships. Nearby points thus represent data sets with low variance and are to be regarded as identical.

In order to determine the influence of the culture medium or the change of the medium caused by the growth of the organism on the identification, 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.

This also shows the crucial difference of the new method according to the invention over conventional impedance methods, where exposed and directly in contact with the organisms electrodes mit- detect the degradation and excretion products of living systems. Furthermore, this makes it possible to eliminate a sample preparation step, since the cell cultures can be measured directly according to the invention.

As an example of the factor analysis approach, 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. For example, 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. The calculated Principle Components PC1 and PC2 were used for the graphic illustration of the respectively obtained pattern. It is clearly evident from the two diagrams of FIGS. 5A and 5B that the dielectric signals obtained by means of the apparatus according to the invention actually make it possible to identify microorganisms.

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.

Figures 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. By increasing the shear forces, caused by an increase in the flow rate, see the solid arrow, it is possible to keep the cell population of the yeast P. pastoris constant in the growth chamber, while " low " flow rates indicate a dynamic growth pattern, recognizable by periodic signals , A 10-fold increase in the flow rate from 0.05 μL / min to 0.5 μL / min initially leads to a marked loss of the cell population of the C. albicans culture, ie to a leaching, followed by a steady increase and establishment of a biofilm. Furthermore, in the clearly synchronous growth pattern, "periods" of the biofilm can be observed, which indicates that some of the new cells are noticeably excreted from the biofilm approximately every 30 minutes.

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.

Of particular biological and medical interest is the detection of subcellular changes, eg the cell wall composition of living cells. The scheme: Z (ohms) at time (h) of Figure 7A shows the dynamic behavior of a Candida Biofilms before and after addition of 0.5 μg / mL amphotericin B (arrow). Shortly after the addition of the fungicide, the impedance signal rises at a rate of 30 ohms / hr for the first two hours, followed by a slightly slower rate of 10 ohms / hr for the remaining 10 hours. In turn, as can be seen from Fig. 7b, the cell count has steadily increased during this period, the measured signal changes directly indicate changes in subcellular structures within the cell population and not a decrease in cell number.

It is known that 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.

It can also be used for long-term measurement of cell cultures and their dynamic analysis, for the detection of toxic effects caused by various chemicals, materials and environmental influences on specific cell types with known identity. In addition, the characterization of genetically engineered and standard

Cell lines, the acquisition of electrophysiological data from cell cultures and the optimization of biotechnological processes in the smallest scale suitable applications.

For all applications, 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.

Subsequently, 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.

Furthermore, 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.

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III. Bochner, B.A., New Technologies to Assess Genotype-Phenotype Relationships. Nature Reviews Genetics 2003, 4, 309-314. IV. Asami, K., Characterization of Biological Cells by Dielectric Spectroscopy Journal of Non-Crystalline Solids 2002, 305, (1-3), 268-277. V. Ciambrone, G.J .; Liu, V.F .; Lin, D.C .; McGuinness, R.P .; Leung, G.K .; Pitchford,

S., Cellular Dielectric Spectroscopy: A Powerful New Approach to Label-Free

Cellular Analysis. Journal Biomolecular Screening 2004, 9, (6), 467-480. VI. Markx, G.H .; Davey, C.L., The dielectric properties of biological cells at radiofrequencies: Applications in biotechnology. Enzymes Microbial Technol. 1999

25, (3-4), 161-171. VII Gerwen, P. V .; Laureyn, W .; Laureys, W .; Huyberechts, G .; De Beeck, M.O .;

Baert, K .; SuIs, J .; Sansen, W .; Jacobs, P .; Hermans, L; Mertens, R., Nanoscaled interdigitated electrode arrays for biochemical sensors. Sensors and Actuators B

1998, 49, 73-80. VIII. Igreja, R .; Dias, C.J., Analytical evaluation of the interdigital electrodes capacitance for a multi-layered structure. Sensors and Actuators A 2004, 112, 291-

301. IX. Dittrich, P.S. Tachikawa, K .; Manz, A., Micro Total Analysis Systems. Latest

Advancements and Trends. Analytical Chemistry 2006, 78, 3387-3907 X. Deutsch, J .; Desai, T.A .; Motlagh, D .; Russell, B., Microfabricated in vitro cell culture Systems for investigating cellular interacitons. Proc. Soc. Photooptical

Instrum. Closely. 2000, 3912, 105-113. Xl. EI-AIi, J .; Sorger, P.K .; Jensen, K.F., Cells on Chips. Nature 2006, 442, (27), 403-

411 XII. Shackman, J. G .; Dahlgren, G .; Peters, J. L .; Kennedy, R.T., Perfusion and chemical monitoring of cells on a microfluidic chip. Lab on a Chip 2005, 5,

56-63. XIII. Tourovskaia, A .; Figueroa-Masot, X .; Folch, A., Differentiation-on-a-chip: A microfluidic platform for long-term cell culture studies. Lab on a Chip 2005, 5, 14-

19. XIV. Xing, W.-L .; Cheng, J., Biochips: Technology and Applications. Springer Verlag:

New York, 2003. Figure-explanations:

Fig. 1: A) graphic representation of the biochip layout, B) image extracts of

Measuring chamber and the micro-dielectrischer sensor, C) experimental

Experimental setup Fig. 2: A) Cyclic voltammetry of (1) unisolated and (2) isolated μlDES

sensors; B) Calculation of the total capacity depending on the coverage

Fig. 3: A) Comparison of the sensor signals (amount of impedance at 50 kHz) with

Cell count concentration of six different organisms; B) Comparison of the sensor signals (impedance at 50 kHz) between Gram-negative (E. coli) and Gram-positive (B. subtilis) bacteria

Fig. 4: A) Block diagram of live, treated with Amphoterizine B and ultrasound

yeasts; B) Raw data of C. albicans before and after ultrasound treatment

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 μl 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 μg / 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

37oC and 0.5 μL / min flow rate was cultivated.

Claims

claims:
1. Device, in particular biochip, for phenotypic examination, preferably for, optionally continuous, direct non-contact
Identification and characterization of organisms, in particular microorganisms, by means of dielectric spectroscopy without the addition of markers using at least one interdigital, metallic comb structure as a dielectric micro-sensor and at least one above derbzw. the same arranged, for receiving the organisms to be examined, especially microorganisms, provided measuring chamber, preferably with supply and discharge channels for fluid media with connections for lines, reservoirs, pumps, heating systems od. Like., And with at least one connected to the micro-sensor, Unit (potentiostat) for the supply of high-frequency alternating fields over a wide frequency range in the micro-sensor and the impedance.
 Impedance change detection, characterized in that - od for a non-invasive recording of dielectric characteristics, data, signals or the like -. With the micro-organisms (8) to be supplied measuring chamber (5) or their with the (micro-) Orga [pi] ismen (8) to be occupied Bodenund measuring surface (50) and thus the (micro) organisms (8) even by a lower thin, bioaffine, preferably multi-layer, for the micro-sensor (4) emitted electric fields and for the response signals from or
 from the micro-organisms (8) in the measuring chamber (5) permeable, but any contact of the to be examined (micro) organisms and the fluid medium containing them with the metallic surface of the interdigital comb structure of the micro-sensor absolutely exclusive, from at least an electrically non-conductive, non-magnetic, non-magnetizable, non-dielectric and impermeable to molecules of any kind material Abschirmbzw. Insulating layer (40) of which the metallization (41) of the interdigitated comb structure (42) of the microsensor (4), facing the measuring chamber (5), in particular its measuring surface (50), is deposited on a substrate (1), preferably a glass substrate ) is disconnected.
2. Device according to claim 1, characterized in that the shielding layer (40) with two to five, preferably with each other, non-dielectric materials or with materials having different physical properties, in particular silicon dioxide, silicon nitride, alumina, spin-on Glasses (SOG) with at least one additive, such as Organosilicates, and / or photoresists, e.g. SU8, formed on the micro-sensor surface (4) and adhering individual layers is formed.
3. Device according to one of claims 1 to 3, characterized in that the shielding layer (40) has a total thickness (ds) of 200 to 500 nm.
4. Device according to one of claims 1 or 2, characterized in that the individual layers of the shielding layer (40) each have a thickness (dl) of 50 to 250 nm, preferably of at most 100 nm. *
5. Device according to one of claims 1 to 4, characterized in that the sum of the thicknesses of the shielding layer (41, ds) and layer thickness of the examined (micro) organisms (8, do) a maximum of 10 mu, preferably 5.5 [mu] m.
6. Device according to one of claims 1 to 5, characterized in that the micro-sensor (4) or the same forming dielectric comb structure (42) made of a noble metal or of a noble metal alloy, in particular of a titanium-gold or titanium -Platin alloy is formed, and said comb structure (42) by means of an adhesive, such as Titanium, is fixed on the substrate (1).
7. Device according to one of claims 1 to 6, characterized in that the measuring chamber (5) and the affiliated fluidic microstructure (7) in one of the micro-sensor (4) and the shielding layer (40) having substrate (1) covalently bonded applied layer (2) is embossed.
8. Device according to one of claims 1 to 7, characterized in that the used reaching biochip two measuring chambers (5, 5 ') arranged below the same, from the Bodenbzw. Measuring surface (50) by a shielding layer (40) isolated micro-sensor (4), wherein one (5) of the measuring chambers (5, 5 ') to be loaded with the micro-organisms (8) identification chamber (5) and the other one, for example, with the suspension fluid fed, Referenzbzw. Compensation chamber (5 ') is.
9. Device according to one of claims 1 to 8, characterized in that the same or the used reaching biochip (100) is sterilizable and reusable.
10. A method for phenotypic examination, preferably for, optionally continuous, identification and characterization of organisms, in particular microorganisms, by dielectric spectroscopy without the addition of markers using at least one interdigital, metallic comb structure as a dielectric micro-sensor and at least one measuring chamber arranged above the same, in which the organisms to be examined, in particular microorganisms, preferably in the form of a suspension, introduced or on the Bodenbzw. Measuring surface as, preferably single-layer, are applied, in particular using a device according to one of claims 1 to 9, characterized
- That the dielectric micro-sensor over a wide frequency range high-frequency signals are emitted to the organisms in the measuring chamber or on the measuring surface, wherein the high-frequency signals and the impedance of the interdigital comb structure changing response signals from the measuring chamber or from the organisms located there a transparent to these signals, the same and the RF signal flow substantially not influencing, thin, the measuring chamber or its measuring surface facing surface of the interdigital comb structure of the micro-sensor of said measuring chamber or of the Organisms located there separating, preferably multi-layered, Abschirmbzw.
 Insulating layer of at least one electrically non-conductive, non-magnetic, non-magnetizable and non-dielectric material, traverse, and that the thus obtained amount of impedance (change) data in the arithmetic unit (according to functional diagram in Figure 1C) subjected to a chemometric data analysis be represented by generating abstract vectors, in particular by means of Principle Component Analysis (PCA) as separate, each characteristic of a phenotypically uniform organism, in particular factor 1 / factor 2-Diagenlagespezifische, Plattern Recognition Plots or -Plot pattern.
A method according to claim 10, characterized in that samples of (micro) organism suspensions are introduced into the measuring chamber of the bio-chip whose individual concentration is more than 10 <7> cfu / ml.
12. The method according to claim 10 or 11, characterized in that a preferably area-covering, one-layer coating of the measuring surface of the measuring chamber with the (micro) organisms is made.
13. The method according to any one of claims 10 to 12, characterized in that the covering of the measuring chambers or of their Bodenbzw. Measuring surface with (micro) organisms 5 to 95%, preferably to 75 to 95%, set or held.
14. The method according to any one of claims 1 to 13, characterized in that the micro-sensor formed with the interdigital comb structure with an alternating RF field having a potential of + - 10 to + - 30 mV, preferably from + - 10 to + - 20 mV, is fed.
15. Use of the device or of the method according to one of claims 1 to 14 for rapid identification of organisms, in particular microorganisms, directly from pure cultures by means of fingerprinting and comparison to or with impedance spectra of databases.
16. Use of the device or the method according to one of claims 1 to 14 for the rapid differentiation and classification of microorganisms in Gram positive and Gram negative bacteria.
17. Use of the device or the method according to one of claims 1 to 14 for the long-term characterization of (micro-) organisms or. Cell populations from standardized samples and pure cultures.
18. Use of the device or of the method according to one of claims 1 to 14 for monitoring and determining the vitality and the growth, in particular the doubling rates, of (micro-) organisms, in particular in the presence of the same influencing media.
19. Use of the device or of the method according to one of claims 1 to 14 for long-term studies of (micro-) organisms under the influence of the same in the measuring chamber brought into contact substances of variable chemical composition and / or toxic substances and / or Flow conditions of the (micro) organisms containing or rinsing fluid medium.
PCT/AT2008/000111 2007-03-27 2008-03-27 Device, particularly a biochip, for identifying microorganisms WO2008116244A1 (en)

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