WO2020020617A1

WO2020020617A1 - Arrangement for recognising the presence of pathogens

Info

Publication number: WO2020020617A1
Application number: PCT/EP2019/068236
Authority: WO
Inventors: Tim Kunze; Wulf Grählert; Sabri Alamri
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date: 2018-07-26
Filing date: 2019-07-08
Publication date: 2020-01-30
Also published as: DE102018212495B3

Abstract

The invention relates to an arrangement for recognising the presence of pathogens on a surface of a planar substrate, wherein a surface of the substrate has, locally defined, a multivariate surface structure having sizes, geometries and/or orientations, which deviate from one another, of 2- to 3-dimensional structural elements, the individual structural elements having a lateral dimension in the range from 10 nm to 50 μm. The arrangement comprises a light source for irradiating the 2- to 3-dimensionally structured surfaces and a plurality of detectors, for spatially and spectrally resolved detection of the electromagnetic radiation scattered, reflected and/or transmitted to the 2- to 3-dimensionally structured surface. The detectors are connected to an electronic evaluation unit which is designed for evaluation of intensities, which are spatially and spectrally-resolved by the detectors, of the scattered, reflected, transmitted electromagnetic radiation which is formed by scattering, absorption, Raman scattering or fluorescence processes. An electronic storage device is present, in which prediction models based on spectra of known pathogens on the same 2- to 3-dimensional structural elements are stored, and these models can be used by means of the electronic evaluation unit for classification and thus for verification of the presence of at least one specific pathogen on the 2- to 3-dimensional structured surface.

Description

ARRANGEMENT FOR DETECTING THE EXISTENCE OF PATHOGENS

The invention relates to the field of identification of pathogens or the selective detection of germs, and in particular to an arrangement for recognizing the presence of pathogens, for example bacteria,

Fungi, protozoa, parasites or parasitic molecules, especially viruses, viroids, transposons or prions.

Prevention through efficient diagnostics is anchored in the research strategy. The rapidly increasing additional expenditure in the health care system due to approx. 10 T € per nosocomial, ie infections acquired in the hospital (= 7 billion € / a in Europe), but also economic costs of up to 3 billion € / a in Germany due to the seasonal Flu must be countered by effective solutions. At the moment there are little efficient and meaningful rapid tests on the market, which achieve a comparable or sufficient reliability compared to conventional laboratory tests at the same time, however, they are (clearly) superior in terms of both time and costs (laboratory tests currently ~ 50-150 € / laboratory test).

The safe, quick and inexpensive identification of bacteria or other pathogens is one of the most important prerequisites for fast and safe treatment of infections today. Has z. B. a bacterium identified, an adequate treatment with optimal antibiotics can be derived and consequently the likelihood of developing

Antibiotic resistance can be reduced.

The main challenges in identifying bacteria are:

The highest possible sensitivity of the process, i.e. the presence of the bacterium / pathogen should be possible even at low concentrations. The highest possible specificity of the process, i.e. the correct detection of the absence of the target bacterium / pathogen.

• The lowest possible analytical detection limit and the selectivity of the method used, which directly affect sensitivity and selectivity.

• A low cross-sensitivity to possible "contaminations" (e.g. also the mixing of living and dead species)

• The realization of a high analysis speed and / or throughput of the method used.

There are a large number of known detection methods which serve to identify pathogens or germs:

1. Biochemical characterization, agglutination test, cultivation of microbes with subsequent morphological or physiological characterization

Identification time: typically at least 24 hours

2. Polymerase chain reaction (PCR test) or real-time PCR

Identification time: several hours

Detection limit for Gram-positive or negative microorganisms in blood: approx. 10 ⁴ - 10 ⁷ per mL

3. Fluorescence-based capillary electrophoresis

PCR single strand conformation polymorphism (SSCP) protocol Identification time: ~ 7 h

4. Bloodstream infections - Fluorescent in situ hybridization (FISH) -

Identification time: 2 - 3 h

5. Rapid identification of pathogens using a combination of PCR amplification and microarray detection

Identification time: approx. 6 hours

Detection limit: 10 cells (e.g. E. coli) to 105 cells (S. aureus) per mL artificial (enriched) blood

The main disadvantages of the methods mentioned are:

1. Long identification times> 30 minutes and / or

2. Insufficient detection specificity and / or

3. Low detection sensitivity

It is therefore an object of the invention to provide possibilities for a safe detection of the presence of certain pathogens, the time required for detection to be reduced.

According to the invention, this object is achieved with an arrangement which has the features of claim 1. Advantageous refinements and developments of the invention can be realized with features designated in the subordinate claims.

The arrangement according to the invention for detecting the presence of pathogens has, on a surface of a flat substrate, locally defined a multivariate surface structure with differing sizes, geometries and / or orientations of 2 to 3-dimensional structural elements, the individual structural elements having a latex dimension have in the range 10 nm to 50 pm.

In addition, there is a light source for irradiating the 2 to 3-dimensional structured surface and several detectors for the spatially and spectrally resolved detection of the surface scattered, reflected and / or transmitted on the 2 to 3-dimensional structured surface Radiation present.

The detectors are connected to an electronic evaluation unit, which is used to evaluate the intensities of the scattered, reflected or transmitted electromagnetic radiation, which are detected with the detectors in a spatially and spatially resolved manner, and which is formed by scattering, absorption, Raman scattering or fluorescence processes.

There is an electronic memory in which prediction models based on spectra of known pathogens are stored on these two to three-dimensional structural elements, which are used for classification and thus for the detection of the presence of at least one specific pathogen by means of the electronic evaluation unit the 2 to 3-dimensional structured surface can be used ..

2- to 3-dimensional structural elements can advantageously be formed as depressions or bulges in the surface of the substrate by means of laser structuring, in particular direct laser interference structuring and / or laser-induced self-organized, periodic surface structuring. 2- to 3-dimensional structural elements can be formed in a point, line shape with different dimensions. In particular, line-shaped 2 to 3-dimensional structural elements can have different widths and / or lengths and different orientations on the surface of the substrate.

This makes it possible to exploit the fact that certain pathogens can use better cultivation conditions in surface areas that are particularly suitable for them, and that particularly favorable locally defined colonization with the respective pathogen can be achieved there. As a result, the concentration or the number of individual pathogens in specific areas can be increased or decreased in a targeted manner, which can have an advantageous effect on improved selectivity when determining the presence of the respective pathogen.

2- to 3-dimensional structural elements can be designed with a multivariate surface topography. 2- to 3-dimensional structural elements can be formed continuously or discontinuously changing in size, shape and / or orientation starting from at least one outer edge of the substrate surface, whereby a gradual change in size, orientation and / or geometry over the surface of the substrate can be reached.

On the surface of the substrate, there can also be a plurality of surface areas on the substrate, each of which is formed with the same three-dimensional structural elements and different 2 to 3-dimensional structural elements are formed in the individual surface areas. In this way, differently structured surface areas can be used and at least one surface area for a certain type of pathogen has better cultivation or settlement conditions than other surface areas and an increased concentration of pathogens of this type of pathogen can be achieved in this surface area.

In particular, in order to detect the intensity of reflected, scattered or transmitted electromagnetic radiation for determining at least one pathogen in a spatially resolved manner, a plurality of detectors which are designed for spatially resolved spectral detection of electromagnetic radiation within a wavelength interval should be arranged in a row or in a row and column arrangement , The detectors are then connected to the electronic evaluation unit and arranged such that electromagnetic radiation emitted by a radiation source is directed onto the surface of the substrate and electromagnetic radiation reflected, scattered or transmitted through the substrate strikes the detectors.

If Raman or fluorescent radiation is used for the detection of at least one pathogen, the surface of the substrate should be irradiated with a laser beam which is emitted by a laser beam source, so that specific Raman or fluorescent radiation is generated on surfaces of existing pathogens , The generated Raman or fluorescence radiation is based on a spectrally and spatially resolved detection Solution of this radiation trained detector array directed.

The detectors of the detector array are designed for spatially and spectrally resolved detection of the electromagnetic radiation reflected or scattered by the pathogens.

An optical filter or a beam splitter is arranged between the surface of the substrate and the detector array and is designed such that electromagnetic radiation with the wavelength of the laser beam does not strike the detectors of the detector array. At least the detectors of the detector array are connected to an electronic evaluation unit. The electronic evaluation unit is designed to evaluate the spatially and spectrally resolved intensities of the Raman or fluorescent radiation detected by the detectors of the detector array and to carry out a data analysis to determine the distribution of the spatially and spectrally resolved intensities representing the pathogens on the surface of the substrate.

The basic idea is to use surfaces with a surface structuring tailored to a bacterium / pathogen in the micrometer range (structure sizes 10 nm - 50 pm)

• a laterally specific adhesion behavior z. B. induced a target bacterium

• enables the use of fast and sensitive area sensors for the detection of pathogens / bacteria

• if necessary, the evidence of the arrangement increased drastically

The customized surface topography serves to optimize the absorption or adhesion behavior of pathogens on the surface, so that a selective coupling between surface shape and

Pathogen attachment is made possible. A specifically optimized, multivariate surface topology can drastically increase the variance of the adsorption and adhesion of the pathogens and thus the sensitivity and selectivity of the pathogenic detection method used, which enables significantly reduced measurement times. The structured test substrates are fundamentally realizable as (low-priced) consumables. The production of the customized, defined and versatile surface topographies with the three-dimensional structural elements can be done, for example, using a direct laser interference method (direct laser

Interference Patterning - DLIP) can be realized. The range of realizable surface structures can be selected by choosing the DLIP setup (e.g.

B. Switching between 2- and 3-beam interference, changing the polarization of the individual partial beams; Process strategy) can be freely selected, as has been explained for example in DE 10 2018 200 036, and the disclosure content of which is referred to in full. As a result, for example, line, point and cross structures can be used directly on substrate surfaces that will be used later, e.g. B. Realize metals. Other surface topographies generated by laser processes (e.g. laser-induced periodic surface structuring, i.e. laser-induced periodic surface structures - LIPSS) can also be used for a targeted setting of the surface pathogen response.

The multivariate surface structures with the three-dimensional structural elements, which are formed in a sufficient variety of structures on the substrate surface, should enable a topography-dependent - and thus locally determined - specific adhesion behavior of the pathogens to be detected. The topography of a surface of a substrate supports or inhibits the growth of certain pathogens / bacteria. The realization of different structures on an analysis surface, which is possible with the DLIP method, and the knowledge of the adhesion behavior specific for these structures as well as their lateral and temporal characteristics can form the basis of a pronounced sensitivity and selectivity of the arrangement.

Hyperspectral imaging (HSI) enables the qualitative / quantitative areal identification of pathogens / bacteria based on their specific spectral signatures. FIG. 2 shows a specific surface topography (monostructure) and associated HSI signals of the adhered bacterial strains.

A light signal is generated after the interaction with the sample (pathogenic microorganisms attached to the surface of the substrate) detected spectrally and laterally resolved. For each point on the sample, the evaluation of the respective spectrum can provide information about sample properties (here, for example, the basic presence of pathogens / bacteria and their classification) or the reaching or exceeding of a minimum number of pathogens / bacteria become. Depending on the excitation mode, the absorption or reflection behavior can be evaluated classically, but also fluorescence behavior and Raman scattering can be used. The measurement time required for this is in the range of seconds to a few minutes (depending on the substrate surface size, the lateral and spectral resolution, exposure / accumulation time).

The HSI detection sensitivity can be drastically increased again in Raman / fluorescence mode in combination with the applied microstructures (prerequisite: sufficiently small peaks on the surfaces) by using the so-called Surface Enhanced Raman Spectroscopy Effect (SERS). This significantly increases the sensitivity and specificity of the detection of pathogen samples compared to previous solutions. This immediately leads to the fact that reliable detection of pathogens is already possible if they are only present in the lowest concentrations, which means that valid results can be obtained after only a short measuring time (a few minutes).

The identification specificity of the pathogen measurement system can be significantly strengthened by using multivariate surface regions (so-called segments), as shown in FIG. 3. The evaluation of the pathogens adsorbed on the surface enables the identification of both individual pathogens and combinations of different pathogens. Due to the defined multivariant structured surface, the evaluation can be carried out in different ways:

Spectral (signal = ί (l) = "pathogen class (n)")

Spectral as a function of time

(Signal = f (, t) = "pathogen class (s)")

Spectral depending on the topology (signal = ί (l, DLIP-

Parameters) = "pathogen class (s)") spectral depending on the Time and topology (signal = ί (l, DUP parameter, t) =

"Pathogenklasse (s)")

By using multivariate surface areas, pathogens / bacteria can be clearly assigned, since there is a direct connection between pathogen / bacterial adherence and the present

Surface topography that matches the 2 to 3 dimensional

Structural elements is formed, is present.

An alternative approach consists in realizing a continuous, multivariate distribution of three-dimensional structural elements on the surface by continuously varying the DLIP structural parameters (see FIG. 5). As a result, the surface of the substrate can be qualitatively / quantitatively examined using HSI and a multivariant adhesion behavior of pathogens / bacteria can be detected. Machine learning / deep learning methods can be used to identify specific pathogen signatures early and accurately.

The advantages compared to the prior art are as follows:

• Increase detection sensitivity

• Increase detection specificity

• Reduce the identification time of bacteria / pathogens

• Simple measurement setup

• Inexpensive test substrates

• Extremely high flexibility -> DLIP parameters, huge potential parameter window

• The invention can be transferred to other pathogens through software-based evaluation (models) such as machine learning / deep learning

The invention will be explained by way of example below. Show:

Figure 1 4 examples of surfaces of substrates with different

Surface structuring for a specific settlement with pathogens; FIG. 2 shows an example of a surface of a substrate which is populated differently with several pathogens, with diagrams which illustrate the relationship between measurement signals which are recorded in a spatially and spatially resolved manner for three different types of bacteria as examples of pathogens.

Figure 3 shows an example of one divided into six surface areas A-F

Surface of a substrate populated with pathogens, in which the surface areas AF are designed with different surface structuring for flexible and parallel recognition of different types of pathogens, and a diagram arranged in the center shows the different spectral behavior in the individual surface areas AF and in the diagram shown on the right that in the Surface areas AF different settlement kinetics is clarified;

Figure 4 shows in the surface areas A-F colonization characteristic for three different types of bacteria, as examples of pathogens to be recognized, depending on the different surface topography in each of the surface areas A-F and

FIG. 5 shows a surface of a substrate with a continuous structural variance due to a continuous variation of the DLIP structural parameters, which, due to the continuous change of the 2 to 3-dimensional structural elements over the entire surface of the substrate, results in an area-specific adhesion behavior of Bak that can be detected qualitatively / quantitatively by HSI leads as an example of pathogens.

Claims

claims

1. Arrangement for detecting the presence of pathogens on a surface of a flat substrate, in which a surface of the substrate (1) locally defines a multivariate surface structure with mutually different sizes, geometries and / or alignments of 2 to 3-dimensional structural elements , wherein the individual structural elements have a lateral extent in the range from 10 nm to 50 pm and a light source for irradiating the 2 to 3-dimensionally structured surface and several detectors for the spatially and spectrally resolved detection of the 2 to 3 -dimensionally structured surface scattered, reflected and / or transmitted electromagnetic radiation are present and the detectors are connected to an electronic evaluation unit, which is used to evaluate the spatially and spectrally resolved intensities of the scattered, reflected, transmitted electromagnetic radiation , which is formed by scattering, absorption, Raman scattering or fluorescence processes, and an electronic memory is available, in which prediction models based on spectra of known pathogens are stored on these 2 to 3-dimensional structural elements, which are used by the electronic evaluation unit for a classification and thus can be used for the detection of the presence of at least one specific pathogen on the 2 to 3-dimensional structured surface.

2. Arrangement according to claim 1, characterized in that 2 to 3-dimensional structural elements as depressions or bulges are formed in the surface of the substrate by means of laser structuring, in particular direct laser interference structuring and / or laser-induced, self-organized, periodic surface structuring.

3. Arrangement according to one of the preceding claims, characterized in that 2- to 3-dimensional structural elements, starting from at least one outer edge of the substrate surface, continuously or discontinuously changing in size, shape and / or direction or several surface areas on the substrate are present, which are each designed with the same 2- or 3-dimensional structural elements.

4. Arrangement according to one of the preceding claims, characterized in that a plurality of detectors, which are designed for spatially and spectrally resolved analysis of electromagnetic radiation within a wavelength interval, arranged in a row or a row and column arrangement and

the detectors are connected to the electronic evaluation unit and are arranged such that electromagnetic radiation emitted by a radiation source is directed onto the surface of the substrate and electromagnetic radiation reflected, scattered or transmitted through the substrate hits the detectors.

5. Arrangement according to one of the preceding claims, characterized in that the surface of the substrate is irradiated with a laser beam which is emitted by a laser beam source, so that a generation of Raman or fluorescent radiation takes place by pathogens present on the substrate surface and the generated Raman or fluorescent radiation on a detector designed for the spatially and spectrally resolved detection of this radiation torarray is directed and

Detectors of the detector array are designed for spatially and spectrally resolved detection of electromagnetic radiation reflected or scattered by the pathogens, and an optical filter or a beam splitter is arranged between the surface of the substrate and the detector array and is designed in such a way that electromagnetic radiation with the wavelength of the laser beam does not strike the detectors of the detector array; wherein at least the detectors of the detector array (4) are connected to an electronic evaluation unit and the electronic evaluation unit for evaluating spatially and spectrally resolved intensities of the Raman or fluorescence radiation detected with the detectors of the detector array and for carrying out a data analysis taking into account the distribution the spatially and spectrally resolved intensities representing the pathogens are formed on the surface of the substrate.

6. Arrangement according to one of the preceding claims, characterized in that electromagnetic radiation, which is emitted by a broadband radiation source, is directed to the surface of the sub strate with the structural elements and thereby a specific for respective pathogens by absorption, refraction and scatter Reflected or transmitted electromagnetic radiation influenced by detectors of the detector array can be detected using an electronic evaluation unit for evaluating the spatially and spectrally resolved intensities of the reflected or transmitted radiation detected by the detectors of the detector array and for performing a data analysis taking into account the distribution the spatially and spectrally resolved intensities representing the pathogens are connected on the surface of the substrate.

7. Arrangement according to one of the preceding claims, characterized in that the electronic evaluation unit for data analysis se, a spectral evaluation depending on the time, a spectral evaluation depending on the surface topography or a spectral evaluation depending on the time and Surface topography for detecting the presence of pathogens is formed.