NL2031130B1 - Clinical sample preparation and handling for activity monitoring of single living micro- and nano-organism - Google Patents

Clinical sample preparation and handling for activity monitoring of single living micro- and nano-organism Download PDF

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NL2031130B1
NL2031130B1 NL2031130A NL2031130A NL2031130B1 NL 2031130 B1 NL2031130 B1 NL 2031130B1 NL 2031130 A NL2031130 A NL 2031130A NL 2031130 A NL2031130 A NL 2031130A NL 2031130 B1 NL2031130 B1 NL 2031130B1
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sample
organisms
nano
liquid
living
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Japaridze Aleksandre
Alijani Farbod
Gerard Steeneken Peter
Eugeniusz Roslon Ireneusz
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Univ Delft Tech
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1023—Microstructural devices for non-optical measurement
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0038—Investigating nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1027—Determining speed or velocity of a particle

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Abstract

The present invention is in the field of measuring micro- and nano organisms, in partic- ular by determining their physical, chemical, or biological properties, more in particular to 5 clinical sample preparation and handling for activity monitoring of single living micro- and nano-organism. Therein use is made of an advanced detector adapted to detect activity of eX- tremely small scale organisms, such as micro-organisms, such as bacteria and fungi, and even of vi-ruses and genetic material, such as DNA and RNA. The motion detector is capable of detecting nano-motion, that is, in the order of nanometers or less.

Description

P100762NL00
Clinical sample preparation and handling for activity monitoring of single living micro- and nano-organism
FIELD OF THE INVENTION
The present invention is in the field of measuring micro- and nano organisms, in partic- ular by determining their physical, chemical, or biological properties, more in particular to clinical sample preparation and handling for activity monitoring of single living micro- and nano-organism. Therein use is made of an advanced detector adapted to detect activity of ex- tremely small scale organisms, such as micro-organisms, such as bacteria and fungi, and even of viruses and genetic material, such as DNA and RNA. The motion detector is capable of de- tecting nano-motion, that is, in the order of nanometers or less.
BACKGROUND OF THE INVENTION
The present invention makes use of a motion detector adapted to detect micro- or na- nometer motion of small scale objects.
Techniques are available to detect cells and bacteria using micro- and nanosystems.
These are however of limited use for biology, as they often destroy the live specimen, typi- cally by requiring a vacuum environment. This limitation may pose a problem for advance- ment in further study and advancement in biology since it is not possible to look into pro- cesses that occur in live specimens, such as a metabolism thereof, growth thereof, and self- assembly and response to external stimuli or drugs.
These motion detectors are typically provided with an oscillator. Recently detectors have been developed having a flexible sample support in the form of a cantilever, or an opti- cal fiber, or a piezoelectric system, capable of fluctuating, such as in US 2018/312898 Al.
The displacement of the cantilever, typically flexing thereof, can be measured quite accurately using e.g. an optical system, typically comprising a mirror a laser, and photodiodes, which is capable of measuring a deflection of the cantilever. Movement detection is limited to a na- noscale or larger scale motion.
So, these prior art detectors and sensors, for certain applications, are not sensitive enough. Typically, they can not detect motion of a smaller living specimen, such as a single live bacterium or a virus. For certain application also a faster response is required such as re- vealing the status of the living organism in few seconds after a drug susceptibility test. Some- times cost, size, multiplication, and complexity may be a challenge as well, in addition.
Thereto the present applicant filed a patent application WO 2021/112666 Al, relating to an improved motion detector. However, clinical sampling, preparation, and handling are still relatively time consuming. For instance, in the clinic a sample 1s taken, the sample typically comprising, cells, biochemical constituents, DNA, RNA, and fragments thereof. From the sample, for instance a cell is taken and re-grown, typically in a larger volume. A liquid me- dium may be used to that end. The re-grown cells are then transferred to e.g. a microfluidic cartridge, in order to monitor further growth. The process takes a relatively long time, is la- bour intensive, and expensive.
The present invention relates to an improved method for clinical sample preparation and handling for activity monitoring of a single living micro-organism or nano-organism, and further aspects thereof, which overcomes one or more of the above disadvantages, without jeopardizing functionality and advantages.
SUMMARY OF THE INVENTION
The present invention relates in a first aspect to a method for clinical sample prepa- ration and handling for activity monitoring of a single living micro-organism or nano-organ- ism. In particular the present invention relates to a method for clinical sample preparation and handling for activity monitoring of a single living micro-organism or nano-organism, com- prising providing a quantity of living micro-organisms and/or nano-organisms 101, optionally selecting a sub-sample 102 thereof, sampling the quantity of living micro-organisms and/or nano-organisms therewith forming a sample, in particular by using a sampler 103, such as a cotton swab, or a microneedle, transferring the sample to a container 104, providing a liquid to the container and resuspending or suspending the sample in said liquid, in particular into a volume of 0.1-10 ml liquid, from the liquid obtaining a limited number of living micro-organ- isms or nano-organisms, in particular a single living organism, in particular by using a micro- pipet or nano-pipet 105, providing a sensor assembly 200 for activity monitoring, wherein said sensor assembly comprises a 2D microscale motion detector 201 adapted to act as a sam- ple receiver, comprising at least one sample receiver, in particular an array of sample receiv- ers microwell, drums, an inert suspended layer 107, wherein the suspended layer is 1-5 atoms thick, at least one support 109 for the suspended layer, and a read-out system 203 adapted for measuring alteration of the suspended layer, transferring said limited number or single living organism 106 to the sensor assembly, and analysing said activity of limited number or single living organism by measuring said alteration.
In a second aspect the present invention relates to a sensor assembly for activity moni- toring of living micro-organisms and/or nano-organisms, wherein said sensor assembly 200 comprises a 2D microscale motion detector 201 adapted to act as a sample receiver, compris- ing an array of sample receivers, in particular microwells, or drums, wherein the array com- prises 102-10° sensors/mm?, wherein the array has a size of 1-10° mm?, an inert suspended layer 107 per sample receiver, wherein the suspended layer is 1-5 atoms thick, at least one support 109 for the suspended layer, and a read-out system 203 adapted for measuring altera- tion of the suspended layer.
The present invention further relates to a sample obtained by a method according to the invention, wherein the sample comprises a liquid, and in the liquid a living single micro- or nano organism.
The present invention therewith provides a high parallelization. The small size of the sample receivers, in particular referred to as (graphene) drums, enables massive paralleliza- tion, allowing millions of cells or the like to be monitored in parallel, such as in the presence of antibiotics. The density of sensors is at the order of 10% sensors/mm? (figure 2). Further a controllable deposition of cells on the sensors is provided. In particular a suspension contain- ing the bacteria is confined between the substrate containing traps and a top plate (110 mov- ing surface). The top plate is pulled across the substrate and the particles are accumulated at the edge of the suspension. The particles are contained in the traps due to the capillary forces and the evaporation, which is influenced by the temperature control. In an exemplary embodi- ment pores (112 Porous Inert suspended layer) of the same or varying sizes (nanometer up to micron) are provided into the graphene, such as by focused 10n beam lithography. In order to provide the organisms in de sample receivers a pressure may be applied across the membrane (layer with holes) to drive the liquid through the pores (typical flow rates of about 5 10°!3 m/s - Pa are obtained). The pressure leads to precise cell deposition on the membranes, in the sam- ple receivers, as the flowing liquid drives the cells to the pores. A further way to deposit cells ina controllable way and multiplexing is by using microfluidics. A sample insertion tube 112 flows the cells through microfluidics channels 114 into smaller compartments 113 where dif- ferent chemicals/drugs/stimuli may be present. This transfer is achieved by applied pres- sure/capillary drag/gravity/electrical potential. Further advantages are that measurements can be performed on individual organisms, under identical or different circumstances, allowing a relatively fast result, in particular in a hospital environment, cells of interest can easily be sep- arate and subsequently be studied, no (intermediate) division of samples is required, the num- ber of organisms per unit volume is easily controllable, a (very) limited number of organisms per sample can be taken, and stimuli can be applied to the organisms. As mentioned, multi- plexing, such as on a chip, is easily done with the present method and assembly.
The present invention is also subject of a scientific publication expectedly in Nature
Nanotechnology, entitled "Probing nanomotion of single bacteria with graphene drums”, which publication and it’s contents are hereby incorporated by reference.
Thereby the present invention provides a solution to one or more of the above men- tioned problems. Advantages of the present invention are detailed throughout the description.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates in a first aspect to a method for clinical sample prep- aration and handling for activity monitoring of a single living micro-organism or nano- organism.
In an exemplary embodiment of the present method the quantity of living micro-or- ganisms and/or nano-organisms is provided from urine, sputum, blood, or is provided on a culture plate 101, such as a bacterial culture plate.
In an exemplary embodiment of the present method a sub-sample is selected, and wherein said sub-sample relates to a single sub-population, such as a single bacterial colony.
In an exemplary embodiment of the present method the liquid is a growth medium,
in particular selected from a Muller-Hinton broth, a Lysogeny broth (LB), and a minimal me- dium.
In an exemplary embodiment of the present method a living organism is selected from a human or animal cell, or part thereof, or fragment thereof, a microorganism, and a vi- rus.
In an exemplary embodiment of the present method the activity is selected from movement, in particular by nano-motion detection.
In an exemplary embodiment of the present method the sample comprises a pharma- ceutical composition, or a human or animal liquid or tissue, an aqueous composition, such as a waste water composition, a food composition, a to be researched composition, and a good manufacturing practice sample.
In an exemplary embodiment of the present method the volume of the sample is from 109-107 dm’.
In an exemplary embodiment of the present method before providing the quantity of living micro-organisms and/or nano-organisms, said quantity of living micro-organisms and/or nano-organisms is cultured.
In an exemplary embodiment of the present method said quantity of living micro- organisms and/or nano-organisms is cultured during 2-12 hours, in particular 4-8 hours, such as overnight, in particular under ambient culture conditions.
In an exemplary embodiment of the present method resuspending into the liquid is done in a period of resuspending time of 1- 60 minutes, such as 10-30 minutes, in particular at a resuspension temperature of 293-315 K.
In an exemplary embodiment of the present method resuspending into the liquid is done until an OD600 of 0.02-1 1s obtained, in particular an OD600 of 0.04-0.2, more in par- ticular an OD600 of 0.05-0.1.
In an exemplary embodiment of the present method resuspending into the liquid 1s done until the limited number of living micro-organisms or nano-organisms is 105-108, in par- ticular wherein said limited number is 5*10°-10".
In an exemplary embodiment of the present method an amount of liquid is 0.01-1 ml, in particular 0.02-0.1 ml.
In an exemplary embodiment of the present method after resuspending at least one living organism fixating compound is added to the liquid, such as APTES, in particular during a fixating period of 3-30 minutes, such as 5-15 minutes, in particular at a fixation temperature of 293-315 K.
In an exemplary embodiment of the present method transferring said limited number or single living organism 106 to the sensor assembly comprises confining individual living micro-organisms or nano-organisms between the at least one sample receiver and a top plate by pulling the top plate over and in contact with the surface of the 2D microscale motion de- tector, or wherein the inert suspended layer 111 is a membrane comprising at least one pore,
wherein a cross-section of the at least one pored is 10-5000 nm, wherein the suspended layer is provided over a cavity in the support, and wherein the support comprises at least one micro- channel in fluidic contact with the cavity, and wherein transferring comprises providing an under-pressure over the membrane therewith providing a flow of the liquid comprising the 5 living micro-organisms and/or nano-organisms, or wherein the sensor assembly comprises at least one sample insertion tube 112 in fluidic contact with the at least one sample receiver 113, in particular in fluidic contact with each individual sample receiver, more in particular in fluidic contact with the at least one sample receiver by at least one microfluidic channel 114, wherein transferring comprises applying a physical force causing the liquid comprising the living micro-organisms and/or nano-organisms to move to the at least one sample receiver, wherein the physical force in particular is selected from pressure, capillary drag, gravity, elec- tric potential, and a combination thereof.
In an exemplary embodiment the present method comprises providing a volume of liquid, the volume being < 10 pl, the volume comprising a microorganism, or living cell con- stituent, or virus, and measuring motion of the microorganism, or living cell constituent, or virus, over time.
In an exemplary embodiment of the present sensor assembly the inert suspended layer is a membrane comprising at least one pore, wherein a cross-section of the at least one pored 1s 10-5000 nm, wherein the suspended layer is provided over a cavity in the support, and wherein the support comprises at least one microchannel in fluidic contact with the cav- ity.
In an exemplary embodiment the present sensor assembly comprises at least one sample insertion tube 112 in fluidic contact with the at least one sample receiver, in particular in fluidic contact with each individual sample receiver, more in particular in fluidic contact with the at least one sample receiver by at least one microfluidic channel 114, a physical actu- ator for moving the liquid comprising the living micro-organisms and/or nano-organisms to the at least one sample receiver, in particular a pump, and electric power source.
The below provides further details of the present sensor assembly.
In an exemplary embodiment of the present sensor assembly material of the sus- pended layer is a two-dimensional crystal providing interlayer van der Waals interactions in a direction perpendicular to the layer surface, and is preferably selected from graphene, hexago- nal-BN, black phosphorus, transition metal dichalcogenides, wherein the metal is preferably selected from Mo, W, Nb, and wherein the chalcogen is preferably selected from S, Se and
Te, such as MoS;, NbSe:, and WSe:, and combinations thereof.
In an exemplary embodiment of the present sensor assembly the read-out system is selected from a Fabry-Perot interferometer, a Michelson interferometer, an optical interferom- eter, a laser Doppler vibrometer, one or more capacitor electrodes, a piezoelectrical element, a piezoresistive element, an impedance analyser, and combinations thereof, and/or wherein al- teration of the suspended layer changes at least one physical characteristics thereof selected from deflection, resonance frequency, reflection spectrum, transmission spectrum, optical ad- sorption, orientation of at least part of the suspended layer, optical interference, 2D crystal structure, electromagnetic properties, such as resistivity, conductivity, and combinations thereof.
In an exemplary embodiment of the present sensor assembly the read-out system comprises a laser for providing light, an optical system for directing light from the laser to the sample, an optical system for directing reflected light from the sample to a photo detector, such as a photo diode, optionally an amplifier for amplifying detected light response, and a recorder for representing motion, such as an oscilloscope.
In an exemplary embodiment of the present sensor assembly the suspended layer is 1-3 atoms thick, and/or wherein the suspended layer is 0.1-50 um wide, such as 1-2 um wide, and/or wherein the suspended layer is 0.1-50 um broad, such as 1-2 um broad.
In an exemplary embodiment of the present sensor assembly the suspended layer has a stiffness of <10 N/m, such as <1 N/m.
In an exemplary embodiment of the present sensor assembly the suspended layer has a Youngs modulus of >100 GPa, such as >500 GPa (ASTM E1111).
In an exemplary embodiment of the present sensor assembly the suspended layer has a weight of <10 kg, preferably <10°!° kg, such as <107" kg.
In an exemplary embodiment of the present sensor assembly under the suspended layer a cavity of > 100 nm height is provided, such as > 250 nm.
In an exemplary embodiment of the present sensor assembly the cavity is filled with a fluid, such as a gas or liquid.
In an exemplary embodiment of the present sensor assembly the at least one support comprises an electrically insulating material, such as with an electrical conductivity ¢ (20°C) of <10° S/m, preferably <10°% S/m, such as silicon oxide, silicon nitride, and silicon carbide.
In an exemplary embodiment of the present sensor assembly the at least one support has a height of 20-1000 nm, such as 100-300 nm, and/or wherein the at least one support is provided on or in a substrate , such as a silicon substrate.
In an exemplary embodiment of the present sensor assembly the suspended layer, the at least one support , and substrate , are each individually non-toxic, and at least partly support organism activity.
In an exemplary embodiment the present sensor assembly further comprises a humid- ity chamber for receiving the suspended layer and a sample.
In an exemplary embodiment the present sensor assembly comprises an array of sam- ple receivers.
The invention further relates to a chip comprising at least one 2D microscale motion de- tector according to the invention.
The invention further relates to an electronic device comprising a sensor assembly according to the invention or a chip according to the invention, and at least two channels each individually in electrical connection with the read-out system , such as 5-200 channels, and at least one readout line.
The invention is further detailed by the accompanying examples, which are exem- plary and explanatory of nature and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.
SUMMARY OF THE FIGURES
Figs. 1-4 show an example of the present method.
DETAILED DESCRIPTION OF THE FIGURES
Figs. 1-4 show an example of the present method.
In the figures:
For description:
Renumbering of elements: 101 bacteria culture 102 single bacteria culture 103 sampler 104 liquid container 105 pipet 106 individual microorganism 107 inert suspended layer 108 cavity 109 support 110 moving surface 111 porous inert suspended layer (membrane) 112 insertion tube 113 microwell 114 microfluidic channel 200 sensor assembly 201 2D microscale motion detector 203 read-out system 300 chip 400 electronic device
Figure 1 shows on the left side a bacterial culture 101, having at least one single colony of bacteria. A sample of said single colony is take, such as with a microneedle or cotton swab 103. The sample is transferred to a container 104. Liquid is provided to the container. Using a pipet 105 a small amount of liquid, comprising in this case mi- cro-organisms 106 is transferred to an inert suspended layer 107, being part of the measurement device.
Figure 2 is to highlight the device is highly parallelized and the ability to use a specific method to deposit cells precisely on the devices. The method allows precisely deposition of microparticles, as well as live cells, and to detect nanomotion. It com- prises a sandwiched droplet between the bottom plate and a moving top plate. When one moves the top plate 110 with respect to the bottom one the droplet slowly evapo- rates and drags the sample along the liquid front, till the particles (cells in our case) get positioned into the microwells 108, provided in support 109.
Figure 3 depicts another way of depositing single cells in a controllable way on the devices. One uses porous suspended layer 111 through which one pushes the liquid (by applying external pressure). The pressure leads to cell deposition on the membranes where the pores are, as the liquid flows through till a cell is physically blocking it.
Figure 4: Another way of depositing cells in a controllable way is by using micro- fluidics. A sample insertion tube 112 flows the cells through microfluidics channels 114 into smaller compartments 113 where different chemicals/drugs/or other stimuli are present. This transfer is achieved by applied pressure /capillary drag /gravity /electrical potential.
EXAMPLES/EXPERIMENTS
The invention although described in detailed explanatory context may be best un- derstood in conjunction with the accompanying examples.
For the purpose of searching the following section is added, which may be considered embodiments of the present invention, and of which the subsequent section represents a trans- lation into Dutch. 1. Method for clinical sample preparation and handling for activity monitoring of a single liv- ing micro-organism or nano-organism, comprising providing a quantity of living micro-organisms and/or nano-organisms (101), optionally selecting a sub-sample (102) thereof, sampling the quantity of living micro-organisms and/or nano-organisms therewith form- ing a sample, in particular by using a sampler (103), such as a cotton swab, or a microneedle, transferring the sample to a container (104), providing a liquid to the container and resuspending the sample in said liquid, in partic- ular into a volume of 0. 1-10 ml liquid, from the liquid obtaining a limited number of living micro-organisms or nano-organ- isms, in particular a single living organism, in particular by using a micro-pipet or nano-pipet (105), providing a sensor assembly (200) for activity monitoring, wherein said sensor assem- bly comprises a 2D microscale motion detector (201) adapted to act as a sample receiver, com- prising at least one sample receiver, in particular an array of sample receivers, an inert sus- pended layer (107), wherein the suspended layer is 1-5 atoms thick, at least one support (109) for the suspended layer, and a read-out system (203) adapted for measuring alteration of the suspended layer, transferring said limited number or single living organism (100) to the sensor assembly, and analysing said activity of limited number or single living organism by measuring said alteration.
2. Method according to embodiment 1, wherein the quantity of living micro-organisms and/or nano-organisms is provided from urine, sputum, blood, or is provided on a culture plate (101), such as a bacterial culture plate.
3. Method according to any of embodiments 1-2, wherein a sub-sample is selected, and wherein said sub-sample relates to a single sub-population, such as a single bacterial colony. 4. Method according to any of embodiments 1-3, wherein the liquid is a growth medium, in particular selected from a Muller-Hinton broth, a Lysogeny broth (LB), and a minimal me- dium. 5. Method according to any of embodiments 1-4, wherein a living organism is selected from a human or animal cell, or part thereof, or fragment thereof, a microorganism, and a virus. 6. Method according to any of embodiments 1-5, wherein the activity is selected from move- ment, in particular by nano-motion detection. 7. Method according to any of embodiments 1-6, wherein the sample comprises a pharmaceu- tical composition, or a human or animal liquid or tissue, an aqueous composition, such as a waste water composition, a food composition, a to be researched composition, and a good manufacturing practice sample. 8. Method according to any of embodiments 1-7, wherein the volume of the sample is from 107-107 dm’. 9. Method according to any of embodiments 1-8, wherein, before providing the quantity of living micro-organisms and/or nano-organisms, said quantity of living micro-organisms and/or nano-organisms is cultured. 10. Method according to embodiment 9, wherein said quantity of living micro-organisms and/or nano-organisms is cultured during 2-12 hours, in particular 4-8 hours, such as over- night, in particular under ambient culture conditions. 11. Method according to any of embodiments 1-10, wherein resuspending into the liquid is done in a period of resuspending time of 1- 60 minutes, such as 10-30 minutes, in particular at a resuspension temperature of 293-315 K. 12. Method according to any of embodiments 1-11, wherein resuspending into the liquid is done until an OD600 of 0.02-1 is obtained, in particular an OD600 of 0.04-0.2, more in par- ticular an OD600 of 0.05-0.1, and/or wherein resuspending into the liquid is done until the limited number of living micro-organ- isms or nano-organisms is 10°-10%, in particular wherein said limited number is 5%10°-107, and/or wherein an amount of liquid is 0.01-1 ml, in particular 0.02-0.1 ml. 13. Method according to any of embodiments 1-12, wherein after resuspending at least one living organism fixating compound is added to the liquid, such as APTES, in particular during a fixating period of 3-30 minutes, such as 5-15 minutes, in particular at a fixation temperature of 293-315 K. 14. Method according to any of embodiments 1-13, wherein transferring said limited number or single living organism (106) to the sensor assembly comprises confining individual living micro-organisms or nano-organisms between the at least one sample receiver and a top plate by pulling the top plate over and in contact with the surface of the 2D microscale motion de- tector, or wherein the inert suspended layer (111) is a membrane comprising at least one pore, wherein a cross-section of the at least one pored is 10-5000 nm, wherein the suspended layer is pro- vided over a cavity in the support, and wherein the support comprises at least one microchan- nel in fluidic contact with the cavity, and wherein transferring comprises providing an under- pressure over the membrane therewith providing a flow of the liquid comprising the living micro-organisms and/or nano-organisms, or wherein the sensor assembly comprises at least one sample insertion tube (112) in fluidic con- tact with the at least one sample receiver (113), in particular in fluidic contact with each indi- vidual sample receiver, more in particular in fluidic contact with the at least one sample re- ceiver by at least one microfluidic channel (114), wherein transferring comprises applying a physical force causing the liquid comprising the living micro-organisms and/or nano-organ- isms to move to the at least one sample receiver, wherein the physical force in particular is se- lected from pressure, capillary drag, gravity, electric potential, and a combination thereof. 15. Sample obtained by a method according to any of embodiments 1-14, wherein the sample comprises a liquid, and in the liquid a living single micro- or nano organism. 16. Sensor assembly for activity monitoring of living micro-organisms and/or nano-organ- isms, wherein said sensor assembly (200) comprises a 2D microscale motion detector (201) adapted to act as a sample receiver, com- prising an array of sample receivers, wherein the array comprises 102-10° sensors/mm}, wherein the array has a size of 1-10° mm? an inert suspended layer (107) per sample receiver, wherein the suspended layer 1s 1-5 atoms thick, at least one support (109) for the suspended layer, and a read-out system (203) adapted for measuring alteration of the suspended layer. 17. Sensor assembly according to embodiment 16, wherein the inert suspended layer is a membrane comprising at least one pore, wherein a cross-section of the at least one pored is 10-5000 nm, wherein the suspended layer is provided over a cavity in the support, and wherein the support comprises at least one microchannel in fluidic contact with the cavity. 18. Sensor assembly according to any of embodiments 16-17, wherein the sensor assembly comprises at least one sample insertion tube (112) in fluidic contact with the at least one sam- ple receiver, in particular in fluidic contact with each individual sample receiver, more in par- ticular in fluidic contact with the at least one sample receiver by at least one microfluidic channel (114), a physical actuator for moving the liquid comprising the living micro-organ- isms and/or nano-organisms to the at least one sample receiver, in particular a pump, and elec- tric power source.

Claims (18)

ConclusiesConclusions 1. Werkwijze voor de bereiding en behandeling van klinische monsters voor de monito- ring van de activiteit van een enkel levend micro-organisme of nano-organisme, bestaande uit het verstrekken van een hoeveelheid levende micro-organismen en/of nano-organis- men (101), het naar keuze selecteren van een deelmonster (102) daarvan, het bemonsteren van de hoeveelheid levende micro-organismen en/of nano-organis- men, waarbij een monster wordt gevormd, met name met behulp van een monsternemer (103), zoals een wattenstaafje, of een micronaald, het overbrengen van het monster in een vat (104), het voorzien van een vloeistof aan het vat en het monster opnieuw in deze vloeistof op lossen, met name in een volume van 0,1-10 ml vloeistof, het uit de vloeistof verkrijgen van een beperkt aantal levende micro-organismen of nano-organismen, in het bijzonder één levend organisme, met name met behulp van een micropipet of nano-pipet (105), het verstrekken van een sensorassemblage (200) voor activiteitsmonitoring, waarin de sensorassemblage omvat een 2D-microschaaldetector (201) die is aangepast om te fungeren als monster- ontvanger, omvattend ten minste één monsterontvanger, in het bijzonder een reeks mon- sterontvangers, een inerte opgehangen laag (107), waarbij de opgehangen laag 1-5 atomen dik 1s, ten minste één drager (109) voor de opgehangen laag, en een afleessysteem (203) dat is aangepast voor het meten van de verandering van de opgehangen laag, het overbrengen van bovengenoemd beperkt aantal of enkel levend organisme (106) naar de sensorassemblage, en het analyseren van de activiteit van een beperkt aantal of een enkel levend orga- nisme door het meten van de verandering.1. Method for the preparation and treatment of clinical samples for monitoring the activity of a single living micro-organism or nano-organism, consisting of providing a quantity of living micro-organisms and/or nano-organisms (101), optionally selecting a sub-sample (102) thereof, sampling the quantity of living micro-organisms and/or nano-organisms, whereby a sample is formed, in particular with the aid of a sampler (103) , such as a cotton swab, or a microneedle, transferring the sample into a vessel (104), supplying the vessel with a liquid and redissolving the sample in this liquid, in particular in a volume of 0.1-10 ml of liquid, obtaining from the liquid a limited number of living micro-organisms or nano-organisms, in particular one living organism, in particular using a micropipette or nano-pipette (105), providing a sensor assembly (200 ) for activity monitoring, wherein the sensor assembly comprises a 2D microscale detector (201) adapted to function as a sample receiver, comprising at least one sample receiver, in particular an array of sample receivers, an inert suspended layer (107), wherein the suspended layer 1-5 atoms thick 1s, at least one support (109) for the suspended layer, and a reading system (203) adapted to measure the change of the suspended layer, transferring the above-mentioned limited number or only living organism (106) to the sensor assembly, and analyzing the activity of a limited number or a single living organism by measuring the change. 2. Werkwijze volgens conclusie 1, waarbij de hoeveelheid levende micro-organismen en/of nano-organismen is verschaft uit urine, sputum, bloed, of is verschaft op een kweek- plaat (101), zoals een bacteriële kweekplaat.A method according to claim 1, wherein the amount of living microorganisms and/or nanoorganisms is provided from urine, sputum, blood, or is provided on a culture plate (101), such as a bacterial culture plate. 3. Werkwijze volgens een van de conclusies 1-2, waarbij een deelmonster wordt gekozen, en waarbij dat deelmonster betrekking heeft op een enkele subpopulatie, zoals een enkele bacteriekolonie.Method according to any of claims 1-2, wherein a sub-sample is selected, and wherein that sub-sample relates to a single subpopulation, such as a single bacterial colony. 4. Werkwijze volgens een van de conclusies 1-3, waarbij de vloeistof een groeimedium is, in het bijzonder gekozen uit een Muller-Hinton groeimedium, een Lysogeny groeimedium (LB), en een minimaal medium.Method according to any one of claims 1-3, wherein the liquid is a growth medium, in particular selected from a Muller-Hinton growth medium, a Lysogeny growth medium (LB), and a minimal medium. 5. Werkwijze volgens een van de conclusies 1-4, waarbij een levend organisme wordt ge- kozen uit een menselijke of dierlijke cel, of deel daarvan, of fragment daarvan, een micro-A method according to any one of claims 1 to 4, wherein a living organism is selected from a human or animal cell, or part thereof, or fragment thereof, a micro- organisme, en een virus.organism, and a virus. 6. Werkwijze volgens een van de conclusies 1-5, waarbij de activiteit wordt gekozen uit beweging, in het bijzonder door nano-bewegingsdetectie.Method according to any one of claims 1-5, wherein the activity is selected from movement, in particular by nano-motion detection. 7. Werkwijze volgens een van de conclusies 1-6, waarbij het monster omvat een farma- ceutische samenstelling, of een vloeistof of weefsel van mens of dier, een waterige samen- stelling, zoals een samenstelling van afvalwater, een samenstelling van levensmiddelen, een te onderzoeken samenstelling, en een monster van goede fabricagepraktijken.A method according to any one of claims 1 to 6, wherein the sample comprises a pharmaceutical composition, or a human or animal fluid or tissue, an aqueous composition, such as a waste water composition, a food composition, a composition to be examined, and a sample of good manufacturing practice. 8. Werkwijze volgens een van de conclusies 1-7, waarbij het volume van het monster 10% - 103 dm’ bedraagt.Method according to any one of claims 1-7, wherein the volume of the sample is 10% - 103 dm'. 9. Werkwijze volgens een van de conclusies 1-8, waarbij, voordat de hoeveelheid levende micro-organismen en/of nano-organismen wordt verstrekt, deze hoeveelheid levende mi- cro-organismen en/of nano-organismen wordt gekweekt.A method according to any one of claims 1 to 8, wherein, before the quantity of living micro-organisms and/or nano-organisms is provided, this quantity of living micro-organisms and/or nano-organisms is cultured. 10. Werkwijze volgens conclusie 9, waarbij de hoeveelheid levende micro-organismen en/of nano-organismen wordt gekweekt gedurende 2-12 uur, in het bijzonder 4-8 uur, zo- als 's nachts, in het bijzonder onder omgevingscultuuromstandigheden.Method according to claim 9, wherein the quantity of living microorganisms and/or nanoorganisms is cultured for 2-12 hours, in particular 4-8 hours, such as overnight, in particular under ambient culture conditions. 11. Werkwijze volgens een van de conclusies 1-10, waarbij het resuspensieproces in de vloeistof plaatsvindt in een resuspensietijd van 1-60 minuten, bijvoorbeeld 10-30 minuten, in het bijzonder bij een resuspensietemperatuur van 293-315 K.Method according to any one of claims 1-10, wherein the resuspension process in the liquid takes place in a resuspension time of 1-60 minutes, for example 10-30 minutes, in particular at a resuspension temperature of 293-315 K. 12. Werkwijze volgens een van de conclusies 1-11, waarbij het resuspensieproces in de vloeistof wordt uitgevoerd totdat een OD600 van 0,02-1 wordt verkregen, in het bijzonder een OD600 van 0,04-0,2, meer in het bijzonder een OD600 van 0,05-0,1, en/of wanneer het resuspensieproces in de vloeistof wordt uitgevoerd totdat het beperkte aantal levende micro-organismen of nano-organismen 10°-105 bedraagt, in het bijzonder wan- neer dit beperkte aantal 5*10°-107 bedraagt, en/of waarbij een hoeveelheid vloeistof 0,01-1 ml bedraagt, in het bijzonder 0,02-0,1 ml.Method according to any one of claims 1-11, wherein the resuspension process is carried out in the liquid until an OD600 of 0.02-1 is obtained, in particular an OD600 of 0.04-0.2, more in particular an OD600 of 0.05-0.1, and/or when the resuspension process in the liquid is carried out until the limited number of living micro-organisms or nano-organisms is 10°-105, in particular when this limited number is 5 *10°-107, and/or where an amount of liquid is 0.01-1 ml, in particular 0.02-0.1 ml. 13. Werkwijze volgens een van de conclusies 1-12, waarbij na resuspensie ten minste één levend organisme fixerende verbinding aan de vloeistof wordt toegevoegd, zoals APTES, in het bijzonder gedurende een fixatieperiode van 3-30 minuten, zoals 5-15 minuten, in het bijzonder bij een fixatietemperatuur van 293-315 K.Method according to any one of claims 1-12, wherein after resuspension at least one living organism fixing compound is added to the liquid, such as APTES, in particular during a fixation period of 3-30 minutes, such as 5-15 minutes, in especially at a fixation temperature of 293-315 K. 14. Werkwijze volgens een van de conclusies 1-13, waarbij het overbrengen van het be- perkt aantal of slechts één levend organisme (106) naar de sensorconstructie omvat het op- sluiten van individuele levende micro-organismen of nano-organismen tussen de ten min- ste één monsterontvanger en een bovenplaat door de bovenplaat over en in contact met het oppervlak van de 2D-microscale bewegingsdetector te trekken, of waarbij de inerte opgehangen laag (111) een membraan is dat ten minste één porie omvat, waarbij de doorsnede van ten minste één porie 10-5000 nm bedraagt, waarbij de opgehan- gen laag over een holte in de drager is aangebracht, en waarbij de drager ten minste één microkanaal omvat dat in vloeistofcontact staat met de holte, en waarbij het overbrengen omvat het aanbrengen van een onderdruk over het membraan waardoor een stroom van de vloeistof met de levende micro-organismen en/of nano-organismen op gang wordt ge- bracht, of waarbij de sensoropstelling ten minste één buisje voor monsterinbrenging (112) omvat dat in vloeibaar contact staat met ten minste één monsterontvanger (113), in het bijzonder in vloeibaar contact met elke afzonderlijke monsterontvanger, meer in het bijzonder in vloei- baar contact met ten minste één monsterontvanger door ten minste één microfluidisch ka- naal (114), waarbij het overbrengen omvat het uitoefenen van een fysische kracht waar- door de vloeistof die levende micro-organismen en/of nano-organismen omvat, zich naar de ten minste één monsterrecipiént beweegt, waarbij de fysische kracht in het bijzonder wordt gekozen uit druk, capillaire weerstand, zwaartekracht, elektrische potentiaal, en een combinatie daarvan.The method of any one of claims 1 to 13, wherein transferring the limited number or only one living organism (106) to the sensor assembly includes trapping individual living microorganisms or nanoorganisms between the ten at least one sample receiver and a top plate by pulling the top plate over and into contact with the surface of the 2D microscale motion detector, or wherein the inert suspended layer (111) is a membrane comprising at least one pore, wherein the cross-sectional area of at least one pore is 10-5000 nm, wherein the suspended layer is disposed over a cavity in the support, and wherein the support comprises at least one microchannel in fluid contact with the cavity, and wherein the transfer comprises applying a negative pressure across the membrane which initiates a flow of the liquid containing the living micro-organisms and/or nano-organisms, or where the sensor arrangement comprises at least one sample introduction tube (112) in liquid contact with at least one sample receiver (113), particularly in liquid contact with each individual sample receiver, more particularly in liquid contact with at least one sample receiver through at least one microfluidic channel (114), the transferring comprising exerting a physical force causing the liquid containing living micro-organisms and/or nano-organisms to move towards the at least one sample recipient, wherein the physical force is selected in particular from pressure, capillary resistance, gravity, electrical potential, and a combination thereof. 15. Een monster verkregen met een werkwijze volgens één van de conclusies 1-14, waar- bij het monster een vloeistof omvat, en in de vloeistof een levend enkel micro- of nano- organisme.A sample obtained by a method according to any one of claims 1-14, wherein the sample comprises a liquid, and in the liquid a living single micro- or nano-organism. 16. Sensorassemblage voor het monitoren van activiteit van levende micro-organismen en/of nano-organismen, waarbij de sensorassemblage (200) omvat een 2D-microschaalbewegingsdetector (201) aangepast om te fungeren als monsterontvanger, omvattend een array van monsterontvangers, waarbij de array 107-105 sensors/mm? omvat, en de array een grootte heeft van 1-10° mm’, een inerte opgehangen laag (107) per monsterontvanger, waarbij de opgehangen laag 1-5 atomen dik is, ten min- ste één drager (109) voor de opgehangen laag, en een afleessysteem (203) dat is aangepast voor het meten van de verandering van de opgehangen laag.16. Sensor assembly for monitoring activity of living microorganisms and/or nanoorganisms, wherein the sensor assembly (200) includes a 2D microscale motion detector (201) adapted to function as a sample receiver, comprising an array of sample receivers, the array 107-105 sensors/mm? comprises, and the array has a size of 1-10° mm', an inert suspended layer (107) per sample receiver, the suspended layer being 1-5 atoms thick, at least one support (109) for the suspended layer , and a reading system (203) adapted to measure the change in the suspended layer. 17. Sensorassemblage volgens conclusie 16, waarbij de inerte opgehangen laag een mem- braan is dat ten minste één porie omvat, waarbij een doorsnede van de ten minste één po- rie 10-5000 nm bedraagt, waarbij de opgehangen laag over een holte in de drager is aan- gebracht, en waarbij de steun ten minste één microkanaal omvat dat in vloeibaar contact staat met de holte.The sensor assembly of claim 16, wherein the inert suspended layer is a membrane comprising at least one pore, wherein a diameter of the at least one pore is 10-5000 nm, wherein the suspended layer extends over a cavity in the carrier is provided, and wherein the support comprises at least one microchannel that is in fluid contact with the cavity. 18. Sensorassemblage volgens een van de conclusies 16-17, waarbij de sensorassemblage ten minste één buis voor monsterinbrenging (112) omvat die in fluïde contact staat met ten minste één monsterontvanger, in het bijzonder in fluïde contact met elke afzonderlijke monsterontvanger, meer in het bijzonder in fluïde contact met ten minste één monsteront- vanger via ten minste één microfluidisch kanaal (114), een fysische actuator om de vloei- stof die de levende micro-organismen en/of nano-organismen omvat naar ten minste één monsterontvanger te bewegen, in het bijzonder een pomp, en een elektrische stroombron.Sensor assembly according to any one of claims 16 to 17, wherein the sensor assembly comprises at least one sample introduction tube (112) that is in fluid contact with at least one sample receiver, in particular in fluid contact with each individual sample receiver, more specifically specifically in fluid contact with at least one sample receiver via at least one microfluidic channel (114), a physical actuator to move the fluid comprising the living microorganisms and/or nanoorganisms to at least one sample receiver, in particular a pump, and an electrical power source.
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