WO2018167106A1 - Procédé et dispositif microfluidique pour le traitement de virus et de bactéries d'un échantillon - Google Patents

Procédé et dispositif microfluidique pour le traitement de virus et de bactéries d'un échantillon Download PDF

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Publication number
WO2018167106A1
WO2018167106A1 PCT/EP2018/056314 EP2018056314W WO2018167106A1 WO 2018167106 A1 WO2018167106 A1 WO 2018167106A1 EP 2018056314 W EP2018056314 W EP 2018056314W WO 2018167106 A1 WO2018167106 A1 WO 2018167106A1
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WO
WIPO (PCT)
Prior art keywords
bacteria
viruses
retaining element
sample
chamber
Prior art date
Application number
PCT/EP2018/056314
Other languages
German (de)
English (en)
Inventor
Jochen Hoffmann
Original Assignee
Robert Bosch Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Robert Bosch Gmbh filed Critical Robert Bosch Gmbh
Publication of WO2018167106A1 publication Critical patent/WO2018167106A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
    • B01L2400/0439—Moving fluids with specific forces or mechanical means specific forces vibrational forces ultrasonic vibrations, vibrating piezo elements

Definitions

  • Microfluidic analysis systems allow an automated, reliable, compact and cost-effective processing of biochemical substances for medical diagnostics, for example for the detection of pathogens in biological samples, for example in body fluids.
  • the causative agents may be of viral and / or bacterial origin.
  • Flow logs include a compromise between harsh ones
  • Lysis conditions especially for the digestion of gram-positive bacteria, and subtle conditions to minimize damage, especially strand breaks, to viral nucleic acids. Disclosure of the invention
  • the invention relates to a method for processing viruses and bacteria of a sample with a microfluidic device.
  • a first step the sample is rinsed via a retaining element of the device, wherein the retaining element is designed to fix viruses and bacteria from the sample.
  • a second step a flushing of a first fluid via the retaining element for detachment from the through
  • a second fluid is purged via the
  • Retaining element for detachment of bacteria fixed by the retaining element and transfer of the bacteria-enriched second fluid into a second chamber for further processing of the bacteria.
  • the third step can take place before or after the second step.
  • the purging of the first fluid and / or of the second fluid can in principle in the same direction, thus in the same flow direction, or in a different direction as the direction of the purging of the sample on the
  • the purging of the second fluid may be in the opposite direction to the direction of purging the sample via the retention member.
  • viruses or bacteria By fixing viruses or bacteria by the retaining element, in particular a recording of viruses or bacteria via immobilization and / or binding, for example a chemical or electrostatic as well as positive or non-positive immobilization
  • a surface of the retaining element to be understood, for example, an adsorption on the surface.
  • it can be understood as a receptacle in one or more cavities, for example in the form of pores, of the retaining element.
  • Retaining element retained by the retaining element in particular against the direction of the flush.
  • the purging of the second fluid for dissolving the bacteria fixed on the retaining element may preferably be opposite to the direction of purging the sample over the
  • the viruses are previously removed by rinsing with the first fluid in the same direction as the direction of the rinsing of the sample.
  • the further processing of the viruses or bacteria can be part of a detection method of the viruses or bacteria, in particular with the aid of a polymerase chain reaction, which is carried out, for example, in the microfluidic device set up for this purpose.
  • the method according to the invention has the advantage that viruses and bacteria can be isolated particularly efficiently and in a particularly simple manner from the sample for further, separate processing.
  • the viruses and bacteria may in particular be pathogens of
  • further processing of the viruses comprises lysis of the viruses or denaturation of protein envelopes of the viruses.
  • processing may include performing a polymerase chain reaction (PCR) of RNA or DNA, optionally after
  • This processing may preferably take place in the first chamber, wherein in the first chamber preferably for the other
  • the upstream substances may in particular comprise substances for lysing the viruses or denaturing proteins of the viruses, as well as substances for carrying out a (reverse transcriptase) polymerase chain reaction of the viruses, ie in particular substances adapted to the further processing of viruses.
  • the substances may include enzymes, primers and probes for detecting genetic features of the viruses.
  • the further processing of the bacteria comprises carrying out a polymerase chain reaction of DNA of the bacteria, preferably in the second chamber of the device, wherein required in the second chamber for further processing
  • Preceding substances for example in freeze-dried form as a lyophilizate. This has the advantage that the further processing of the bacteria can take place immediately after isolation from the sample, without the bacteria first having to be transported further.
  • the upstream substances may in particular substances for carrying out the
  • Polymerase chain reaction of the bacteria include, ie in particular on the further processing of bacteria tuned substances. Further, the substances may comprise enzymes for detecting genetic characteristics of the bacteria.
  • the sample can be mixed before the rinsing of the sample via the retaining element with a binding buffer for binding the virus to the retaining element. This advantageously increases the proportion of viruses isolated from the sample.
  • the retention element for lysing the bacteria is exposed to a lysis buffer, in particular during a predetermined period of time.
  • the lysis buffer can be substances for chemical lysis of the bacteria such as chaotropic salts and
  • the retaining element can be surrounded with a fluid comprising water, in particular flooded, to the liberated DNA by lysis in a neutral medium without inhibiting substances for a
  • the filter may be a silica filter or a silica membrane.
  • This has the advantage that the complexity of the microfluidic device is low, since the specific retention of the two species, viruses and bacteria, is achieved by two different properties of the retention element, namely the retention of bacteria via size exclusion and the retention of viruses via affinity binding.
  • a silica filter a complex functionality of the device can be achieved with minimal space.
  • the flushing of the second fluid can be carried out preferably in the opposite direction to the direction of the flushing of the sample via the retaining element, in particular for detachment of the bacteria fixed on the filter. This causes an advantageously easier detachment.
  • a selective lysis of human cells in the sample is carried out before the rinsing of the sample via the retaining element. This is particularly advantageous in the case of a sample in which viruses can be present in human host cells.
  • the invention also provides a microfluidic device for
  • the device comprises a retaining element, wherein the retaining element is designed for fixing viruses and bacteria from the sample. Furthermore, the device comprises a first chamber for receiving fixed by the retaining element viruses and a second chamber for receiving bacteria fixed by the retaining element, wherein the first chamber comprises a first reaction mix for further processing of the viruses and the second chamber comprises a second reaction mix for further processing of the bacteria.
  • the device comprises a first valve and a second valve for the fluidic separation of the first chamber and the second chamber from the retaining element.
  • Contamination of the two chambers can be prevented in particular by other components of the sample.
  • the device comprises a heater disposed adjacent to the retaining element, for example, a resistance heater or a Peltier element.
  • a thermal lysis of the bacteria on the retaining element can advantageously be carried out.
  • FIG 1 shows an embodiment of the device according to the invention
  • FIG. 2 shows a flowchart of an associated embodiment of the method according to the invention.
  • FIG. 1 shows an exemplary embodiment of the microfluidic device 100 according to the invention.
  • FIG. 2 shows a flow chart 500 of an associated microfluidic device 100
  • Embodiment of the method 500 according to the invention Embodiment of the method 500 according to the invention.
  • the microfluidic device 100 shown in FIG. 1 is the microfluidic device 100 shown in FIG. 1
  • the retaining member 150 for fixing viruses and bacteria from a sample, for example, a sample of human body fluid such as blood or sputum is formed.
  • the retaining element 150 comprises a filter 151, wherein at least part of the surface of the filter 151 comprises a material for fixing nucleic acids and / or predetermined proteins. It may be a silica filter with exemplary pore sizes between 0.025 and 10 microns, preferably between 0.1 and 1 microns.
  • the device 100 comprises a first chamber 110 for receiving viruses bound by the retaining element 150 and a second chamber 120 for receiving bacteria bound by the retaining element 150.
  • the two chambers 110, 120 may be fluidically connected to the retaining element 150 via a first channel 131, the device 100 in this example having a first valve 191 and a second valve 192 for fluidically separating the first chamber 110 and the second chamber 120 from the retaining element 150 includes.
  • a connection 130 may be lockable from the retaining element 150 to the first channel 131 via a third valve 193.
  • the sample can be introduced via a closable opening 160 of the device 100 into a second channel 132 connected to the retaining element 150, wherein the retaining element 150 can be fluidically separated from the second channel 132 via a fourth valve 194.
  • the first chamber 110 comprises a first reaction mix 111 for further processing of the viruses and the second chamber 120 a second reaction mix 121 for further processing of the bacteria.
  • the first and second reaction mixes 111, 121 can be viruses
  • the first reaction mix 111 may comprise substances for a reverse transcriptase polymerase chain reaction (RT-PCR), for example a QIAGEN® OneStep RT-PCR kit or a NEB® OneTaq® RT-PCR kit, for the RNA of the virus for further
  • Detection reactions preferably a polymerase chain reaction, first to rewrite in cDNA. Both the first and the second reaction mix can be in freeze-dried form, so-called lyophilisate. Furthermore, the reaction mixtures may comprise enzymes, primers or probes for detecting genetic characteristics of the bacteria or viruses.
  • FIG. 2 shows a flowchart 500 of a
  • a first step 501 the sample is passed over the
  • Retention element 150 in this example the above-mentioned silica filter
  • the device 100 rinsed to fix the viruses and bacteria from the sample on the filter.
  • the sample can with a
  • Binding buffer for binding the virus to the retaining element 150 are mixed.
  • the binding buffer may be, for example, a
  • Phosphate buffer having a pH around 5.5 which may contain between 1 and 3 molar guanidinium hydrochloride or guanidinium thiocyanate, preferably between 1.75 and 2.25 M guanidinium hydrochloride or
  • the binding buffer may contain ethanol for the precipitation of the DNA. The fixation of the bacteria takes place via a size exclusion, that is, passages, in particular pores, of the filter 150 are smaller than the size of the
  • selective lysis of these human cells may occur since viruses to be detected may be contained in the human cells.
  • Such selective lysis can be achieved, for example, with the use of 0.05% TWEEN® 80 (final concentration) or 0.2% saponin (final concentrations).
  • TWEEN® 80 final concentration
  • saponin final concentrations
  • a flushing of a first fluid via the retaining element 150 is carried out to detach viruses bound by the retaining element 150 and to transfer the virus-enriched fluid into the first chamber 110 for further processing of the viruses.
  • the first fluid may, in particular, be an elution buffer for detaching the viruses from the retention element 150.
  • the elution buffer may comprise water, preferably double-distilled water.
  • an elution buffer can be used which is based on a mixture of Tris (hydroxymethyl) aminomethane and hydrochloric acid, present in a molarity of between 3 and 7 millimolar, ideally 5 millimolar and having a pH of 8.5.
  • a second fluid is flushed via the retention element 150 to detach bacteria bound by the retention element 150 and transfer the bacteria-enriched fluid into the second chamber 120 for further processing of the bacteria.
  • the second fluid may in particular also be an elution buffer, it being possible, for example, to use a similar elution buffer as in the second step described above. If, as in this embodiment, the bacteria are retained by a filter 150 from the sample and thereby fixed on the filter 150, the flushing of the second fluid through the filter, preferably against the direction of the sample's rinse, can effectively effect the bacteria from the filter 150 to solve again.
  • the device 100 may comprise an ultrasound source 170 or an interface 170, in particular a membrane, for example an elastic polymer membrane, for an ultrasound source, wherein the
  • Ultrasonic source 170 and the interface 170 for a direct action of ultrasound next to the retaining element 150 are arranged.
  • the retaining element 150 can thereby with a water
  • surrounding fluid for receiving the released by the lysis DNA for example, be flooded.
  • a chemical lysis of the bacteria on the retaining element 150 can also be carried out using a lysis buffer before rinsing 503.
  • the lysis buffer may comprise detergents such as Tween® or Triton®-X and / or ethylenediaminetetraacetic acid, EDTA for short.
  • EDTA ethylenediaminetetraacetic acid
  • To the inhibitory effect of EDTA for a To neutralize subsequent polymerase chain reaction can be added after lysis magnesium chloride, preferably in one
  • This additional magnesium chloride is similar to EDTA inactivated magnesium chloride in a typical reaction mix for one
  • the device 100 may be adjacent to the retaining element 150
  • arranged heating element 180 include, for example, a

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé (500) pour le traitement de virus et de bactéries d'un échantillon à l'aide d'un dispositif microfluidique (100). Dans une première étape (501), l'échantillon est versé sur un élément de retenue (150) du dispositif (100), l'élément de retenue (150) étant conçu pour fixer les virus et les bactéries de l'échantillon. Dans une deuxième étape (502), un premier fluide est versé (502) sur l'élément de retenue (150) pour détacher les virus fixés par l'élément de retenue (150) et transférer le premier fluide enrichi en virus dans une première chambre (110) pour un traitement (504) supplémentaire des virus. Dans une troisième étape (503), un deuxième fluide (503) est versé sur l'élément de retenue (150) pour détacher les bactéries fixées par l'élément de retenue (150) et transférer le deuxième fluide enrichi en bactéries dans une deuxième chambre (120) pour un traitement (504) supplémentaire des bactéries.
PCT/EP2018/056314 2017-03-14 2018-03-14 Procédé et dispositif microfluidique pour le traitement de virus et de bactéries d'un échantillon WO2018167106A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102017204195.1A DE102017204195A1 (de) 2017-03-14 2017-03-14 Verfahren und mikrofluidische Vorrichtung zur Prozessierung von Viren und Bakterien einer Probe
DE102017204195.1 2017-03-14

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WO2018167106A1 true WO2018167106A1 (fr) 2018-09-20

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Cited By (1)

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WO2022214340A1 (fr) * 2021-04-07 2022-10-13 Robert Bosch Gmbh Procédé de détection de virus actifs

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DE102021212645A1 (de) 2021-11-10 2023-05-11 Robert Bosch Gesellschaft mit beschränkter Haftung Vorrichtung und Verfahren zur Durchführung mikrofluidischer Prozessschritte

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