US20230279324A1 - Autonomous microfluidic device for sample preparation - Google Patents
Autonomous microfluidic device for sample preparation Download PDFInfo
- Publication number
- US20230279324A1 US20230279324A1 US17/941,237 US202217941237A US2023279324A1 US 20230279324 A1 US20230279324 A1 US 20230279324A1 US 202217941237 A US202217941237 A US 202217941237A US 2023279324 A1 US2023279324 A1 US 2023279324A1
- Authority
- US
- United States
- Prior art keywords
- liquid
- reservoir
- sample
- grid
- stain
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title description 75
- 239000007788 liquid Substances 0.000 claims abstract description 302
- 238000000034 method Methods 0.000 claims abstract description 93
- 239000012530 fluid Substances 0.000 claims abstract description 30
- 238000004891 communication Methods 0.000 claims abstract description 27
- 239000000126 substance Substances 0.000 claims abstract description 22
- 238000010521 absorption reaction Methods 0.000 claims description 60
- 239000012528 membrane Substances 0.000 claims description 50
- 238000011144 upstream manufacturing Methods 0.000 claims description 17
- 238000001035 drying Methods 0.000 claims description 15
- 239000000523 sample Substances 0.000 description 213
- 239000002245 particle Substances 0.000 description 99
- 238000004627 transmission electron microscopy Methods 0.000 description 69
- 239000010408 film Substances 0.000 description 65
- 230000009102 absorption Effects 0.000 description 56
- 239000004372 Polyvinyl alcohol Substances 0.000 description 47
- 229920002451 polyvinyl alcohol Polymers 0.000 description 47
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 44
- 229940068984 polyvinyl alcohol Drugs 0.000 description 44
- 238000001179 sorption measurement Methods 0.000 description 23
- 241000702421 Dependoparvovirus Species 0.000 description 19
- 239000000243 solution Substances 0.000 description 16
- 238000001514 detection method Methods 0.000 description 15
- 238000003384 imaging method Methods 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 241000700605 Viruses Species 0.000 description 11
- 238000004626 scanning electron microscopy Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 7
- 239000002390 adhesive tape Substances 0.000 description 6
- 238000010191 image analysis Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000003570 air Substances 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 239000000835 fiber Substances 0.000 description 5
- 239000010409 thin film Substances 0.000 description 5
- 208000002267 Anti-neutrophil cytoplasmic antibody-associated vasculitis Diseases 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 4
- 238000003917 TEM image Methods 0.000 description 4
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 229920001872 Spider silk Polymers 0.000 description 3
- 239000000853 adhesive Substances 0.000 description 3
- 230000001070 adhesive effect Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000001045 blue dye Substances 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- AJDUTMFFZHIJEM-UHFFFAOYSA-N n-(9,10-dioxoanthracen-1-yl)-4-[4-[[4-[4-[(9,10-dioxoanthracen-1-yl)carbamoyl]phenyl]phenyl]diazenyl]phenyl]benzamide Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C=CC=C2NC(=O)C(C=C1)=CC=C1C(C=C1)=CC=C1N=NC(C=C1)=CC=C1C(C=C1)=CC=C1C(=O)NC1=CC=CC2=C1C(=O)C1=CC=CC=C1C2=O AJDUTMFFZHIJEM-UHFFFAOYSA-N 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 238000005464 sample preparation method Methods 0.000 description 3
- 230000001960 triggered effect Effects 0.000 description 3
- 239000001043 yellow dye Substances 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 108010085220 Multiprotein Complexes Proteins 0.000 description 2
- 102000007474 Multiprotein Complexes Human genes 0.000 description 2
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 102000034238 globular proteins Human genes 0.000 description 2
- 108091005896 globular proteins Proteins 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 239000012678 infectious agent Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000010954 inorganic particle Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000010030 laminating Methods 0.000 description 2
- 238000007648 laser printing Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- LSGOVYNHVSXFFJ-UHFFFAOYSA-N vanadate(3-) Chemical compound [O-][V]([O-])([O-])=O LSGOVYNHVSXFFJ-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 201000011001 Ebola Hemorrhagic Fever Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 208000020329 Zika virus infectious disease Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000012080 ambient air Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- -1 delivery vesicles Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000002864 food coloring agent Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 230000005660 hydrophilic surface Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005661 hydrophobic surface Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000003475 lamination Methods 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000011170 pharmaceutical development Methods 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000001394 sodium malate Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000012906 subvisible particle Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012783 upstream development Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 244000052613 viral pathogen Species 0.000 description 1
- 238000007794 visualization technique Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
Images
Classifications
-
- 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
- B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
-
- 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
- B01L3/502738—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 characterised by integrated valves
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- 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
- B01L2200/16—Reagents, handling or storing thereof
-
- 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
- B01L2300/069—Absorbents; Gels to retain a fluid
-
- 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
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
-
- 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
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
-
- 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
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0688—Valves, specific forms thereof surface tension valves, capillary stop, capillary break
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Clinical Laboratory Science (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Sustainable Development (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Sampling And Sample Adjustment (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Abstract
The method is for preparing a sample in a microfluidic device. A microfluidic device is provided that has a first reservoir in fluid communication with a second reservoir in fluid communication with and adjacent to a draining unit that has a first absorbing member disposed therein. The first reservoir contains a first liquid that is held in the first reservoir by a capillary stop valve connecting the first and second reservoirs. The second reservoir has a sample support disposed therein. A second liquid, containing substances, is added to the second reservoir. The second liquid contacts the first liquid and the first absorbing member. The first absorbing member absorbs the second liquid and the first liquid. The substances adhere to the sample support.
Description
- This is continuation patent application that claims priority from a continuation-in-part (CIP) patent application Ser. No. 17/025,390, filed 18 Sep. 2020 that claims priority from U.S. patent application Ser. No. 17/023,922, filed 17 Sep. 2020.
- The present invention generally relates to a device and method for consistent and user-independent preparation of particulate or elongate fiber-like samples, such as micro-particles, nanoparticles and/or micro/nano-sized fibers, for subsequent analysis using microscopy or other inspection techniques. In particular, this is useful for applications in transmission electron microscopy (TEM) or scanning electron microscopy (SEM).
- A consistent, user-independent and repeatable sample preparation method is necessary for objective analysis of liquid samples of micro and nano-sized particles such as virus particles, virus-like particles, proteins, protein complexes, fibers, delivery vesicles, pharmaceuticals and inorganic particles.
- For example, modified virus vectors are commonly used in gene therapy applications. Determining the ratio of infectious to un-infectious particles and debris/other material in the sample provides invaluable information about the quality and efficacy of the final gene therapy product and the upstream development processes.
- SEM (Scanning Electron Microscopy) and nsTEM (Negative Stain Transmission Electron Microscopy) application are clinical diagnostic devices where SEM and nsTEM are used to detect and analyze infectious agents, such as viruses, for diagnostic purposes. Additionally, SEM and nsTEM are widely used in the characterization of biological and inorganic particles and materials in research, development, quality control of vaccines, pharmaceuticals and materials. The main advantage of SEM/nsTEM over chemical and bio-chemical characterization techniques is the possibility of directly visualizing the sample of interest. This makes it possible to determine, for example, the cell morphology or to identify the virus family of a pathogenic organism. In nsTEM, the image contrast is achieved through a heavy metal stain solution (uranyl acetate, phosphotungstic acid, etc.) that embeds and preserves the particles of interest.
- The value of TEM as a first screening tool to identify viral pathogens in infectious diseases was demonstrated during the SARS epidemic 2003 where diagnostic TEM first indicated that the causative virus was a member of the coronavirus family. Considering the ability of emerging infectious agents, such as Ebola, Zika or SARS-COV2, to spread rapidly on an intercontinental level as a result of globalized trade and travel, and the risks of bioterrorist attacks due to the instability of the global political scene, it is clear that access to efficient TEM analysis is a vital part of our emergency preparedness, management and civil defence. This is in addition to TEM's routine clinical use and its use in process design and quality control in pharmaceutical development and production.
- When imaged using TEM, the stain scatters more electrons than the particles in the sample. This results in an image where the particles appear bright on a dark background with a resolution in the order of a few nanometers. Conventionally, TEM grids are prepared by following a manual preparation protocol. This involves pipetting 3-5 μl of the sample liquid onto a TEM grid and then letting it adsorb for about 10-60 seconds depending on the specimen. Excess sample is then manually blotted off the grid by using blotting paper. Immediately after blotting the sample, 3-5 μl of an aqueous stain solution is added to the grid.
- Excess stain is then blotted off ideally leaving a uniform thin layer or thin film of stain liquid covering the adsorbed specimen. This thin film is left to dry. The film embeds the specimen for TEM imaging and protects it from dehydration. The stain also increases the contrast. One problem is that this manual procedure is highly dependent on the skill of the operator which affects the preparation consistency and leads to unreliable results. Inconsistent timing of the manual steps and the final blotting are often the cause for bad TEM grid preparations.
- Alternative methods for trying to obtain a consistent nsTEM sample preparation employ contact pin-printing techniques where pipetting robots automatically dispense liquids onto the TEM grid. These approaches have some advantages over the manual preparation such as reduction of liquid volumes and the possibility for automation. However, they require special instrumentation and are significantly more complex and time-consuming than the manual preparation protocol.
- Also, a microfluidic device for nsTEM grid preparation has been described. The TEM grid is confined in a microfluidic channel and the liquid handling for the sample preparation is controlled by an external pressure pump. While this improves the preparation consistency over manual preparations, the approach requires significantly more liquid volume than the manual procedure. It also requires special equipment and involves the user to control the timing of every preparation step which makes the preparation method unreliable and inconsistent.
- There are several hurdles that must be overcome to reach the feasibility of using electron microscopy in time and resource limited situations such as the development and quality control in the production of pharmaceuticals, material synthesis and routine clinical diagnostics. As indicated above, the expert task of preparing the sample for analysis is associated with extreme complexity. This makes the use of the TEM technology a craftsmanship limited to a small number of experts. A sample preparation method can be learnt in a month for a person that has basic laboratory skills but to master it takes about 10 years while still generating a significant expert variability. This means that even experts in the field cannot produce consistent results without undesirable variability.
- Sample preparation is normally performed according to a standardized procedure. First the sample is supplied onto a sample support (which in the case of TEM is a metal grid that is about 3 mm in diameter) and left to adhere to the sample support. In the next step, excess sample solution is removed, and a stain to protect the particles and/or increase the contrast is instantly added. In the case of negative stain TEM, this stain is a heavy metal salt solution. Excess stain is then removed. The removal of excessive fluid/stain is done by blotting with a filter paper. An additional washing step subsequent to the removal of excessive fluid is sometimes done after the sample addition and prior to adding the stain. Alternatively, the addition of liquids can be done by dipping the grid into droplets of the liquids. These steps are typically carried out manually by the instrument operator and hence the results strongly depend on the operator's ability to consistently perform the correct procedure.
- Also, regardless of how consistent and skilled the operator is, it is not possible to consistently control the forces the different preparation steps induce on the particles. This affects the quality of the prepared sample and limits the reliability of subsequent analysis results.
- As indicated above, some automatic or semi-automated preparation methods have been suggested in the past. They rely on robotic dispensers, microfluidics using special equipment or special sample holders connected to a pipetting device. The robotic dispensers require only minute sample volume but instead rely on highly specialized equipment. The microfluidic-based sample preparation approach results in more consistent preparations but again rely on special equipment (special grid holder and external pressure pump) and require about 10-times larger sample volumes compared to manual preparation. A method using a special pipette tip with a pocket/slit holding the grid has also been suggested. This mprep-based approach also requires larger sample volumes and involves manual timing steps. In addition, the liquids are flushed on both sides of the grid that increase the risk for poor quality preparations.
- Hence, there is a need for a more reliable and consistent nsTEM sample preparation method. The present invention provides a solution to the above-described problems without having the user-bias and consistency problems associated with manual preparation, and without the drawbacks of quality, large sample volumes, expensive and special equipment associated with conventional automated approaches.
- More particularly, the device and method of the present invention provides a consistent objective (user-independent) and reproducible preparation of samples of sub-visible particles for subsequent imaging and analysis. The method of the present invention is based on microfluidic technology combined with dissolvable films that act as delay valves and absorption membranes. It is all built into a disposable sample preparation device or card, and hence does not require any special equipment or large sample volumes. The different liquids flow over the grid in a sequential fashion with a certain delay and speed that is defined by the dissolvable films and design of the absorption membranes (filters).
- This combination allows for a highly automated procedure where different sample preparation liquids are automatically flushed over the sample grid in a controlled and well-defined manner. Once the user has added the sample liquid, the entire grid preparation process is self-driven, self-contained or automatic because the various liquids are automatically driven through the device of the present invention, without requiring any additional input, by relying on capillary forces and other surface tension effects. It should be understood that the use of stain and sample liquids are merely illustrative examples of suitable liquids to be used in the device of the present invention. A wide variety of other liquids may be used, as required. The user interaction is reduced to just adding the sample liquid after preloading the stain to specific positions of the device in a non-time sensitive manner. The addition of the sample triggers a sequence of flushing steps over the sample grid with liquids (such as stains) which are either pre-added or pre-stored in the card/device. When the automatic preparation is completed, the operator then simply transfers the correctly prepared sample grid into the TEM or SEM microscope or even transfers the card itself into the SEM or light microscope.
- The device of the present invention preferably constitutes or is realized as a disposable paper-based kit, consisting of containers for adding liquids and absorption membranes and where the grid onto which the sample is loaded is either pre-fitted or added by the user. In case of a pre-fitted grid, the user-input consists of only pipetting the stain (unless the stain is pre-loaded) and then the sample liquid into different containers in the device. The addition of the last liquid triggers the start of the autonomous preparation process where microfluidic forces drives the flow of the two liquids (i.e. the stain and the sample liquid) over the grid and where dissolvable valves control the timing of the process. The grid may be coated or covered with a thin carbon layer onto which the particles in the sample liquid are permitted to adsorb or adhere until the dissolvable membrane in the draining or unit is dissolved, so that the particles remain on the grid and are subsequently embedded by the stain liquid, as explained in detail below.
- More particularly, the autonomous microfluidic device of the present invention is, preferably, for microscopy sample preparation. It should be understood that the use of laminates in the device is merely an illustrative example and the device of the present invention is not limited to using laminates. Any other fabrication method could be used such as molding.
- The microfluidic device of the present invention has a first reservoir that preferably includes a first liquid or into which a first liquid is added. The first liquid is being held by a capillary stop valve in the first reservoir. A second reservoir is in fluid communication with the first reservoir. The second reservoir has a second liquid and a sample support disposed therein. The second reservoir has an inlet opening defined therein. A draining unit is adjacent to the second reservoir. The draining unit is being in fluid communication with the second reservoir. The draining unit has a first absorption member disposed therein.
- In an alternative embodiment of the present invention, the microfluidic device has a channel defined therein and the first reservoir is in fluid communication with the second reservoir via the channel.
- In yet an alternative embodiment of the present invention, the channel extends to an edge at the second reservoir.
- In another alternative embodiment of the present invention, the sample support has a first width and the opening has a width that is substantially similar to the first width.
- In an alternative embodiment of the present invention, the draining or blotting unit has a dissolvable membrane disposed therein below the first absorption member.
- In another embodiment of the present invention, the draining unit has a second absorption member located below the dissolvable membrane so that the dissolvable membrane is disposed between the first absorbing member and the second absorbing member.
- In yet another embodiment of the present invention, the first reservoir is a preloaded stain reservoir containing a stain liquid.
- In an alternative embodiment of the present invention, the first liquid in the capillary stop valve extends between the edge and another surface edge of the channel.
- In another embodiment of the present invention, the first absorption member is a first filter or paper and the second absorption member is a second filter or paper.
- In yet another embodiment of the present invention, the dissolvable membrane is a film based on poly-vinyl-alcohol (PVA).
- In another embodiment of the present invention, the sample support is a grid for negative-stain transmission electron microscopy preparation.
- In an alternative embodiment of the device of the present invention, the first liquid is a stain.
- In yet another embodiment, the device has an additional reservoir upstream of the first reservoir.
- In another embodiment of the device of the present invention, the draining unit has a second dissolvable member below the second absorption member, and a third absorption member below the second dissolvable member.
- In an alternative embodiment, the draining unit has a vent opening defined therein.
- In yet an alternative embodiment, the draining unit has a second dissolvable member and a third absorption member disposed below the first dissolvable member.
- The method of the present invention is for preparing a sample in a microfluidic device. A microfluidic device is provided having a first reservoir in fluid communication with a second reservoir in fluid communication with and adjacent to a draining unit having a first absorbing member disposed therein. The first reservoir contains a first liquid that is being held in the first reservoir by a capillary stop valve connecting the first and the second reservoirs. The second reservoir has a sample support disposed therein. A second liquid, containing substances, is added to the second reservoir. The second liquid contacts the first liquid and the first absorbing member. The first absorbing member absorbs the second liquid and the first liquid. The substances adhering to the sample support.
- In an alternative method, the draining unit is provided with a dissolvable membrane upstream of the first absorbing member. The second liquid or the first liquid dissolving the dissolvable membrane prior to the first absorbing member absorbing the first and second liquids.
- In another method, the substances adhere to the sample support while the second liquid or the first liquid dissolves the dissolvable membrane.
- In yet another method, the capillary stop valve holding the first liquid in the first reservoir preventing the first liquid from flowing into the second reservoir prior to adding the second liquid to the second reservoir.
- In another method, a portion of the first liquid embedding the substances adhered to the sample support.
- In yet another method, the capillary stop valve is provided with an edge that separates the first reservoir from the second reservoir and the edge holding the first liquid in the first reservoir.
- In another method, the dissolvable membrane is provided downstream of the first absorption member and a second absorption member downstream of the dissolvable membrane and the first absorption member absorbing the second liquid and permitting the second liquid to come into contact with the dissolvable member.
- In yet another method, the second absorption member absorbing the second liquid and the first liquid after the dissolvable membrane has been dissolved.
- In another method, the second liquid breaking a surface tension of the first liquid upon contact with the first liquid held in the capillary stop valve.
- In yet another method, a time period required to dissolve the dissolvable membrane controlling a permitted time period for the substances to adhere to the sample support.
- In another method, the second liquid contacting the absorbing member before the first liquid.
- In yet another method, the first portion of the first liquid drying on the sample support.
- In an alternative method, a portion of the first liquid forming a liquid film on the sample support, wherein the liquid film has a film thickness of less than 1 mm but more than 10 nm.
- In yet another method, the sample support is dried within three minutes at an ambient temperature and 50% relative humidity.
- In another method, the first liquid has a volume of between 0.1-50 μl.
- In yet another method, the second liquid has a volume of between 0.1-50 μl.
- In an alternative method of the present invention, the method is for preparing a sample in a microfluidic device. A microfluidic device is provided having a first reservoir in fluid communication with a second reservoir in fluid communication with and adjacent to a draining unit having a first absorbing member disposed therein. The first reservoir containing a first liquid. The first liquid being held in the first reservoir by a capillary stop valve connecting the first and second reservoirs. A user of the microfluidic device adding a sample support into the second reservoir. The user adding a second liquid, containing substances, to the second reservoir. The user waiting a waiting period of at least 20 seconds before removing the sample support from the second reservoir. During the waiting period, the second liquid contacting the first liquid and the first absorbing member. During the waiting period, the first absorbing member absorbing the second liquid and the first liquid. During the waiting period, the substances adhering to the sample support. At the end of the waiting period, the user removing the sample support from the second reservoir.
- In another method, the draining unit is provided with a dissolvable membrane upstream of the first absorbing member and the second liquid dissolving the dissolvable member during the waiting period.
- In yet another method, the first liquid forming a film on the sample support and embedding substances adhered to sample support.
- In another method, the film drying on the sample support.
- The present invention is now described, by way of example, with reference to the accompanying drawings, in which:
-
FIG. 1A is an elevational cross-sectional side view of the device of the present invention showing sample addition; -
FIG. 1B is an elevational cross-sectional side view of the device of the present invention showing time-controlled sample adsorption; -
FIG. 1C is an elevational cross-sectional side view of the device of the present invention showing automatic draining of excessive sample and stain; -
FIG. 1D is an elevational cross-sectional side view of the device of the present invention showing film drying before grid removal; -
FIG. 2A is a top view of the device shown inFIG. 1A ; -
FIG. 2B is a top view of the device shown inFIG. 1B ; -
FIG. 2C is a top view of the device shown inFIG. 1C ; -
FIG. 2D is a top view of the device shown inFIG. 1D ; -
FIG. 3 is a schematic cross-sectional view of the device of the present invention; -
FIG. 4 is a schematic top view of the device shown inFIG. 3 ; -
FIG. 5 is a top view of the device of the present invention; -
FIG. 6 is a schematic view showing microfluidic timing results for five different devices of the present invention; -
FIG. 7 is a schematic view illustrating measurement showing the average dissolving time of components of the present invention; -
FIG. 8 is a schematic view illustrating five grids, five grid squares per grid and nine images per grid square results in 225 images of the present invention; -
FIG. 9A is a magnified view of a TEM grid prepared by using the device of the present invention; -
FIG. 9B is a magnified view of a sample area of the same size as the area marked inFIG. 9A ; -
FIG. 9C is a magnified view of a sample area of the same size as the area marked inFIG. 9B ; -
FIG. 10A is an example of an image from a first grid prepared by using the device of the present invention; -
FIG. 10B is an example of an image from a second grid prepared by using the devoice of the present invention; -
FIG. 10C is an example of an image from a third grid prepared by using the device of the present invention; -
FIG. 10D is an example of an image from a fourth grid prepared by using the device of the present invention; -
FIG. 10E is an example of an image from a fifth grid prepared by using the device of the present invention; -
FIG. 11 is a schematic illustration of the average diameter of the particles and the number of particles on each grid of the present invention; -
FIG. 12 is a table showing results of a manual subset testing with five images per grid and the ratio of true and false positives; -
FIG. 13 is a schematic cross-sectional view of the device of the present invention; -
FIG. 14 is a top view of the device shown inFIG. 13 ; -
FIG. 15 is a cross-sectional side view of a first alternative embodiment of the device of the present invention; -
FIG. 16 is a cross-sectional side view of a second alternative embodiment of the device of the present invention; -
FIG. 17A is an image of proteasomes at a first magnification (the length of 200 nm is shown); -
FIG. 17B is an image of proteasomes shown inFIG. 17A at a second magnification (the length of 100 nm is shown); -
FIG. 17C is an image of protein (WPI) fibrils at a first magnification (the length of 1 μm is shown); -
FIG. 17D is an image of the WPI fibrils at a second magnification (the length of 200 nm is shown); -
FIG. 18 is an elevational schematic cross-sectional view of a fourth alternative embodiment of the device of the present invention; -
FIG. 19 is an elevational schematic cross-sectional view of a fifth alternative embodiment of the device of the present invention; and -
FIG. 20 is a cross-sectional view of a sixth alternative embodiment of the device of the present invention. - A capillary-driven microfluidic device of the present invention is presented herein for sample preparation that requires the same small liquid volumes as the conventional manual procedure does, and which requires minimal user-interaction. More particularly, the sample support is preferably a grid, such as a TEM grid. The user merely initiates the autonomous sample preparation process, waits for about one minute and then extracts the TEM grid that is ready for imaging in a TEM or SEM microscope. The autonomous process of the present invention typically requires a film, that is soluble by the sample liquid, such as a PVA (polyvinyl alcohol) film for a water-based sample liquid, that automatically controls the time for sample adsorption and draining of excess liquids. Microfluidic consistency for five microfluidic devices is demonstrated below by comparing the timing and duration of the microfluidic TEM grid preparation events. Furthermore, the adjustability of the time-delay is explained for 15 devices using three different thicknesses of the water-soluble film (12 μm, 24 μm, 36 μm). Sample preparation consistency is examined by imaging five autonomously prepared TEM grids, with AAV (Adeno-associated virus) particles as sample and Methylamine Vanadate as stain.
- A particle detection script, extracting morphological information such as the average particle size, was run on 45 microscopy images per grid to investigate whether the images are suitable for automated image analysis. The device of the present invention may also be used to prepare protein samples and fibers for TEM investigations and other stains may be used.
- The device of the present invention adapts the sample preparation steps of the manual procedure and replaces user-interactions with automated and capillary-driven microfluidic events. The device is preferably, but not necessarily, designed for single-use and does not require special instrumentation.
-
FIG. 1A-1D illustrate the conceptual sequence of the autonomous TEM grid preparation events in the device of the present invention.FIG. 1A shows how the step of adding the sample triggers the autonomous preparation process. - More particularly, the
device 200 has astain reservoir 202 adjacent to a sample reservoir orgrid chamber 204. The sample reservoir is adjacent to a draining orblotting unit 206. Thestain reservoir 202 holds or contains astain liquid 208. Preferably, thestain liquid 208 is preloaded prior to use. Thesample reservoir 204 contains asample liquid 210 that includessubstances 214, such as objects, molecules or particles, to be analyzed. The particles could be virus or virus-like particles or any other type of fibrous or particulate biological or inorganic object. - When the
sample liquid 210 is applied into thesample reservoir 204, the liquid 210 covers asample support 216 such as a TEM grid and connects to thepreloaded stain 208 upstream of the sample support orgrid 216 and to a blotting paper or filter 218 in the draining orblotting unit 206 that is located downstream of thegrid 216. The contact between thesample liquid 210 and the absorption units in thedraining unit 206 starts the time-controlled sample adsorption step (as shown inFIG. 1B ). When thesample liquid 210 is deposited or added into thesample reservoir 204, thesample liquid 210 comes into contact with a first absorption unit 218 (such as a first blotting/filter paper) of thedraining unit 206. The drainingunit 206 has a dissolvable valve ormembrane 220 located below thefirst absorption unit 218. Thesample liquid 210 covers theTEM grid 216 while thedissolvable valve 220, that separates thefirst blotting paper 218 from a second absorption unit such as a second blotting/filter paper 222, is closed. Thevalve 220 is closed until it has been dissolved by the liquid absorbed by thefirst absorption member 218. The time it takes to dissolve the dissolvable valve ormembrane 220 is a critical step of the present invention because during this time, theparticles 214 in thesample liquid 210 are permitted to adhere to or be adsorbed by thegrid 216. - Once
valve 220 is dissolved, excess amounts of both thesample liquid 210 and thestain liquid 208 are autonomously drained or blotted off by the two absorption units, blotting/filter papers FIG. 1C . Theparticles 214 adhere to or are adsorbed by thegrid 216. A remainingthin stain film 224 covers or embeds theparticles 214 on thegrid 216 and dries while thefilm 224 embeds the sample particles 214 (as shown inFIG. 1D ). Thegrid 216 is then ready for imaging and can easily be retrieved by peeling off aflap 226 and extracting thegrid 216 with, for example, a pair of tweezers. -
FIGS. 2A-2D are top views of the device showing the corresponding frames. - The key components of the
device 200 are depicted in the cross-sectional illustration shown inFIG. 3 and in the top view shown inFIG. 4 .FIG. 5 shows a top view of a fabricated device of the present invention. - As indicated above, the
microfluidic device 200 of the present invention consists of the liquid (stain)reservoir 202, the second liquid (sample) reservoir orgrid chamber 204 and thedraining unit 206. The key function of thestain reservoir 202 is to contain thestain liquid 208 until the user adds thesample liquid 210 that includes the particles 214 (best shown inFIGS. 1A-1D ) that eventually come into contact with thestain liquid 208, as described in detail below. - A key enabling feature is a capillary stop valve or liquid pinning mechanism such as a pinning
edge 228, as indicated inFIG. 3 that separates thestain reservoir 202 from thegrid chamber 204. The capillary stop valve or mechanism could be a hydrophobic surface area and/or geometrical structure where surface tension prevents the liquid from going beyond the hydrophobic area and/or the geometrical structure. Preferably, the geometrical stop valve is a sudden divergence of the channel cross-section (e.g. an edge) in the flow direction of the channel or part of the channel. In the preferred embodiment, the capillary stop-valve is located the edge of the channel and ends where the second reservoir starts. It is not necessary to have a channel as long as there is an edge at the second reservoir that stops the first liquid from flowing into the second reservoir. If the edge had been located away from the second reservoir then there is a risk that an air bubble is formed between the first liquid and the second liquid so that no contact between the two liquids can be established. It is very important that the first liquid is easily accessible for the second liquid so that the two liquids can connect and the surface tension of the first liquid is broken. - One purpose of the liquid pinning mechanism of the present invention is to confine a liquid in one reservoir which is connected (in fluid communication) with a second reservoir. The
stain liquid 208 is pinned at or held by the pinningedge 228 and is held to ahydrophilic underside 230 of afirst laminate portion 232 due to surface tension forces between thestain liquid 208 and theunderside 230. - Preferably, the pinning
mechanism 228 is an edge, more particularly a sharp edge such as a 90-degree edge, formed between ahorizontal bottom surface 231 and avertical side wall 233 oflaminate 252 that extends towards a laminate 253 and below theadhesive tape 254 holding thegrid 216 in place. Surface tension at thesurface 234 extending between the pinningedge 228 and theunderside 230 holds thestain liquid 208 in place in thestain reservoir 202 and so that thesurface 234 and thefirst laminate portion 232 extend over the pinningedge 228. The surface tension is caused by intermolecular forces near the surface leading to the apparent presence of a surface film and to capillarity on the surface. The surface of the liquid tends to contract and has properties resembling those of a stretched elastic membrane. The combination of the pinningedge 228, thehydrophilic underside 230 and the surface tension in thesurface 234 thus enables the autonomous sample preparation process to be initiated by the addition of thesample liquid 210. - The
grid chamber 204 has a sample inlet opening 236 defined between aforward edge 238 of aflap laminate 240 and arearward edge 242 of thelaminate portion 232. Thestain reservoir 202 has aninlet opening 203 defined between arearward edge 244 of asecond laminate portion 246 and aforward edge 248 of thefirst laminate portion 232. Preferably, the first andsecond laminate portions flap laminate 240 are part of the same laminate to make the fabrication of thedevice 200 easier. - The
grid chamber 204 contains thegrid 216, such as TEM grid, and is connected to and in fluid communication with thestain reservoir 202 upstream of thegrid 216. Thegrid chamber 204 is also connected to and in fluid communication with the drainingunit 206 downstream of thegrid 216. Preferably, the capillary forces drive the liquid sideways towards and into thedraining unit 206. More particularly, thegrid chamber 204 is in fluid communication with thestain reservoir 202 via achannel 250 defined between the laminate 232 and abottom laminate 252 of thestain reservoir 202. - The
grid 216 is fixated by a low-tack adhesive laminate 254 at the backside grid perimeter so that thegrid 216 is removably held to thelaminate 254. Acavity 256 is formed below thegrid 216 to make sure that no liquid reaches the backside or underside of thegrid 216 which otherwise could lead to TEM imaging artifacts. - The top or opening in the
grid chamber 204 serves as thesample inlet 236 and ensures fast drying of the thin stain film after draining (blotting off) of excessive liquids. Theopening 236 is slightly smaller than the length of theTEM grid 216 because thefirst laminate portion 232 and itsrearward edge 242 extends over thegrid 216. Similarly, theflap portion 240 and itsforward edge 238 extend over thegrid 216. This leaves an overlap between the top hydrophilic layer orlaminate portions grid 216. The overlap ensures that thesample liquid 210 reliably connects with thepreloaded stain liquid 208 and thedraining unit 206. - The draining
unit 206 is, preferably, formed by a stack of two absorption units orfilter paper units PVA film 220 that separates the two absorptions units ormembers top absorption member 218 is to ensure a good contact between the draining unit and the second liquid in the second reservoir. One function of thesecond absorption member 222 is to ensure proper flow of the liquids from the first and second reservoir and into the second absorption member when thedissolvable membrane 220 has dissolved. Preferably, the two liquids flow in a sequence over the sample support orTEM grid 216 so that thesample liquid 210 flows over the sample support first and come in contact with thefirst absorption member 218 followed by thestain liquid 208 so that a portion of thestain liquid 208 remains on the sample support and embeds the substances or objects of thesample liquid 210. This principle applies to all the embodiments of the present invention even if the device only has one absorption member or the absorption member is located downstream of a dissolvable membrane such as a PVA film. PVA is especially suitable for the scope of the invention as biological specimens are typically prepared in aqueous solution. Thetop paper unit 218 provides a stable connection between thegrid chamber 204 and thePVA layer 220. Thepaper unit 218 is in fluid communication with the sample reservoir orgrid chamber 204. Avent 264, located above thetop paper unit 218, ensures that no air is trapped which could block the draining process. The vent is an important feature when using a gas-tight membrane. - An important aspect of the present invention is that the dissolving time of the
PVA layer 220 controls the adsorption time of thesample liquid 210 that has been deposited on theTEM grid 216. The draining or blotting step is triggered when thePVA layer 220 is dissolved by theliquid 210 of the sample and the liquid reaches the second absorption (paper)unit 222. The high capillary (draining) force of the second absorption (paper)unit 222 leads to fast absorption of theliquid volumes device 200. After the sample preparation in complete, theflap portion 240 can be peeled off to collect thegrid 216. Besides grid collection, theflap portion 240 could allow the user to introduce a grid of choice before the preparation procedure. -
FIG. 5 is a top view of a fabricated version of thedevice 200 of the present invention. -
FIG. 6 is aschematic view 400 showing test results of five different devices (device nos. 1-5) of the present invention. The view shows the preloading of stain at different times relative to the addition of the sample liquid containing the particles. More particularly, it shows the microfluidic timing results for five different devices and the times for stain preloaded, i.e., the time period between stain and sample addition, adsorption time, draining/blotting time and drying time of each device. The autonomous TEM grid preparation starts attime 0 with, and is triggered by, the sample addition, as described in detail above. The stain is added about 40 seconds before the sample addition (such as sample liquid 210) in device no. 1. The stain is added about 20 seconds before the sample addition in device no. 2, about 60 seconds in device no. 3, about 40 seconds in device no. 4 and about 50 seconds before the addition of the sample liquid in device no. 5. Although thetime periods time periods 412 to complete the adsorption, draining and drying steps are about the same for all five devices. The steps following the preloading step are all slightly longer than 20 seconds in total. The adsorption time period is thus the same as the time it takes for the sample liquid, absorbed in the first absorption media (such as paper), to dissolve the dissolvable membrane (PVA film). This means it is not time critical when the sample liquid is added relative to the time the stain was added or preloaded which makes the process easier for the user who adds the sample liquid. -
FIG. 7 is aschematic view 420 showing the time required to dissolve three differentdissolvable membranes c having thicknesses 12 μm, 24 μm and 36 μm, respectively.Membrane 220 a required about 15 seconds to dissolve,membrane 220 b about 90 seconds andmembrane 220 c required over 180 seconds to dissolve. -
FIG. 8 is aschematic view 430 showing an imaging scheme with fivegrids grid squares 442 per grid and nineimages 444 pergrid square 442 that results in a total of 225images 446. -
FIG. 9A is an example magnifiedview 600, including afirst portion 602, of a TEM grid prepared by the device of the present invention.FIG. 9B is aview 604, of higher magnification relative to view 600 inFIG. 9A , including asecond portion 606, of the same size as theportion 602 marks in theview 600, shown inFIG. 9A . In the same way,FIG. 9C is aview 608, of higher magnification relative to view 604 inFIG. 9B , of the same size as theportion 606 of theview 604, shown inFIG. 9B . -
FIG. 10A is an example of animage 610 from afirst grid image 610 includes or depictsparticles 614 such as virus particles. Similar toFIG. 10A ,FIG. 10B is an example of animage 616 from asecond grid image 616 includesparticles 620 such as virus particles.FIG. 10C is an example of animage 622 from athird grid image 622 includesparticles 626 such as virus particles.FIG. 10D is an example of animage 628 from afourth grid image 628 includes or depictsparticles 632.FIG. 10E is an example of animage 634 from afifth grid image 634 includes or depictsparticles 638 such as virus particles or any other suitable particle. -
FIG. 11 is agraph 640 of the average diameter of the particles and the number of particles on each grid that has been prepared by using the device and method of the present invention. -
FIG. 12 is a table 642 showing results of a manual subset testing with five images per grid and the ratio of true and false positives. -
FIG. 13 is a cross-sectional view of themicro-fluidic device 100 of the present invention. It should be noted that thedevice 100 is not fabricated from laminates. Thedevice 100 is substantially similar todevice 200 and everything that applies todevice 100 also applies todevice 200 and vice versa. Thedevice 100 has a microfluidic platform with liquid reservoirs in fluid communication, and absorption units and a dissolvable film that act as time-controlled liquid drainage with a delay valve. Thesample support 116, here illustrated as a TEM grid, is positioned at a bottom of thesample reservoir 104. Thestain reservoir 102 has aninlet opening 143 defined between the front end of aback section 141 of the device and the back end of amiddle section 132 of the device. Preferably, if the device is constructed using laminate technology, thesections device 100. - The
sample reservoir 104 has aninlet opening 145 defined between the laminate orsection 132 and the laminate orsection 140. Thesample reservoir 104 is upstream (on one side) connected to and in fluid communication with astain reservoir 102 via amicrofluidic channel 150 that extends between thesample reservoir 104 and theupstream stain reservoir 102. It is to be understood that thechannel 150 may have a pinning edge or a discontinuity at only a portion of the end of thechannel 150 so that, for example, the sidewalls do not have any edges. There may also be an edge of the channel at the upper side of inner surface so that there are two opposite edges at the end of the channel. - Preferably, the
channel 150 is defined between ahydrophilic underside 130 of a first laminate portion orsection 132 and abottom surface 131 of thestain reservoir 102. Thebottom surface 131 extends to a pinningedge 128. Capillary forces between the liquid 108 and theunderside 130 and the surface tension of thesurface 134 hold the liquid 108 in thestain reservoir 102 and prevents the liquid 108 from flowing into thesample reservoir 104. In other words, the pinningedge 128 prevents liquid 108, such as stain liquid, added to thestain reservoir 102 from flowing into thesample reservoir 104. - The
sample reservoir 104 is downstream (on the opposite side relative to the upstream connection to the stain reservoir 102) connected to and in fluid communication with a first filter orabsorption media 158 which is separated from a second filter orabsorption media 160 by a dissolvable film, membrane orvalve 162. Thefirst filter 158 is also connected to avent 164. Thevent 164 serves as an emergency exit for potentially trapped air and gas which would otherwise hinder the flow of the liquid 108, 110 into and to be absorbed by the first andsecond filters - With reference to
FIG. 14 , theremovable flap 126 is, upon completion of the grid preparation, removed from thedevice 100 prior to removing thesample grid 116 from thesample reservoir 102. -
FIG. 15 shows adevice 300 that is substantially similar to thedevice 100 shown inFIG. 13 but includes anadditional reservoir 302 and an additional dissolvable film or membrane orvalve 320 and anadditional absorption unit 322. Only the main differences betweendevice 100 anddevice 300 are here described. Thedevice 300 is used when additional liquids are to be flushed over thegrid 116 in a sequential and time-controlled manner. The additionalliquid reservoir 302 is placed upstream of the stain orsecond reservoir 102 and in fluid communication with and connected thereto via achannel 304 that is defined between abottom surface 306 of thereservoir 302 and ahydrophilic underside 308 of a laminate portion orsection 310. Between thereservoirs edge 312 that prevents liquid 314 in theupstream reservoir 302 from flowing into thestain reservoir reservoir 302 in the same way as the liquid 108 in thereservoir 102 i.e., by capillary forces to thehydrophilic underside 308 and by surface tension in thesurface 316. - The order of the reservoirs corresponds to the order in which the liquids flow over the
grid 116. That is, if the liquids are sample, wash, stain then the stain liquid should be added to theupstream reservoir 302. The wash liquid should be added to the middle orsecond reservoir 102, which upon addition connects to the liquid in theupstream reservoirs 302. The sample liquid should be added to thefirst reservoir 104 on top thegrid 116, which upon addition connects to the upstream liquid train ofwash 305 andstain 314 and downstream connects to thedraining unit 318. - The draining
unit 318 has the absorption members (filter papers) 158, 160 anddissolvable film 162 and is located downstream of thesample reservoir 104. The drainingunit 318 has anadditional dissolvable film 320 and another filter orabsorption member 322 to illustrate how the timing of the additional liquid can be controlled. The thickness of thefirst dissolvable film 158 decides how long thefirst liquid 110 added to thesample reservoir 104 sits or stays on top of thegrid 116 i.e., how long thesample liquid 110 and particles 114 are permitted to adhere to thegrid 116. Thesecond filter 160 should be big enough to absorb and store the amount of liquid corresponding to the volume of thesample liquid 110. Once the liquid 110 reaches the second dissolvable film ormembrane 320, the flow of the liquid over thegrid 116 stops until thefilm 320 has been dissolved and thelast filter 322 in this setup with 3 liquids pulls thesecond liquid grid 116 by absorbing all the volumes of all threeliquids -
FIG. 16 shows adevice 380 which includes modifications of the devices shown inFIGS. 13 and 15 and illustrates how the shapes of thefilter paper 382 in thedraining unit 384 can be modified in order to steer the flow-speed of the liquids. Everything else indevice 380 is identical to the components ofdevices filter paper 382 with aneck 386 slows down the flow speed over thegrid 116 whereas a wide and thick filter increases the flow speed. -
FIGS. 18-19 show alternative embodiments of thedevices device 100 shown inFIG. 13 except that the draining orblotting units blotting unit 106. Preferably, drainingunit 706 has only onefirst absorption member 758 but no dissolvable membrane or a second absorption member, as shown inFIG. 13 . The operation ofdevice 700 is substantially similar to that ofdevice 100 in that the liquids in the first and second reservoirs are absorbed by the absorbingmember 758 during a suitable time period so that there is enough time for the particles in the sample liquid to adhere to thegrid 116, as explained in detail with reference todevices - Similarly,
device 800 is substantially similar to that ofdevice 100 except that the drainingunit 806 has adissolvable membrane 862 and afirst absorption member 858. The drainingunit 806 does not have an absorption member between thedissolvable membrane 862 and thesecond reservoir 104 so that thedissolvable member 862 comes into direct contact with the second liquid without the second liquid having to pass through an absorption member before coming into contact with the dissolvable member to start dissolving thedissolvable membrane 862. Except for the differences of the drainingunits unit 106 all other features and method steps ofdevices devices -
FIG. 20 is yet another embodiment of thedevice 900 that is substantially similar to thedevice 100 shown inFIG. 13 except that it has acapillary channel 958 in draining orblotting unit 906 instead of thefirst absorption member 158. The capillary forces in thechannel 958 urges the second liquid 110 from thesecond reservoir 104 into thechannel 958 so that the liquid 110 comes into contact with thedissolvable membrane 162 to dissolve the membrane, as described in detail in connection withdevices - In operation, the method of the present invention comprises the steps of providing the
stain reservoir 202 connected to and in fluid communication with the grid chamber or sampleliquid reservoir 204 that has apre-mounted grid 216. Thereservoir 204 is in turn connected to, and in fluid communication with, the drainingunit 206. Thestain liquid 208 is added to thestain reservoir 202 which is contained in thereservoir 202 until the user adds thesample liquid 210 including theparticles 214 into thesample liquid reservoir 204. - This key feature is enabled through a capillary stop valve or pinning mechanism, here in the form of an
edge 228 located at the end ofchannel 250 that separates thestain reservoir 202 from thegrid chamber 204. Thestain liquid 208 is pinned at the pinningedge 228 due to capillary forces so that the liquid 208 adheres to theunderside 230 and extends over the pinningedge 228 and the surface tension at thesurface 234 prevents the liquid 208 from flowing into thegrid chamber 204 although there is fluid communication between thestain reservoir 202 and the sample reservoir orgrid chamber 204 viachannel 250. The fact that thestain liquid 208 is held inside thestain reservoir 202 in this way enables the sample preparation process to be initiated by adding thesample liquid 210 including theparticles 214 into thesample reservoir 204. The liquid 210 is added in such an amount so that the liquid 210 comes into contact withsurface 234 to break the surface tension of the liquid 208 between the pinningedge 228 and theunderside 230. When the surface tension of thesurface 234 is broken, the twoliquids sample liquid 210 including theparticles 214 are added to thegrid chamber 204 via theopening 236 from above thedevice 200, the liquid 210 also flows into and connects with the drainingunit 206 that is downstream of thesample reservoir 204. Theopening 236 through which thesample liquid 210 andparticles 214 are added is slightly smaller than the width of thegrid 216 to make thesample liquid 210 reliably connects to thestain liquid 208 and theblotting unit 206. The cavity 257 located below thegrid 216 makes sure that no liquid flows and attaches to the wrong side of thegrid 216 and interferes with the quality of the preparation. - The draining
unit 206 has two absorption units (such as filter papers) 218 and 222 and thesoluble PVA film 220 located between the twofilters top filter 218 makes sure that thesample liquid 210 reliably connects to thePVA film 220 by absorbing the liquid 210 so that the liquid travels from a top side of thefilter 218 to a bottom of thefilter 218 that is in contact with thedissolvable film 220. - The
vent 264 above the top filter serves as the emergency exit for potentially trapped air, which could otherwise block the connection between thesample liquid 210 and thedraining unit 206. Thesample liquid 210 flows through thetop filter 218 and upon contact with the PVA film orlayer 220 dissolves thePVA layer 220 so that the liquid can flow into thefilter 222 located below thefilter 218. The time is takes for thesample liquid 210 to dissolve thePVA layer 220 is critical because it controls the time theparticles 214 in thesample liquid 210 are permitted to adhere to and adsorb into thegrid 216. Once thePVA layer 220 is dissolved, the liquid 210 followed by flow into and connect to thebottom filter 222 which absorbs all the liquid 210, 208 in thedevice 200. The filter orabsorption member 222 first absorbs thesample liquid 210 and then thestain liquid 208. Thebottom filter 222 hence corresponds to the manual blotting step. Theopening 236 over thegrid 216 through which thesample liquid 210 and theparticles 214 were added, now ensures rapid drying of thethin stain film 224 that remains left after the draining/blotting by the two absorption members or filters 218, 222 have absorbed all excess liquid 210, 208. Finally, theflap 226 can be peeled off thedevice 200 to provide easy access to thegrid 216 that is easily extracted from thedevice 200 for subsequent imaging in, for example, a ns-TEM device. - If more liquids need to be added in a sequential manner, for example a
washing liquid 305 in a washing step before thestain liquid 314 is added to thesample liquid 110, anotherliquid reservoir 302 connected to and in fluid communication with themiddle reservoir 303 via thechannel 304 and separated by the pinningedge 312 can be added, as shown and described in connection withFIG. 15 . Thewash liquid 305 should then be added to themiddle reservoir 303 after thestain liquid 314 is added to the mostupstream reservoir 302. If the incubation time of the additional liquid (here the wash liquid 305) need to be controlled, an additional layer ofsoluble film 320 andfilter paper 322 can be added to thedraining unit 318. - The speed of the flow over the
grid 116 can be controlled by the shapes and thicknesses of the filter papers in the draining unit. Less amount of available absorption media (i.e. filter/paper) or lower capillarity (also known as Wicking rate) of the filter results in a slower flow/drainage and vice versa. For example: a thin, narrow and long filter after the soluble film results in a slower liquid flow and drainage pace. - Instead of adding a droplet of a pre-mixed stain solution, the salt constituting the stain can be dried at the bottom of the
stain reservoir 102 and then only water or another dissolvent/buffer is added to thereservoir 102 when preparing the stain reservoir and the grid. The stain salt is then dissolved when the dissolvent is added to create thestain liquid 208. - Instead of adding a hydrophilized grid to the preparation assembly kit, a hydrophilization liquid such as Alcian-Blue can be flushed over the grid before the sample liquid is added. In this way, the stain and sample liquids are loaded in two separate reservoirs upstream of the
grid chambers FIG. 15 ) connected viamicrofluidic channels edges grid 116 in thegrid chamber 104 to start the sequence of liquids flowing over thegrid 116, i.e., initiating the grid preparation process. - An alternative use of the method of the present invention is to use it for controlled deposition or a matrix on to of the grid. 116. The same method as described above applies with the exception that only one liquid, i.e. the substrate, is used and it is added to the
grid chamber 104. For example, fibers, such as spider silk, may be permitted to polymerize in the air-liquid interface on the droplet f the sample (substrate) added onto thegrid 116. When thesoluble layer 162 is dissolved, the spider silk gently falls down on the EM grid while thefilter paper 160 drains the device. The fiber network (in this case the spider silk) disposed on top of the grid then acts as a matrix forcing a protein which is later added to be placed in a random orientation on the grid, before subsequent single particle reconstruction in (cryo- or negative stain) TEM. Adding a protein direct the grid. 116 often results in that the protein orients itself in a preferred orientation (i.e., laying down when elongated and/or flat), which limits the resolution that can be achieved in the reconstruction. - As described above, the microfluidic device of the present invention consists of several layers of different materials, as particularly indicated in, for example,
FIG. 3 It was fabricated from hydrophilic sheets (Type C laser printing transparency, Xerox, Elmstock, UK), adhesive tape 1 (64620, Tesa, Norderstedt, Germany) and adhesive tape 2 (300LSE, 3M, VWR, Sp{dot over (a)}nga, Sweden). Low-tack adhesive tape (Scotch® 928, 3M, Amazon, Koblenz, Germany) was used to fixate the 400 mesh TEM grids (01754-F, Ted Pella Inc., Redding, Calif.) which are formvar coated copper grids with a continuous carbon film.Ahlstrom grade 238 and 222 (Ahlstrom Filtration LLC, Mt. Holly Springs, Pa.) were used asabsorption paper 1 andabsorption paper 2 in the draining or blotting unit, respectively. The soluble film or membrane/valve was fabricated from granular PVA (360627, Sigma-Aldrich, St. Louis, Mo.). AAV (adeno-associated virus) particles, serotype 2 (AAV2) encapsulated with Cytomegalovirus (CMV) promoter-driven expression of Green Fluorescent Protein (GFP), with a stock concentration of 1×1013 gc/mL (CV10004-50UL, AMS Biotechnology Ltd., Abingdon, UK) was used as the sample. - The AAV sample was diluted with phosphate-buffered saline (DPBS (−/−) 14190-094, Thermo-Fisher, Uppsala, Sweden) to a concentration of 1×1012 gc/mL. 26S proteasome (#: E-350, BostonBiochem, Cambridge, Mass.) was used as a test sample representing a large globular protein complex. The sample with protein fibrils from whey protein isolate (WPI)16, with an initial concentration of 40 mg/ml, was a gift from the Division of Applied Physical Chemistry at the Royal Institute of Technology in Stockholm, Sweden.
- NanoVan®, 2% Methylamine vanadate in solution, (#2011-5ML, Nanoprobes, Yaphank, N.Y.) and
Uranyl Acetate 2% in solution (#2240-2, Electron Microscopy Sciences, Hatfield, Pa.) were used as stain. Aqueous solutions of food color dyes (EAN-codes: 5701073064665 and 5701073064672, Dr. Oetker, Coop, Solna, Sweden) were used as models for sample and stain. - Each device was fabricated using lamination technology where the devices were formed by stacking several layers of different materials, as described previously. The cross section in
FIG. 3 shows the different layers. The denomination, brand name and thicknesses of these layers were as: -
Denomination Brand name Thickness [μm] Hydrophilic sheets Type C laser printing 100 transparency, Xerox Adhesive tape 1 64620, Tesa 170 Adhesive tape 2300LSE, 3M 50 Low-tack adhesive Scotch ® 928, 3M 30 Paper 1Ahlstrom grade 238340 Paper 2Ahlstrom grade 222830 - The adhesives and the hydrophilic sheets were structured using a cutting plotter (CE6000, Graphtec America Inc., Irvine, Calif.).
- The PVA film or membrane was fabricated from an aqueous solution of 20 wt % of granular PVA. Using a thin-film applicator (4340, Elcometer, Manchester, UK) the PVA films were uniformly transferred to laminating pouches (3385694, Office Depot, LA Venlo, Netherlands) and dried at room temperature. The final PVA film thickness was measured using a thickness gauge with 1 μm graduation (2109L Metric Dial Gauge, Mitutoyo, Upplands Väsby, Sweden).
- The PVA film was laminated to
absorption paper 2 at 85° C. using a laminator (Heat Seal Pro H600, GBC, Northbrook, Ill.). The paper-PVA laminates were kept in a humidity chamber at 80% relative humidity until 30 minutes before use. - The paper materials, including the paper-PVA laminate, were cut by a laser cutter (VLS 2.30, Universal Laser Systems, Vienna, Austria). After structuring, the layers were assembled by using alignment pins and laminated at room temperature. For improved particle adhesion, the TEM grids were glow discharged in oxygen plasma with a PELCO easiGlow™ (91000S-230, Ted Pella Inc., Redding, Calif.) before fully assembling the microfluidic device of the present invention. A fully assembled fabricated device is shown in
FIG. 5 . The dimensions of the device are 6×12 mm2. The devices were used within one hour after glow discharging the TEM grids. - One important feature of the microfluidic device of the present invention is that it is designed to minimize user-interactions. To demonstrate the autonomous device operation and microfluidic consistency six devices were evaluated. Five devices were used with AAV particles as sample and NanoVan® as stain. The grids from these five devices were used to collect TEM images for an automated image analysis on a total of 225 images. To better visualize the individual preparation steps of the autonomous device, one device was used with color dye solutions. Blue dye solution and yellow dye solution were used as models for sample and stain, respectively. First, 5 μl of stain liquid was added via the stain inlet into the stain reservoir. Then, the autonomous TEM grid preparation mechanism was triggered by adding 5 μl of sample to the sample inlet of the sample reservoir or grid chamber.
- The TEM grid preparation sequence of all the devices was recorded with a camera with a frame rate of 50 frames per second. To analyze the device performance and consistency of the autonomous preparation steps, the time interval of each step was manually obtained. The time period between the addition of stain and the addition of the sample liquid (including the particles) was defined as the stain preloading time.
- To demonstrate the robustness of the stain reservoir, i.e., stain confinement without leakage, the time between stain and sample addition was varied between 20-60 seconds wherein the stain liquid was held in place by a surface extending between the pinning edge and an underside of a hydrophilic surface i.e., capillary forces and surface tension. As illustrated in
FIGS. 1A-1D , the microfluidic TEM grid preparation steps after sample addition includes sample adsorption, draining/blotting and thin film drying. As critical aspect of the method of the present invention is that the adsorption time of the sample on the TEM grid corresponds to and is the same as the dissolving time of the PVA film. It was defined as the time between wetting ofpaper 1 and the start of the blotting event. The PVA layer thickness was 10 μm. The blotting time is the interval between the start and the end of the draining/blotting event. The start of the blotting event is defined as the moment when the liquid first moves into the draining unit. The end of the blotting event is defined as the moment when the bulk of liquid is drained by the draining unit leaving a thin stain film on the TEM grid. After this, the drying interval starts and lasts until the remaining thin film of stain on the grid was visually dry. - In general, TEM imaging is a powerful visualization technique for many different types of samples. However, the required sample adsorption time varies between different samples. The main reason for this is that sample adsorption depends on the interaction between sample and the carbon surface of the TEM grid. Hence, devices with different adsorption times to account for different sample requirements would be desirable.
- Another key element of the microfluidic device of the present invention is the dissolving time of the water-soluble PVA film, that autonomously controls the timing of the device, corresponds to the sample adsorption time of the sample (i.e., film or layer of particles embedded in stain) on the grid.
- To demonstrate the adjustability of the adsorption time of the sample on the grid, microfluidic devices with three different thicknesses of the water-soluble film (12 μm, 24 μm and 36 μm) were fabricated and investigated. Among the parameters that affect the dissolving time (e.g., temperature, relative humidity), the thickness of the dissolvable film is one of the easiest parameters to tune and adjust. The PVA thicknesses of 24 μm and 36 μm were achieved by stacking multiple layers of 12 μm PVA sheets and laminating them to
paper 2 below the PVA sheets at 85° C. with the laminator. The paper-PVA laminates were kept in a humidity chamber at 80% relative humidity until 30 minutes before use. The adsorption time was evaluated of 15 devices, five devices per film thickness, using 5 μl of blue dye solution and 5 μl of yellow dye solution as a model for sample and stain, respectively. - To assess the sample preparation quality, TEM imaging was performed on the five autonomously prepared TEM grids with AAV particles as sample and NanoVan® as stain. NanoVan® was chosen because it is not radioactive, unlike the commonly used Uranyl Acetate, and can be handled in an ordinary laboratory. For all five grids, it was investigated whether AAVs were successfully adsorbed to the TEM grid and sufficiently embedded in stain. The AAV particles on different magnification levels were inspected, with a field of view (FOV) between 16 μm and 500 nm. The imaging was performed on MiniTEM™ microscopes (Vironova AB, Stockholm, Sweden) with an operating voltage of 25 kV.
- To investigate whether the obtained TEM images were useful for automated image analysis, a particle detection script was applied to the TEM images of the five autonomously prepared grids. A total of 225 images were collected according to the imaging scheme shown in
FIG. 8 . At low magnification, the user manually chose five non-neighboring grid squares. Then, nine high magnification images were acquired per grid square at a FOV of 2 μm, resulting in 45 images per grid. At this magnification, where a pixel represents approximately 1 nm, a number of particles per image can usually be seen and the morphology of the AAVs is typically visible.Grid 1,grid 4 andgrid 5 were imaged on the same microscope, whilegrid 2 andgrid 3 were imaged on a second microscope. The particle detection script was applied to all 225 images. AAVs have an icosahedral capsid that appears round and has an expected diameter of 20-25 nm. However, the script was designed to detect the stain envelope around the AAV particles so that the particles appear larger than the actual virus size. Therefore, the particle detection script was set to detect round objects within a diameter range of 24 nm to 32 nm. From the automated image analysis, the number of detected particles per grid were obtained, where each detected particle is characterized by its position and size. - To quantify the particle detection results, a manual particle detection was performed on a subset of 25 of the images, with five randomly chosen images per TEM grid. The number of particles were manually counted and compared with the results from the detection script. This was done to find the ratio of true and false positives, which both are important measures for the performance of the detection script.
- nsTEM is routinely used as a quality control during the preparation of biological specimens, e.g. protein complexes, for structural biology. To investigate the potential use of the microfluidic device for wider applications and with different stains, proteasomes were prepared and image, as a larger globular protein complex, and protein fibrils from WPI, as a filamentous protein. The PVA films in the used microfluidic devices had a thickness of 15 μm, corresponding to a dissolving time of around 35 seconds. For the proteasomes and fibrils, stock solution of Uranyl Acetate and NanoVan®, was used, respectively.
- As described in detail above, the TEM grid preparation sequence is shown in
FIGS. 2A-2D . For visibility, colored dye solutions were used instead of sample and stain solutions. The first step shows how the preloaded stain 208 (yellow dye solution) is contained in thestain reservoir 202 and thesample 210, 214 (blue dye solution) is added (best shown inFIG. 2A . In the second step, thesample 210 including the particles, 214 cover the TEM grid as long as the PVA film or valve is closed (best shown inFIG. 2B . When the PVA film or valve has dissolved, the stain and sample liquids are blotted (best shown inFIG. 2C . Finally, the bulk of liquids is contained in the draining media (blotting filter/paper) and the stain film, including particles embedded therein, dries (best shown inFIG. 2D ). Compared to a previously reported microfluidic TEM grid preparation, the user interactions were reduced by providing an autonomous microfluidic operation that is controlled by the water-soluble PVA film. Furthermore, a significantly lower liquid consumption was demonstrated with liquid volumes as small as in the manual preparation protocols. - To demonstrate microfluidic consistency, video recordings were analyzed with respect to timing and duration of the microfluidic events on the five devices used with AAVs as sample and NanoVan® as stain.
FIG. 6 presents a bar chart with the time intervals for each of the four sample preparation steps. The results show that regardless of the length of the stain preloading time, all the following steps including adsorption, draining/blotting and drying, are close to identical for the five devices. This demonstrates that the stain reservoir reliably contains the stain until the sample is added irrespective of the stain preloading time. The average adsorption time for the five devices is 10.6±0.3 s, corresponding to a CV of 3%. This indicates a highly consistent autonomous time-control of the microfluidic device of the present invention. The average draining/blotting time is 0.8±0.1 s, corresponding to a CV of 12.5%. While the CV seems high, the absolute deviation is low and confirms the microfluidic consistency of the device of the present invention. The drying step does not end abruptly which makes it difficult to measure the exact drying interval by viewing videos. However, it was observed that all the TEM grids were visually dry within one minute. The reliable and fast drying is enabled by the grid area sized top opening in the grid chamber. - The results show that the microfluidic device of the present invention works as intended although the user input was minimal. Irrespective of the stain preloading time, the autonomous device operation after sample addition is close to identical for the five devices which demonstrates a high microfluidic consistency.
- To demonstrate the adjustability of the sample adsorption time, which corresponds to the PVA dissolving time, three different thicknesses (12 μm, 24 μm and 36 μm) of the water-soluble PVA film were tested in the microfluidic device of the present invention.
FIG. 7 shows the measurement results of the adsorption time. The dissolving time of the PVA films increases with PVA film thickness. For 12 μm, 24 μm and 36 μm thicknesses of the PVA films, the average dissolving time is 14.4±0.9 s (n=5), 89.9±12.0 s (n=5) and 191.6±20.3 s (n=5), respectively. The results show that it is possible to easily adjust the adsorption time by changing the PVA film thickness. The variation of dissolving time increases with increased PVA film thickness. This could be due to small differences in the PVA film thickness between different devices. However, the variation is low enough to conclude that the adsorption times can be controlled by the design of the PVA layer of the present invention. - TEM imaging of the five autonomously prepared TEM grids made it possible to assess the sample preparation quality.
FIGS. 9A-9C show a magnification series with three FOVs: 16 μm, 2 μm and 500 nm. In the largest FOV (16 μm), the AAV particles appear as dark spots. The intermediate FOV (2 μm) shows a higher level of detail. The particles are visible as bright, round objects encircled by dark rings with a radially fading stain gradient. The smallest FOV (500 nm) in this series has the highest level of detail and provides a close-up view of the AAV particles. -
FIGS. 10A-10E show one exemplary image from each of the five grids (FOV 2 μm). It was found that all five TEM grids contain well embedded particles in the stain film on the grid, visible as bright spots surrounded by dark stain envelopes. Variations in the appearance of the stain envelope might be due to local variations of stain thickness. Also, thickness variations of the TEM grid, e.g. caused by local inhomogeneities of the carbon film, can result in variations of the image darkness. Overall, the results from the five microfluidic devices showed consistent preparation of TEM grids with well embedded AAV particles. - To further demonstrate sample preparation consistency, 225 TEM images were collected and an automated particle detection was performed. The particle detection script detected 5171 particles in all 225 images. Every grid, with 45 images, contained an average of 1034±65 particles, corresponding to a CV of 6%. This indicates a reproducible and consistent AAV particle spreading over five independently prepared TEM grids. Using the results of the automated particle detection, the detected size of the particles was extracted.
-
FIG. 11 is agraph 640 that shows the average particle diameter for the detected particles in each grid. Two different microscopes were used and even though the calibrations might be slightly different, the average particle size for each grid is well within the error bars of the other samples. The average size of all detected particles is 28±2 nm (n=5171), corresponding to a CV of 7%. This low variation means that, irrespective of the grid, all detected particles have a similar detected size. The real size of AAV particles is 20-25 nm but appears larger when imaged in nsTEM due to the stain envelope. The detection script is designed to outline and measure particles at the stain layer, i.e., outside the actual particle. Therefore, the detected particle size is well within the expected size window. - The result of the manual particle detection in a subset of 25 images allowed to quantify the automated detection results.
FIG. 12 summarizes the results of the subset test. The manual count resulted in 605 particles in the subset. The automated particle script found 557 of these particles correctly (True positives), which corresponds to a success rate of 92%. The script found 29 objects that were not correct (False positives), which corresponds to 4.9%. With true positives above 90% and false positives around 5%, it can be concluded that the images and the autonomous sample preparation have sufficient quality for simple automated image analysis. - To broaden the scope of applications proteasomes and protein fibrils from WPI were prepared and imaged. The results of those two
samples FIGS. 17A-17D , reveal an even spreading of the proteins on the TEM grid with well-embedded areas suitable for TEM investigations.FIGS. 17A- 17 B show images 500 of 265 proteasomes at two different magnifications. Atop view 504 and aside view 506 of the proteasomes can be observed.FIGS. 17C-17D show images of WPI fibrils 508 at two different magnifications. The analysis of the proteasome specimen (best shown inFIGS. 17A-17B ) shows that individual proteasomes can clearly be identified, and structural features such as details of the subunits can be distinguished. Different projections can be observed on the images, with the top view appearing as a circular particle and the side view appearing rectangular. The analysis of the WPI fibrils (best shown inFIGS. 17C-17D allows the observation and characterization of well-defined individual fibrils of various lengths. Overall, the morphological observations are in line with reported data of similar samples prepared with conventional manual nsTEM. The preparation of these two protein samples did not require further adjustments of the microfluidic device, hence demonstrating the versatility and robustness of the method. - Below is yet another possible application in the field for the method of the present invention that would require a modified device. The possible nsTEM sample preparation application example is immunogold-labelling where four liquids need to be flushed over the sample. The sequence of preparation steps would be:
-
- 1) A grid, with the sample already attached thereto, is added to the device;
- 2) A primary antibody is permitted to adhere to the grid (so added directly onto the grid as the sample liquid in the description above);
- 3) Once the binding has occurred, a blocking liquid is flushed over the grid (e.g., BSA=bovine serum albumin, or desiccated milk);
- 4) Then a second antibody connected to gold particles is flushed over to bind to and hence mark the primary antibody positions; and
- 5) Finally, non-bound gold particles are washed off with the last washing step.
- In summary, a capillary-driven single-use device of the present invention for autonomous TEM sample preparation has been presented. To avoid operator bias and error-prone manual steps, the device of the present invention is designed to minimize user-interactions. The key design elements are the stain and sample reservoirs combined with the water-soluble valve or PVA film and the absorption membranes. These key elements enable the starting of the autonomous TEM grid preparation with only one non-critical user-interaction. The device consistency both for the microfluidic performance and the sample preparation quality have been demonstrated. The consistency of the microfluidic performance was shown by five microfluidic devices with close to identical TEM grid preparation sequences. The sample preparation consistency was demonstrated by five TEM grids that all exhibit well embedded AAV particles. This preparation consistency was further highlighted by the results of the automated particle detection. From a subset test with true positives above 90% and false positives around 5%, it was concluded that the images and the autonomous sample preparation hold sufficient quality for image analysis. The additional preparation of two protein samples demonstrated the versatility of the microfluidic device for a wider scope of applications. Furthermore, the adjustability of timing of the microfluidic events was demonstrated by changing the thickness of the water-soluble valve or PVA film. This allows to account for different sample adsorption requirements. To account for TEM sample preparation requiring different staining times, the device of the present can be extended by a second draining unit. In conclusion, the demonstrated microfluidic device of the present invention presents a promising, effective and reliable solution to alleviate the problems associated with human inconsistency in manual TEM grid preparations.
- The evaporation arrangement of the present invention is important and that the sample support is exposed to air for proper evaporation. Another aspect is that the width of the opening and the width of the sample support should be about the same. The humidity condition immediately above the sample support is higher than the humidity outside the opening i.e. above the device. At the liquid boundary of the liquid sample on the sample support the humidity is 100% while the relative humidity in the ambient air outside the device is lower which promotes evaporation of the liquid in the stain layer through the opening. It should be understood that the first liquid can be held in the first reservoir without using an edge. The two liquids can connect without the use of the edge but, for example, putting pressure on one of the liquid droplets. The edge, however, stops the first liquid from flowing into the second reservoir. This is because the edge is a discontinuity in the channel between the two reservoirs and the travel of the capillary force along the wall of the channel is stopped. The first liquid, such as the stain liquid, could be held in the first reservoir i.e. the stain reservoir without using an edge. The pinning edge keeps the stain liquid in place i.e. stops the stain liquid from flowing into the sample reservoir while adding the liquid to the sample reservoir. The expansion (bulging out) of the first liquid between the edge and the hydrophilic upper surface is beneficial but not necessary. It makes the device more robust.
- It should also be understood that it is not necessary to use capillary forces to hold the first liquid in the first reservoir. The device preferably, but not necessarily, has a channel going from the first reservoir to the second reservoir. Preferably, the two reservoirs should be in fluid communication and that the first liquid should be held in the first reservoir. This confinement of the first liquid in the first reservoir is preferably but not necessarily based on capillary forces and/or surface tension which are easily broken when the second liquid is added and connects to the first liquid. It is not necessary that the first reservoir is at a higher elevation compared to the second reservoir and the blotting unit. All three units could be located on a common surface at the same elevation.
- Preferably, the dissolvable member decides the timing of the adsorption of sample particles on the sample support but this could also be adjusted by using different filters that absorb fluids at different rates. For example, small narrow filter slows down the absorption rate. The drainage in micro channels could also acts as a delay mechanism and drainage speed control. It may also be necessary to have a minimum speed or a certain delay time to give enough time for the sample particles to adhere to the sample support or grid. It may also be necessary to have a high speed when draining the first liquid (stain) in order to leave a stain layer. If the draining of the first liquid is too slow, too much stain will be drained leaving the particles unprotected. This results in a poor-quality preparation. It should be noted that although the dissolvable membrane or film delays the liquid flow and once the membrane is dissolved the flow rate is quite rapid as opposed to a very slow constant flow rate.
- While the present invention has been described in accordance with preferred compositions and embodiments, it is to be understood that certain substitutions and alterations may be made thereto without departing from the spirit and scope of the following claims.
Claims (20)
1. A method of preparing a sample in a microfluidic device, comprising:
providing a microfluidic device having a first reservoir in fluid communication with a second reservoir in fluid communication with a draining unit having a first absorbing member disposed therein, the first reservoir containing a first liquid, the second reservoir having a sample support disposed therein;
adding a second liquid, containing substances, to the second reservoir;
the second liquid contacting the first liquid and the first absorbing member;
the first absorbing member absorbing the second liquid and the first liquid; and
the substances adhering to the sample support.
2. The method of claim 1 wherein the method further comprises the steps of providing the draining unit with a dissolvable membrane upstream of the first absorbing member, the second liquid dissolving the dissolvable membrane prior to the first absorbing member absorbing the first and second liquids.
3. The method of claim 1 wherein the method further comprises the steps of the substances adhering to the sample support while the second liquid dissolving a dissolvable membrane.
4. The method of claim 1 wherein the method further comprises the step of a capillary stop valve holding the first liquid in the first reservoir preventing the first liquid from flowing into the second reservoir prior to adding the second liquid to the second reservoir.
5. The method of claim 1 wherein the method further comprises the step of a portion of the first liquid embedding the substances adhered to the sample support.
6. The method of claim 1 wherein the method further comprises the steps of providing the capillary stop valve with an edge that separates the first reservoir from the second reservoir, the edge holding the first liquid in the first reservoir.
7. The method of claim 2 wherein the method further comprises the steps of providing the dissolvable membrane downstream of the first absorption member and a second absorption member downstream of the dissolvable membrane, the first absorption member absorbing the second liquid and permitting the second liquid to come into contact with the dissolvable member.
8. The method of claim 7 wherein the method further comprises the step of the second absorption member absorbing the second liquid and the first liquid after the dissolvable membrane has been dissolved.
9. The method of claim 1 wherein the method further comprises the step of the second liquid breaking a surface tension of the first liquid upon contact with the first liquid held in the capillary stop valve.
10. The method of claim 1 wherein the method further comprises the step of a time period required to dissolve the dissolvable membrane controlling a permitted time period for the substances to adhere to the sample support.
11. The method of claim 1 wherein the method further comprises the step of the second liquid contacting the absorbing member before the first liquid.
12. The method of claim 5 wherein the method further comprises the step of the first portion of the first liquid drying on the sample support.
13. The method of claim 1 wherein the method further comprises a portion of the first liquid forming a liquid film on the sample support, wherein the liquid film has a film thickness of less than 1 mm.
14. The method of claim 1 wherein the sample support is dried within three minutes at an ambient temperature and 50% relative humidity.
15. The method of claim 1 , wherein the first liquid has a volume of between 0.1-50 μl.
16. The method of claim 1 wherein the second liquid has a volume of between 0.1-50 μl.
17. A method of preparing a sample in a microfluidic device, comprising:
providing a microfluidic device having a first reservoir in fluid communication with a second reservoir in fluid communication with a draining unit having a first absorbing member disposed therein, the first reservoir containing a first liquid;
a user of the microfluidic device adding a sample support into the second reservoir;
the user adding a second liquid, containing substances, to the second reservoir;
the user waiting a waiting period of at least 20 seconds before removing the sample support from the second reservoir;
during the waiting period, the second liquid contacting the first liquid and the first absorbing member;
during the waiting period, the first absorbing member absorbing the second liquid and the first liquid;
during the waiting period, the substances adhering to the sample support; and
at the end of the waiting period, the user removing the sample support from the second reservoir.
18. The method of claim 17 wherein the method further comprises the steps of providing the draining unit with a dissolvable membrane upstream of the first absorbing member and the second liquid dissolving the dissolvable member during the waiting period.
19. The method of claim 17 wherein the method further comprises the steps of the first liquid forming a film on the sample support and embedding substances adhered to sample support.
20. The method of claim 19 wherein the method further comprises the step of the film drying on the sample support.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/941,237 US20230279324A1 (en) | 2020-09-17 | 2022-09-09 | Autonomous microfluidic device for sample preparation |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/023,922 US11484882B2 (en) | 2020-09-17 | 2020-09-17 | Autonomous microfluidic device for sample preparation |
US17/025,390 US11485945B2 (en) | 2020-09-17 | 2020-09-18 | Autonomous microfluidic device for sample preparation |
US17/941,237 US20230279324A1 (en) | 2020-09-17 | 2022-09-09 | Autonomous microfluidic device for sample preparation |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/025,390 Continuation US11485945B2 (en) | 2020-09-17 | 2020-09-18 | Autonomous microfluidic device for sample preparation |
Publications (1)
Publication Number | Publication Date |
---|---|
US20230279324A1 true US20230279324A1 (en) | 2023-09-07 |
Family
ID=80625811
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/025,390 Active 2041-05-24 US11485945B2 (en) | 2020-09-17 | 2020-09-18 | Autonomous microfluidic device for sample preparation |
US17/941,237 Pending US20230279324A1 (en) | 2020-09-17 | 2022-09-09 | Autonomous microfluidic device for sample preparation |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/025,390 Active 2041-05-24 US11485945B2 (en) | 2020-09-17 | 2020-09-18 | Autonomous microfluidic device for sample preparation |
Country Status (9)
Country | Link |
---|---|
US (2) | US11485945B2 (en) |
EP (1) | EP4255627A1 (en) |
JP (1) | JP2023542086A (en) |
KR (1) | KR20230066273A (en) |
CN (1) | CN115803603A (en) |
AU (1) | AU2021344255A1 (en) |
CA (1) | CA3184969A1 (en) |
IL (1) | IL301093A (en) |
WO (1) | WO2022060537A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11485945B2 (en) * | 2020-09-17 | 2022-11-01 | Intelligent Virus Imaging Inc | Autonomous microfluidic device for sample preparation |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11485945B2 (en) * | 2020-09-17 | 2022-11-01 | Intelligent Virus Imaging Inc | Autonomous microfluidic device for sample preparation |
US11484882B2 (en) * | 2020-09-17 | 2022-11-01 | Intelligent Virus Imaging Inc. | Autonomous microfluidic device for sample preparation |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2626884A1 (en) * | 2012-02-10 | 2013-08-14 | Danmarks Tekniske Universitet - DTU | Microfluidic chip for high resolution transmission electron microscopy |
US9958362B1 (en) * | 2015-10-02 | 2018-05-01 | The Florida State University Research Foundation, Inc. | Microscope sample preparation device |
-
2020
- 2020-09-18 US US17/025,390 patent/US11485945B2/en active Active
-
2021
- 2021-08-23 CN CN202180048649.9A patent/CN115803603A/en active Pending
- 2021-08-23 CA CA3184969A patent/CA3184969A1/en active Pending
- 2021-08-23 IL IL301093A patent/IL301093A/en unknown
- 2021-08-23 WO PCT/US2021/047085 patent/WO2022060537A1/en unknown
- 2021-08-23 AU AU2021344255A patent/AU2021344255A1/en active Pending
- 2021-08-23 JP JP2023514110A patent/JP2023542086A/en active Pending
- 2021-08-23 EP EP21869963.5A patent/EP4255627A1/en active Pending
- 2021-08-23 KR KR1020227043394A patent/KR20230066273A/en unknown
-
2022
- 2022-09-09 US US17/941,237 patent/US20230279324A1/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11485945B2 (en) * | 2020-09-17 | 2022-11-01 | Intelligent Virus Imaging Inc | Autonomous microfluidic device for sample preparation |
US11484882B2 (en) * | 2020-09-17 | 2022-11-01 | Intelligent Virus Imaging Inc. | Autonomous microfluidic device for sample preparation |
Non-Patent Citations (1)
Title |
---|
Kinahan, David J., et al. "Laboratory unit operations on centrifugal lab-on-a-disc cartridges using dissolvable-film enabled flow control." (2014). (Year: 2014) * |
Also Published As
Publication number | Publication date |
---|---|
KR20230066273A (en) | 2023-05-15 |
WO2022060537A1 (en) | 2022-03-24 |
US11485945B2 (en) | 2022-11-01 |
US20220081662A1 (en) | 2022-03-17 |
CA3184969A1 (en) | 2022-03-24 |
AU2021344255A1 (en) | 2023-02-02 |
EP4255627A1 (en) | 2023-10-11 |
CN115803603A (en) | 2023-03-14 |
IL301093A (en) | 2023-05-01 |
JP2023542086A (en) | 2023-10-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6782296B2 (en) | Plasma separation microfluidic device | |
DE60035199T2 (en) | ANALYSIS CASSETTE AND LIQUID CONVEYOR CONTROLLER | |
JP5684757B2 (en) | Lateral flow and flow-through bioassay devices based on patterned porous media, methods of manufacturing the devices, and methods of using the devices | |
JP7120515B2 (en) | Blood staining patch, method and device for blood testing using same | |
JP2010539907A (en) | Microfluidic instruments for handling, imaging and analyzing cells | |
US20230279324A1 (en) | Autonomous microfluidic device for sample preparation | |
US11060127B2 (en) | Imaging cartridge, pipette, and method of use for direct sputum smear microscopy | |
JP2009122082A (en) | Blood diluting and quantifying instrument | |
KR20180053686A (en) | Holder for cell-holding substrate for observation sample preparation, kit containing the same, and method for producing observation sample | |
US11484882B2 (en) | Autonomous microfluidic device for sample preparation | |
CN103502819A (en) | Blood typing devices and methods for testing blood type | |
Hauser et al. | A microfluidic device for TEM sample preparation | |
CN102203581B (en) | Microfluidic apparatus and method for preparing cytological specimens | |
JP2006038512A (en) | Blood filtering glass fiber filter, blood filtering implement and blood analyzing element | |
KR102418963B1 (en) | Apparatus and method for microparticle analysis | |
JP4385084B2 (en) | Blood component separation method and blood component separation device | |
US20230264193A1 (en) | System for analysis | |
TWM362993U (en) | Analytical strip | |
JP2018522596A (en) | Apparatus and method for identifying the migration ability of amoeba-like migratory cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |