WO2022013457A1 - Procédé de détection optique - Google Patents

Procédé de détection optique Download PDF

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Publication number
WO2022013457A1
WO2022013457A1 PCT/EP2021/070185 EP2021070185W WO2022013457A1 WO 2022013457 A1 WO2022013457 A1 WO 2022013457A1 EP 2021070185 W EP2021070185 W EP 2021070185W WO 2022013457 A1 WO2022013457 A1 WO 2022013457A1
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Prior art keywords
dma
cell
cells
droplet
drops
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German (de)
English (en)
Inventor
Pavel Levkin
Anna Popova
Lei Wenxi
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Aquarray GmbH
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Aquarray GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0346—Capillary cells; Microcells
    • G01N2021/035—Supports for sample drops
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity

Definitions

  • the invention relates to a method according to claim 1 and a use according to claim 15.
  • the object of the invention is to provide a fast and inexpensive method with which, for example, substances or activities of cells can be examined and detected.
  • the method can be used in the context of personalized medicine, for example to screen anti-cancer drugs and to screen antibiotics. It is particularly useful for screening drug libraries.
  • the invention is characterized, among other things, by its speed; the procedure delivers a result after just a few minutes.
  • the invention can be implemented, for example, in medical diagnostics as a “bedside” or “bench-to-bed” method, that is to say virtually directly at the patient's bed.
  • the procedure can also be carried out as a "point-of-care" procedure
  • the method is characterized by a high sensitivity, in particular compared to test methods in microtiter plates, which use much higher volumes of liquid than the method according to the invention.
  • the method is useful for many applications. Another advantage is that the array can be archived after the procedure has been performed after drying.
  • the method according to the invention is an optical method that allows the detection of a (bio)chemical reaction in a liquid volume of a few nanoliters without having to use molecular markings ("labels"), which are frequently used in particular when examining biological systems. No further detection steps have to be used either.
  • the method can be carried out without fluorescence and luminescence detection.
  • the method is based on the detection of fluorescence, a change in the light transmittance (turbidity) or the color of a droplet that serves as an aqueous reaction space within the scope of the invention.
  • this reaction space may contain cells that carry out the reaction. At least one property of the cells can be examined.
  • some embodiments of the method relate to chemical reactions that take place in the droplets as a reaction space; other embodiments of the method relate to biochemical reactions taking place by means of organisms; in particular reactions that take place in the organisms.
  • the turbidity A can be determined as follows:
  • A -log (l/lo) with I: intensity of the light after exiting a drop of DMA, lo : intensity of light before entering the drop of DMA.
  • the turbidity or color or depth of color of the droplet can be a direct result of a chemical reaction that occurs without an organism involved. In such cases, no biochemical, but a chemical reaction is detected.
  • the method for determining the pH value can be used (cf. FIG. 15 and the associated description).
  • the scanning can be carried out using a simple scanner, in particular a digital camera or a commercially available paper scanner. In one embodiment, the term scanner includes photography here. If the change in turbidity or color of the drop is to be quantified, commercial software can be used for this purpose, such as B. ImageJ.
  • a microarray, droplet microarray (DMA) or array in the sense of the present invention means a solid substrate with a patterned surface layer comprising hydrophilic areas, so-called spots, each of which is surrounded by hydrophobic areas (hydrophobic borders).
  • the surface layer can be in the form of a foil, a film or a coating, for example.
  • the substrate is translucent and solid at room temperature.
  • the solid substrate can be selected from glass or plastic, for example.
  • the solid transparent substrate is glass.
  • the solid substrate suitably has a flat surface, without protrusions, so that scanning can be done more easily, especially with a paper scanner.
  • the substrate should be thin, in particular a maximum of 1 mm high, so that it can be scanned with a paper scanner without any problems.
  • the substrate is formed in a single layer. There are also no channels in the substrate.
  • the array can be shaped in such a way that a pattern of separated homogeneous aqueous liquid microdroplets of a desired size is arranged.
  • a patterned substrate can be provided by the following steps: a) providing a patterned substrate having a solid layer comprising a porous polymeric layer which (i) has superhydrophilic regions with water contact angles (WCAs) of less than 20° ( with desired size and shape) surrounded by (ii) hydrophobic regions with a WCA greater than 130° and a width of 200 pm or less and
  • hydrophilic spots on a superhydrophobic (or hydrophobic) background are produced using photolithographic methods.
  • a fluorinated rough surface modified with nanoparticles can be ablated using UV light irradiation and using a photomask to shade parts of the UV light.
  • the (super)hydrophobic parts can be prepared by modifying a vinyl-bearing micro-nano raw surface using a fluorinated hydrophobic thiol via a thiol-ene or thio-yne reaction. Again, the surface can be irradiated with UV light through a quartz photomask to produce a pattern. The unmodified portions can then be hydrophilized with reactive vinyl groups containing hydrophilic thiols such as mercaptoethanol or other hydrophilic thiols.
  • the invention relates to an optical detection method.
  • the optical detection method in particular for detecting a substance or an activity (e.g. an enzyme or a cell), has the following steps:
  • DMA transparent droplet microarray
  • the DMA used in the process has a plurality of hydrophilic spots on a superhydrophobic base.
  • the DMA should be translucent.
  • the volume of the drops of DMA is 5 nl to 200 nl, in particular 20 nl to 150 nl, preferably approximately 90 nl. In some embodiments of the method, the volume of the drops is less than 100 nl, in particular 90 nl, 80 nl, 70 nl, 60 nl, 50 nl, 40 nl, 30 nl, 20 nl, 10 nl or 5 nl.
  • the spots have diameters of 0.5 mm to 3 mm.
  • performing a reaction includes adding a dye to the plurality of drops.
  • a dye is in particular a substance that changes its color through a chemical reaction. The detection is then based on the color change.
  • the turbidity of the droplets changes as a basis for detection.
  • the method is characterized by the fact that the scanning process does not have to meet any major requirements.
  • the scan can be carried out with a standard document scanner or a camera, in particular with a digital camera.
  • the scanner preferably allows the light intensity to be adjusted and the resolution to be adjusted, in particular up to 6400 dpi.
  • a document scanner shines light on the object to be examined and directs the reflected light (usually by means of mirrors and/or lenses) onto a photosensitive element, creating a digital copy of the entire object. It is therefore much simpler in terms of design than, for example, a spectrophotometer.
  • the detection method serves to detect glucose, hydrogen peroxide or beta-galactosidase activity.
  • chlorophenol red galactoside chlorophenol red galactoside
  • CPRG chlorophenol red galactoside
  • the yellow CPRG is cleaved by beta-galactosidase into galactose and chlorophenol red. In this way, the activity of the beta-galactosidase can be detected or determined.
  • the procedure for detecting glucose can be as follows. A solution of potassium iodide (KI), glucose oxidase and peroxidase (horseradish peroxidase) is placed on the spots of a DMA and dried. A glucose solution is then applied to the DMA spots. After a few minutes of incubation, a color change (yellow/orange) occurs.
  • KI potassium iodide
  • glucose oxidase glucose oxidase
  • peroxidase horseradish peroxidase
  • the method can be used to detect hydrogen peroxide.
  • a solution of potassium iodide (KI) is placed on the spots of a DMA and dried.
  • a hydrogen peroxide solution is then applied to the superhydrophilic spots of the DMA. After a few minutes of incubation, a color change (yellow/orange) occurs, which can be scanned and optionally quantified.
  • the reaction is carried out using a cell.
  • the reaction can result in a change in an optical property of the droplets.
  • the invention relates to an in vitro method for optically determining at least one property of a cell.
  • the method can include at least the following steps:
  • a droplet microarray (DMA) is provided, which has a plurality of droplets with at least one cell.
  • a reaction is carried out in the majority of the droplets of the DMA, which reaction can result in a change in an optical property of the droplets depending on the at least one property of the cell.
  • a scan of the optical properties of the majority of the drops is then carried out, with the scan being able to relate to properties such as color, including color saturation and/or turbidity of the drops.
  • properties such as color, including color saturation and/or turbidity of the drops.
  • Various properties of the cells contained in the droplets can be examined using the method. Possible properties are the viability of the cell or the number of cells, including living or metabolizing cells in a suspension or cell culture liquid. As explained elsewhere, the property can also be determined after the cells have been treated with an active substance, in particular as part of an active substance screening.
  • the method relates to a method for colorimetrically determining the viability of a cell, which has the following steps:
  • DMA droplet microarray
  • performing a reaction includes adding a dye to the plurality of drops, wherein the dye is metabolized by a living cell in a color-changing manner.
  • a dye examples include resazurin (CAS No. 550-82-3; 62758-13-8 (sodium salt)) or WST-8 (CAS No. 193149-74-5).
  • Resazurin WST-8 The viability method with Resazurin and WST-8 is particularly fast and sensitive compared to known microtiter assays. With it, a small number of cells can be detected after a short time.
  • resazurin or WST-8 is added to droplets of DMA that have 40 to 300 cells and metabolize the dyes to produce color changes. These can be scanned using a paper scanner.
  • the cells present in the drops in the method can be eukaryotic cells or prokaryotic cells.
  • the method can be carried out with 2- and 3-dimensional cell cultures.
  • prokaryotic cells in certain embodiments these are selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp, all of which are facultative pathogenic bacteria for humans.
  • Enterococcus faecium Staphylococcus aureus
  • Klebsiella pneumoniae Klebsiella pneumoniae
  • Acinetobacter baumannii Acinetobacter baumannii
  • Pseudomonas aeruginosa Pseudomonas aeruginosa
  • Enterobacter spp all of which are facultative pathogenic bacteria for humans.
  • animal-pathogenic or non-pathogenic bacteria can also be examined in the method.
  • the number of cells in a droplet of DMA is measured as a property.
  • the turbidity of a droplet increases with the number of bacterial cells in a droplet (from translucent to turbid). This turbidity can be detected optically, even with the naked eye in some embodiments of the method.
  • the method thus allows the number of cells in a drop to be determined without the bacteria having to be labeled or further detection steps being required.
  • This method is therefore suitable, for example, for screening drug candidates, e.g. B. for antibiotics.
  • the method is particularly suitable as a platform technology for screening methods, i.e. when searching for suitable drug candidates from a large number of possible substances and compounds.
  • the method then additionally has the step of adding a substance to be tested into a drop of the DMA, the substance to be tested being an active substance, for example a drug, in particular the substance to be tested being part of a library of substances to be tested.
  • the method can be used as part of a high-throughput screening.
  • the substance to be tested in the method can be of different nature.
  • the substance to be tested is selected from the group consisting of active substance, small molecule (small molecule), nucleic acid (in particular DNA, RNA, mRNA or siRNA), surface materials, etc.
  • the active ingredient may be a medicament for a human or animal disease, for example cancer.
  • the drug is selected from the group consisting of pazopanib, vorinostat, nilotinib and dasatinib, cytarabine, gemcitabine, etoposide, tamoxifen, tretinoin, dasatinib, pazopanib, nilotinib, gefitinib, bicalutamide, vorinostat, vinblastine, carboplatin, oxalitacin, and alendronate, all of which are well-known anti-cancer drugs.
  • eukaryotic cells such as a human cell line
  • a DMA using resazurin or WST-8, which are metabolized by living cells such that a color change occurs. Scanning the color change can be done with the viewer's eye.
  • the DMA can be scanned, e.g. B. using a commercially available paper scanner, followed by the determination of the color depth.
  • a freely available program such as ImageJ can be used for this purpose.
  • eukaryotic cells are assayed in conjunction with an antibiotic drug such as ciprofloxacin, vancomycin, or others.
  • an antibiotic drug such as ciprofloxacin, vancomycin, or others.
  • 1 to 100 cells can be introduced per drop for this purpose.
  • the turbidity of the drops changes. After drying the DMA, the turbidity can e.g. B. be determined by a digital camera.
  • the invention relates to the use of a DMA in an optical detection method, in particular in an optical biological or medical method, in particular in a method for optically determining at least one property of a cell of the type described here.
  • the use relates to a DMA that allows an optical evaluation, in particular based on the turbidity or the color or color depth of the drops examined on the DMA.
  • the invention relates to a kit for carrying out an optical determination of at least one property of a cell of the type described here.
  • a kit has a carrier for a droplet microarray (or a droplet microarray) of the type described here on.
  • the kit can have at least one substance that can be metabolized by a living cell in a color-changing manner and/or a scanner for scanning an optical property of the plurality of drops of the droplet microarray, for example a document scanner or a camera, in particular a digital camera.
  • the invention relates to the use of said kit, in particular for carrying out the method described here.
  • Figure 1 Schematic representation of the mechanism of the resazurin or WST-8 viability assay and the workflow of the colorimetric method on the drop microarray (DMA) platform
  • DMA drop microarray
  • Figure 2 On-chip colorimetric analysis with a cell gradient using resazurin and WST-8 viability assays compared to calzein AM staining, followed by microscopy and cell quantification.
  • (b) Graph describing the distribution of cell number stained with calzein AM (left) and graph showing the measured depth of color after staining of cells with resazurin (middle) and WST-8 (right).
  • FIG. 3 Comparison of Resazurin and WST-8 colorimetric viability assays performed on a droplet microarray and in a 384-well microtiter plate (a) and (b) The graphs show a comparison of the signal change of WST-8 (a) and Resazurin (b) viability assays , which were developed using 300 cells per well/spot over a period of 5 hours. 300 cells were seeded at 30 microliters per well and incubated at 150 nL per well overnight.
  • the appropriate volume of viability assay reagent was added to each well/spot and the absorbance at 450 nm for WST-8 and at 570/600 nm for resazurin in the 384-well plates and the depth of color on the droplet microarray at 5 hours and 1 hour both in the plates as well as on the array measured.
  • the error bars in the graph represent the standard deviation. 24 and 16 replicates, respectively, were performed for each measurement of the droplet microarray and 384-well plate, respectively.
  • FIG. 4 Comparison of resazurin and WST-8 viability assays and microscopy-based methods (read-out) for estimating a response to a specific drug dose.
  • a table summarizing the IC50 values of doxorubicin obtained using the three read-out methods is shown on the right.
  • Figure 5 Comparison of the performance of resazurin and WST-8 viability assays with microscopy-based readouts in randomized treatment with one drug on a droplet microarray.
  • the graphs show a dose-dependent response of HeLa cells to pazopanib (b), vorinostat (c), nilotinib (d ) and dasatinib (e) and a table summarizing the IC50 values for the named drugs obtained using the three read-out methods.
  • FIG. 6 On-chip colorimetric analysis of spheroids using resazurin and WST-8 viability assays.
  • the graphs show the size distribution of the grown spheroids (middle) and the analysis of the color depth for Resazurin (left) and WST-8 (right) viability assays.
  • the error bars of the graph represent the standard deviations. A total of 24 repetitions were performed for each measurement.
  • Figure 7. Optimization of the imaging process of the whole DMA using food coloring (a) A digital photograph of a 14x14 array of 100 nL droplets containing a serial dilution of food coloring. The length of the scale bar is 2 mm. (b) The scan of 100 nL volume droplets on the DMA containing different dilutions of a food coloring. The length of the scale bar is 0.5 mm. (c) Graphs showing mean gray values (left) and normalized mean gray values (right) after image analysis of scans of the DMA containing a serial dilution of food coloring. Image analysis was performed using ImagJ software, which converts the images into 8-bit images before determining the mean gray values. The error bars represent the standard deviation.
  • Figure 8 Colorimetric analysis of cell gradients performed on an array using resazurin and WST-8 viability assays compared to calzein AM staining with subsequent cell quantification using microscopy
  • Figure 9 Depth of color analysis of the entire droplet microarray containing HeLa cells treated with different concentrations of doxorubicin and stained with WST-8 and resazurin, respectively.
  • FIG. 10 Scans of droplet microarrays containing HeLa cells treated with different concentrations of dasatinib (a), pazopanib (b), nilotinib (c) and vorinostat (d) randomly distributed on the microarray and treated with resazurin and WST- 8 (left) were stained. Tables show the concentrations of each drug in mM/L in random positions corresponding to the scan on the left (right).
  • FIG. 11 Growth of P. aeruginosa PA01 on a DMA support
  • e Fluorescence images of the P. aeruginosa PA01 GFP incubated for 24 hours on the DMA support and in a 96-well plate. 500pm, 1mm and 3mm are the side lengths of the hydrophilic rectangle.
  • aeruginosa PA01 GFP in 96-well plates and on a DMA surface detected by measuring the mean fluorescence intensity per pixel of the cultured bacteria. All fluorescence intensity values were normalized against P. aeruginosa PA01 GFP cultured for 24 hours in 96-well plates. Data are presented as the median ⁇ standard error of three experiments, each with three replicates. P-values ⁇ 0.05 were considered statistically significant (g) bacterial density in 96-well plates and on the DMA surface after incubation for 24 hours. Data are presented as the median ⁇ standard error of three experiments, each with three replicates.
  • Droplet microarray as a screening platform (a) Scheme of the sandwiching method for screening antibiotics (b) Pattern of printed antibiotics on fluorinated glass slides (c) Image of the green fluorescence of the bacteria on DMA with 25 (5 x5) spots treated sequentially with vancomycin (13.5 pmol /l) Ciprofloxacin (40 pmol/l).
  • the DMA slides were placed on black paper (h) Grayscale scan of the yellow line shown in (g) (i.) Scanning electron microscope (SEM) image of the transparent hydrophilic spot on the DMA surface shown in (g) (j) SEM image of the line shown in ( g) shown opaque hydrophilic spots of the DMA surface.
  • SEM Scanning electron microscope
  • FIG. 13 (ae): Minimum Inhibitory Concentration (MIC) of Ciprofloxacin, Ceftazidime, Tobramycin, Ampicillin and Tetracycline for P. aeruginosa PA01 GFP assayed in 96-well plates and on DMA surfaces (DMA support: read by fluorescence intensity and the intensities were in Optical density (OD) values converted (96-well plates: read by OD measurements). All results were relative to a blank control (0 pM in 96-well plates). Data are presented as the median ⁇ standard deviation of three experiments, each with 10 replicates. (f) Time course experiment of antibacterial activity of polymyxin B on P. aeruginosa PA01 on DMA supports. Data are presented as the median ⁇ standard deviation of three experiments, each with 5 replicates.
  • MIC Minimum Inhibitory Concentration
  • FIG. 15 Result of a pH measurement using cresol red as a pH indicator.
  • Cresol red is a well-known triphenylmethane dye from the sulfonephthalein group.
  • the figure shows the result of a pH measurement on a drop microarray (DMA) in drops with a volume of 150 nL as an example of the method in which the optical property of the drop is the color. Length of scale bar shown: 1 mm.
  • the inventors have developed a simple method for determining a colorimetric viability assay on a droplet microarray (DMA), whereby, for example, a commercially available document scanner or an image acquisition system based on a smartphone camera provided by a smartphone app can be used for the evaluation.
  • the DMA platform consists of an array of hydrophilic spots on a superhydrophobic background and enables the formation of arrays of hundreds of separated, stable droplets with a volume in the nanoliter range, in particular from 5 nL to 200 nL, preferably from 10 nL to 150 nL. These droplets can be used to cultivate living cells. It is also possible to use such a droplet microarray to carry out a so-called high-throughput screening.
  • Resazurin and WST-8 are dyes that can be used to determine cell variability, usually performed in microtiter plates ( Figure 1a).
  • Resazurin is a phenoxazine dye with a dark blue color and absorbance at 600 nm. It is reduced by aerobic respiration of living cells to resorophine, which shows a light pink color and absorbance at 470 nm ( Figure 1a, top).
  • WST-8 is a colorless tetrazole salt, which is reduced by living cells to the bright yellow WST-8 formazan, which absorbs at 450 nm ( Figure 1a).
  • a simple imaging method was developed using food coloring that was printed on a DMA, by means of which high-resolution homogeneous color images of an entire DMA array can be obtained (FIG. 7).
  • a commercially available document scanner can be used, for example with a resolution of 6400 dpi and 80% exposure (Fig 7a, Figure 1a, images of the arrays).
  • An automated algorithm was developed for image analysis and estimation of color changes in the droplets.
  • An embodiment of the method according to the invention is shown in FIG. 1b.
  • a viability dye is added to the DMA, which has cells that have been treated with the molecules of a suitable library (e.g. drugs, small molecules, siRNAs, etc.), using, for example, a commercially available "non-contact low volume dispenser “ is used (Figure 1b step 1). Spreading 100 nL of dye across the array takes approximately one minute.
  • the DMA is incubated in a cell culture incubator for 20 minutes so that the cells can metabolize the viability dye.
  • the DMA is scanned, for example using a commercially available document scanner, which takes about 6 minutes with the necessary resolution.
  • the array scan is analyzed using an automated algorithm, which takes about two minutes per scan. Thus, it is possible to carry out the method according to the invention within only about 30 minutes.
  • both resazurin and WST-8 were used to estimate cell viability, using a gradient of cells with 0, 40, 80, 120, 160, 200, 250 and 300 cells per Spot in 150 nL droplets were applied to the DMA as shown in Figure 2a and incubated overnight. Thereafter, 100 nL of resazurin or WST-8 dye was spread over the entire DMA, incubated in a cell culture incubator for 20 minutes and scanned using a commercial document scanner ( Figure 2a). A control DMA was stained using calzein AM, evaluated microscopically and the number of living cells was determined (FIG. 2a).
  • HepG2 spheroids are incubated on a DMA using the “hanging droplet” method.
  • different numbers of cells between 40 to 300 cells per spot were seeded in blocks of 6x14 spots ( Figure 2a).
  • the DMA was inverted with the cells and incubated on a holder within a Petri dish in a cell culture incubator. After three days of cultivation, HepG2 spheroids of different diameters had formed on the DMA (FIG. 6a).
  • the human cervical adenocarcinoma cell line HeLa CCL2 and the human liver cancer cell line HepG2 were cultured in Dulbecco's modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FBS) and 1% penicillin/streptomycin. The cells were cultured in 10 ml cell culture dishes and passaged every 2 to 3 days. 2D cell cultures on DMA
  • DMEM Dulbecco's modified Eagle Medium
  • FBS fetal calf serum
  • penicillin/streptomycin penicillin/streptomycin
  • DMA plates were purchased from Aquarray GmbH. The DMA south contained 672 rectangular hydrophilic spots in a 14 x 48 array format with hydrophilic spots having a side length of 1 mm. Before culturing cells, the DMA plates were sterilized with 70% ethanol for 5 minutes followed by drying under sterile conditions for 15 minutes. Cells were trypsinized prior to seeding according to the standard protocol, counted and brought to the desired concentration using cell culture medium. The cells were seeded onto the DMA plate using the low volume non-contact dispenser I-DOT-One (Dispendix GmbH) in a volume of 150 nL per spot. Humidity was adjusted to 70% during cell seeding.
  • Staining was performed by dispensing 100 nL of either WST-8 (Kit-8, Dojindo EU GmbH, Germany) or Resazurin (Sigma-Aldrich, Germany) directly into each cell-containing droplet using a low-volume, non-contact dispenser volume non-contact dispensers I-DOT-One l-DOT One (Dispendix GmbH). After adding the reagent, the DMA was placed in the cell culture incubator and incubated for between 20 minutes and 5 hours, depending on the experiment. After incubation, the DMAs were scanned with a commercial paper scanner using the following settings: color: 80%, light 6700 dpi. Scanning the entire array took 6 minutes.
  • cells were distributed with calzein AM by dispensing 50 nL of a solution containing 1.5 pg/mL calzein AM (Thermo Scientific) in Dulbecco's PBS using an l-DOT One dispenser (Dispendix GmbH) has been used. Thereafter, the cells were incubated for 15 minutes in a cell culture incubator before imaging was performed. First, the entire slide was imaged at 2x magnification using a Keyence BZ-9000 microscope (KEYENCE, Osaka, Japan). microscopically. Each spot was then examined at 10X magnification with an Olympus 1X81 automated screening microscope (Olympus, Tokyo, Japan).
  • WST-8 and resazurin viability testing in 384-well plates was performed according to the manufacturer's protocol.
  • the cells were seeded by hand in 30 pL of culture medium and incubated for 24 hours. Viability tests were performed by adding 10 pL of either WST-8 (Kit-8, Dojindo EU GmbH, Germany) or Resazurin (Sigma-Aldrich, Germany). After addition of the reagent, the plates were incubated in a cell culture incubator for 20 minutes to 5 hours depending on the type of experiment.
  • the absorbance at 450 nm was measured in the case of the WST-8 and at 570 and 600 nm for resazurin. For analysis, the absorbance of control medium was subtracted for each well. For resazurin, the absorbance at 570 nm was subtracted from that at 600 nm. drug treatment
  • doxorubicin doxorubicin
  • pazopanib doxorubicin
  • vorinostat nilotinib
  • dasatinib Solutions of these active ingredients with a concentration of 4 mmol/L were prepared in DMSO. Thereafter, stock solutions were prepared in DMSO at concentrations of 7.5 mM and 750 mM. The active ingredients were distributed on sterile and dry DMAs and applied directly to the hydrophilic spots using a sciFLEXARRAYER S11 liquid dispenser (Scienion, Berlin, Germany).
  • HepG2 cells were seeded onto DMAs as described. Immediately after seeding, the DMAs were placed on a 10 cm petri dish with the cells of a holder in an inverted position. The spheroids formed hanging droplets on the DMA within 72 hours before being stained with WST-8 and resazurin.
  • Test solutions consisted of either deionized water (for UV-released compounds and negative controls) or an aqueous 17ß-estradiol (17ß-estradiol) standard at concentrations of 400 ng/L, 200 ng/L, 100 ng /L, 40ng/L, 20ng/L, 10ng/L and 5ng/L. Subsequently, 50 nL of the yeast suspension was added to each spot and the DMA was incubated in a humid atmosphere for 24 h at 30°C.
  • LacZ substrate reaction mix (comprising chlorophenol red galactoside, chlorophenol red galactoside (CPRG)) was added (10 nL per spot) and the color of the droplets was measured after 30 minutes of incubation using a document scanner.

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  • Physics & Mathematics (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
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  • Spectroscopy & Molecular Physics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détection optique comprend les étapes consistant : à produire une puce à gouttelettes (DMA) translucide/transparente comprenant une pluralité de gouttelettes ; à réaliser une réaction dans la pluralité de gouttelettes ayant pour conséquence de modifier une propriété optique des gouttelettes ; à balayer une propriété optique de la pluralité de gouttelettes de la puce à gouttelettes, en particulier la couleur ou la turbidité et à déduire la réaction ayant eu lieu dans les gouttelettes.
PCT/EP2021/070185 2020-07-17 2021-07-19 Procédé de détection optique Ceased WO2022013457A1 (fr)

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WO2015042583A2 (fr) 2013-09-23 2015-03-26 Anand Srinivasan Systèmes, dispositifs et procédés de détection et d'identification de microbes, test de sensibilité aux antimicrobiens
EP3612638A4 (fr) 2017-04-19 2020-12-16 Cap Diagnostics, LLC, DBA Pathnostics Dosage pour l'identification complète de la sensibilité aux antibiotiques

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