WO2019008136A1 - Transducer for the monitoring of metabolic status of a biological entity - Google Patents

Transducer for the monitoring of metabolic status of a biological entity Download PDF

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Publication number
WO2019008136A1
WO2019008136A1 PCT/EP2018/068362 EP2018068362W WO2019008136A1 WO 2019008136 A1 WO2019008136 A1 WO 2019008136A1 EP 2018068362 W EP2018068362 W EP 2018068362W WO 2019008136 A1 WO2019008136 A1 WO 2019008136A1
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WO
WIPO (PCT)
Prior art keywords
transducer
layer
electrically connected
transparent
wells
Prior art date
Application number
PCT/EP2018/068362
Other languages
French (fr)
Inventor
Jean-Emmanuel SARRY
Stéphane ARBAULT
Jérôme LAUNAY
Gabriel LEMERCIER
Fadhila SEKLI BELAIDI
Pierre Temple-Boyer
Emilie Vanhove
Original Assignee
INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique - Cnrs -
Universite Paul Sabatier Toulouse Iii
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Centre National De La Recherche Scientifique - Cnrs -, Universite Paul Sabatier Toulouse Iii filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Publication of WO2019008136A1 publication Critical patent/WO2019008136A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/27—Association of two or more measuring systems or cells, each measuring a different parameter, where the measurement results may be either used independently, the systems or cells being physically associated, or combined to produce a value for a further parameter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp

Definitions

  • Transducer for the monitoring of metabolic status of a biological entity
  • the present invention relates to the technical field of transducers and more particularly of biochemical transducers. More particularly, the present invention relates to the technical field of transducers for the monitoring of metabolic status of biological entities such as organs, cells and organelles.
  • Human mitochondria are typically 1 ⁇ spherical organelles responsible for 90% of the oxygen consumption of the cell and directly involved in mechanisms resulting in production of H 2 0 2 , a member of the ROS family participating in cell signaling and oxidative stress [2, 3].
  • the number of mitochondria contained in a single cell can vary from zero to a few thousand depending on its function.
  • innovative tools that make it possible to decipher cellular heterogeneity of cancer cells by single cell analysis are in constant and fast development [4]. Mitochondria heterogeneity also exists within a single cell [5, 6]. Therefore, devices enabling single mitochondrion analysis would make it possible to observe biological processes at a new scale, to go deeper in the understanding of the mechanisms underlying chemoresistance in leukemia and cancer, and also those involved in many diseases linked to mitochondrial dysfunction.
  • one aim of the invention is to provide biological process observation at a different scale to those currently available.
  • the present invention provides a transducer
  • the first and second insulator layers being perforated so that wells are defined therethrough from a free surface of the second insulator layer to the surface of the transparent microelectrode;
  • the transducer further comprises a layer of annular working electrodes sandwiched between the first and second insulator layers so that each well is provided with an annular working electrode;
  • the transducer further comprises:
  • This transducer makes it possible to monitor the metabolic status of a single biological entity such as a cell or an organelle. In particular, it enables to confine the metabolic biomarkers such as oxygen, hydrogen peroxide, protons, etc., in a small volume of a few picoliters and thus maximizing the transducer' s sensitivity.
  • the transducer makes it possible to both monitor and/or record the combined metabolic status of a plurality (for example 10,000) of mitochondria (multiple mitochondria analysis) or the isolated metabolic status of each of the mitochondria of the plurality thereof (single mitochondrion analysis).
  • the monitoring and/or recording can be made in real-time thus providing both quantitative and kinetic data.
  • the confinement due to the geometric structure of the wells maximize the collection ratio of the transducer since the diffusion layers relative to the annular working electrode polarization almost totally close the microwell.
  • the confining system acts as an electrochemical filter so that any chemical species entering or exiting the wells are detected by the annular working electrodes.
  • the well may have a base with a diameter of 2 to 10 ⁇ .
  • the well may have a base with a diameter of 10 to 50 ⁇ .
  • the material of the transparent microelectrode may be chosen from the group consisting of transparent conducting oxides, preferably indium tin oxide.
  • the material of the first and second insulator may be independently chosen from the group consisting of biocompatible insulators or silicon dioxide free from electrochemical activity.
  • the thickness of the first and/or second insulator layers may be 2 to 40 ⁇ , preferably the first and second insulator layers have same thickness.
  • the annular working electrodes may exhibit protrusions on the surface of the well, preferably of black platinum or another porous electro-active material. Additionally or alternatively the annular working electrodes may be ring nano-electrodes.
  • the adhesion enhancing agent may comprise antibodies trapped in conductive polymers, preferably polyp yrrole and PEDOT.
  • the anti-biofouling agent may comprise poly(L-lysine)-gra/i-poly(ethylene-glycol), or hyperhydrophobic materials.
  • the transducer may further comprise working terminals electrically connected to the annular working electrodes.
  • at least part of the working terminals may be individually electrically connected to only one annular working electrode.
  • one working terminal may be electrically connected to a plurality of annular working electrodes so that the annular working electrodes of the plurality are connected in parallel.
  • the transducer may further comprise a reference electrode, preferably made of a lower layer of platinum and an upper layer of polypyrrole or silver/silver chloride, and optionally a reference terminal electrically connected to the reference electrode.
  • a reference electrode preferably made of a lower layer of platinum and an upper layer of polypyrrole or silver/silver chloride, and optionally a reference terminal electrically connected to the reference electrode.
  • the transducer may further comprise a counter-electrode, preferably made of a lower layer of titanium and an upper layer of platinum, and optionally a counter terminal electrically connected to the counter-electrode.
  • the invention also provides a chip and cable assembly comprising the transducer as described above, further comprising flexible working cables with a first end connector electrically connected to or to be connected to the working terminals and a second end connector.
  • the transducer further comprises a reference electrode, preferably made of a lower layer of platinum and an upper layer of polypyrrole or silver/silver chloride, and optionally a reference terminal electrically connected to the reference electrode
  • the chip and cable assembly may further comprise a flexible reference cable with a first end connector electrically connected or to be connected to the reference terminal and a second end connector.
  • the transducer further comprises a counter-electrode, preferably made of a lower layer of titanium and an upper layer of platinum, and optionally a counter terminal electrically connected to the counter-electrode
  • the chip and cable assembly may further comprise a flexible counter cable with a first end connector electrically connected or to be connected to the counter terminal and a second end connector.
  • the invention also provides an optoelectrochemical system comprising a chip and cables assembly as described above, a fluidic chamber, and a platform with a receiving part for receiving the chip and cables assembly and the fluidic chamber, and optionally at least one of thermistors configured to regulate the temperature of the fluid contained in the fluidic chamber, a cover to be placed on top of the fluidic chamber and a microscope on which the platform is placed for observation, and optionally a camera coupled to the microscope and configured to capture images seen through the camera.
  • FIG. 1 is a schematic cross-section view of a well of a transducer according to the invention.
  • - figure 2 is a schematic top view of a transducer according to the invention
  • - figure 3 is an exploded view of a optoelectrochemical system according to the invention.
  • the transducer 1 comprises a transparent substrate 11, a transparent microelectrode 13, a first insulator layer 14, a second insulator layer 15, layer of annular working electrodes 16, an adhesion enhancing agent 18 and an antibiofouling agent 19.
  • transparent refers to the ability of the material to let at least a portion of light going therethrough.
  • transparent refers to the ability of the material to let at least 90 % of a wavelength of interest go through it.
  • “transparent” refers to the ability of the material to let at least 90 % of a plurality of wavelengths of interest, such as visible light, to go through it.
  • a transparent substrate 11 makes it possible to couple the transducer 1 with an optical system (described hereafter), typically to evaluate the fill rate of the wells during experimentation and to perform simultaneously electrochemical and optical measurements.
  • the transparent substrate 11 may be made of any material that is transparent such as glass, optical plastic.
  • the transparent substrate 11 may advantageously exhibit a thickness of 100 to 600 ⁇ , preferably of 120 to 170 ⁇ to improve focus through the transparent substrate.
  • the transparent microelectrode 13 is positioned on top of the transparent substrate 11, i.e. in contact with the transparent substrate 11.
  • the transparent microelectrode 13 may be chosen from the group consisting of transparent conducting oxides, e.g. indium tin oxide (ITO).
  • ITO indium tin oxide
  • the transparent microelectrode 13 may exhibit a thickness of 320 to 420 nm, preferably 345 to 395 nm, still preferably 360 to 380 nm, for example about 370 nm.
  • the first insulator layer 14 is positioned on top of the transparent microelectrode 13, i.e. in contact with the transparent microelectrode 13.
  • the first insulator layer 14 may be made of a material chosen from the group consisting of biocompatible insulators (such as polymers like PDMS, SU8, parylene, polyimide, dry-films...) or silicon dioxide (Si0 2 ) free from electrochemical activity.
  • the first insulator layer 14 is made of Si0 2 , particularly because of its higher dielectric properties, lower electrochemical activities and higher biocompatibility.
  • the first insulator layer 14 may exhibit a thickness of 2 to 40 ⁇ depending on the type of biological entity.
  • the thickness of the first insulator layer 14 is advantageously 2 to 3 ⁇ , preferably 2.25 to 2.75 ⁇ , more preferably 2.4 to 2.6 ⁇ ; for example about 2.5 ⁇ , for organelles.
  • the thickness of the first insulator layer 14 is advantageously 10 to 40 ⁇ , preferably 20 to 35 ⁇ , more preferably 24 to 31 ⁇ ; for example about 25 ⁇ or 30 ⁇ , for cells.
  • the second insulator layer 15 is positioned on top of the first insulator layer 14.
  • the second insulator layer 15 may be made of a material chosen from the group consisting of biocompatible insulators (such as polymers like PDMS, SU8, parylene, polyimide, dry- films...) or silicon dioxide (Si0 2 ) free from electrochemical activity.
  • the second insulator layer 15 is made of Si0 2 .
  • the second insulator layer 15 may exhibit a thickness of 2 to 40 ⁇ depending on the type of biological entity.
  • the thickness of the second insulator layer 15 is advantageously 2 to 3 ⁇ , preferably 2.25 to 2.75 ⁇ , more preferably 2.4 to 2.6 ⁇ ; for example about 2.5 ⁇ , for organelles.
  • the thickness of the second insulator layer 15 is advantageously 10 to 40 ⁇ , preferably 20 to 35 ⁇ , more preferably 24 to 31 ⁇ ; for example about 25 ⁇ or 30 ⁇ , for cells.
  • the first and second insulator layers 14, 15 have same thickness.
  • the first and second insulator layers 14, 15 are perforated so that wells 17 are defined therethrough from a free surface of the second insulator layer 15 to the surface of the transparent microelectrode 13.
  • the wells 17 may have a base with a diameter of 10 to 50 ⁇ , especially a diameter of 20 to 30 ⁇ (preferably of 21, 22, 23, 24, 25, 26, 24, 28, 29 ⁇ ) when the transducer 1 is intended for analysis of cells, and a diameter of 2 to 10 ⁇ (preferably of 3, 4, 5, 6, 7, 8, 9 ⁇ ) when the transducer 1 is intended for analysis of organelles. More generally, when used for the analysis of a particular biological entity, the diameter of the well 17 is preferably at least twice as big as the size of the biological entity, and at most 4 times as big as the size of the biological entity. Preferably, the diameter of the well is 3 times as big as the average size of the biological entity.
  • the number of wells 17 may range from 1 to 100,000 or even more. Preferably, the number of wells 17 ranges from 100 to 10,000.
  • the wells 17 are preferably separated from one another of a center- to-center distance of 15 to 35 times the diameter of the well (each value with 0.5 increment is contemplated as lower and upper values of ranges), preferably 17.5 to 32.5 times the diameter of the well.
  • the wells 17 may be arranged in a regular pattern such as triangular and squared patterns.
  • the wells 17 may also be arranged in concentric circles with one well 17 at the center, the radius of a bigger circle being equal to the radius of the adjacent smaller circle increased by a constant value, such as the radius of the smallest circle.
  • six wells 17 are disposed on the smallest circle and two adjacent wells 17, 17' on a same circle are separated by a distance equal to the distance separating two adjacent wells 17, 17' on the smallest circle.
  • Each well 17 may have a right cylindrical shape or a right frustoconical shape with its smaller base at the transparent microelectrode side.
  • Other shapes are also contemplated such as parallelepipeds.
  • the base of the cylindrical or frustoconical shape is preferably circular but other shapes may be contemplated such as polygons.
  • These shape definitions encompass the situation where there is a slight discrepancy in diameter and/or longitudinal axis shift between the first and second insulator layers 14, 15 on their sides at which they are opposed to each other.
  • the slight discrepancy is lower than 25 , preferably lower than 20 %, 15 %, 10 %, 5 %, of the average diameter at the opposed sides of the first and second insulator layers 14, 15.
  • the diameter of the well 17 at the side of the first insulator layer 14 opposed to the side of the second insulator 15 is smaller.
  • the slight shift in longitudinal axis is lower than 25 %, preferably lower than 20 %, 15 %, 10 %, 5 %, of the average diameter at the opposed sides of the first and second insulator layers.
  • the diameter is to be understood as the diameter of the circumscribed circle.
  • the wells 17 may be distributed into islets 170.
  • Each islet 170 may comprise 1 to 100,000 wells 17, but at least one islet has more than two wells.
  • the wells 17 are preferably separated from one another of a center-to-center distance of 15 to 35 times the diameter of the well (each value with 0.5 increment is contemplated as lower and upper values of ranges), preferably 17.5 to 32.5 times the diameter of the well.
  • the wells 17 may be arranged in a regular pattern such as triangular and squared patterns.
  • the wells 17 may also be arranged in concentric circles with one well 17 at the center, the radius of a bigger circle being equal to the radius of the adjacent smaller circle increased by the radius of the smallest circle.
  • six wells 17 are disposed on the smallest circle and two adjacent wells 17, 17' on a same circle are separated by a distance equal to the distance separating two adjacent wells 17, 17' on the smallest circle.
  • the shorter distance separating a well 17 of an islet 170 from a well 17 of an adjacent islet 170' is preferably at least 10 times longer than the greater distance separating two adjacent wells 17, 17' within an islet.
  • the islets 170 are preferably also distributed in a regular pattern, such as triangular and squared patterns.
  • the islets may also be arranged in concentric circles with one islet 170 at the center, the radius of a bigger circle being equal to the radius of the adjacent smaller circle increased by the radius of the smallest circle.
  • six islets 170 are disposed on the smallest circle and two adjacent islets on a same circle are separated by a distance equal to the distance separating two adjacent islets on the smallest circle.
  • the layer of annular working electrodes 16 is sandwiched between the first and second insulator layers 14, 15 so that each well 17 is provided with an annular working electrode 16.
  • Each of the annular working electrodes 16 is coaxial with its corresponding well 17. A slight shift of the alignment lower than 25 % of the diameter of the annular working electrode 16 may be observed. In such case, it is considered within the present disclosure that the annular working electrodes 16 are coaxial with its corresponding well 17.
  • the annular working electrodes 16 preferably comprise a working material chosen from the group consisting of platinum, gold, carbon or diamond; preferably platinum.
  • the working material preferably has a thickness of 150 to 250 nm, preferably 175 to 225 nm, more preferably 190 to 210 nm; for example about 200 nm.
  • the annular working electrodes 16 preferably further comprise an interfacial layer on both sides of the working material to ensure adhesion of the working material to the first and second insulator layers 14, 15.
  • the interfacial layer is preferably in a material chosen from the group consisting of titanium, tantalum, chromium..., preferably titanium.
  • the thickness of each interfacial layer is 15 to 25 nm, preferably 17.5 to 22.5 nm, more preferably 19 to 21 nm; for example about 20 nm.
  • Each annular working electrode 16 protrudes from the lateral surface of its corresponding well diminishing the diameter of the well at this level of at least 1 , and/or at most 10 % of the average diameter at the opposed sides of the first and second insulator layers, any digit increment being contemplated as lower and upper values of preferably ranges.
  • the protrusions 161 of the annular working electrode 16 may be made of black platinum or another porous electroactive material. Black platinum is known to enhance the catalytic activity of platinum regarding ⁇ 2 0 2 and other ROS.
  • the protrusions typically present a cauliflower- like shape which increases the surface/volume ratio.
  • the biological entities must be located only inside the wells.
  • Several washes are generally performed to evacuate excess biological entities outside the wells. However, those washes are also susceptible to cause the biological entities to escape from the wells.
  • the adhesion enhancing agent 18 prevents that.
  • the adhesion enhancing agent 18 is disposed at the bottom of the wells on the transparent microelectrode.
  • the adhesion enhancing agent 18 may comprise antibodies 181 trapped in polyp yrrole 182 or other conductive polymers such as poly(3,4- ethylenedioxythiophene (PEDOT).
  • the anti-body 181 is preferably an antibody directed against proteins of the biological entity; for example, in the case the biological entity is mitochondria, against proteins of mitochondrial outer membrane (MOM) e.g. the voltage- dependent anion channel (VDAC), which is the most abundant protein of MOM, when the transducer is intended for analyzing mitochondria.
  • MOM mitochondrial outer membrane
  • VDAC voltage- dependent anion channel
  • Biological entities have the tendency to sediment onto surfaces and form a biofilm. During a typical experiment the normalization is possible through optical observation through the rear face of the transparent substrate 11. If some biological entities are located at the inter- wells surface, they will not be taken into consideration so that normalization and measurements will be distorted.
  • the anti-biofouling agent helps to prevent the biological entity to be analyzed from adhering to the free surface of the second insulator layer 15, especially to ensure that no biological entity remains at the free surface after washing of the transducer. It also enables the use of a milder method for washing the transducer and thus reducing the risk of detachment of the biological entity present in the wells.
  • the anti-biofouling agent 19 is disposed at the free surface of the second insulator layer 15.
  • the anti-biofouling agent 19 may comprise poly(L-lysine)- ⁇ ra i-poly(ethylene- glycol) (PLL-g-PEG), (PLL-g-dextran), or hyperhydrophobic materials such as Teflon® for example.
  • the transducer 1 may further comprise working terminals 22 electrically connected to the annular working electrodes 16. At least part of the working terminals 22 may be electrically connected to only one annular working electrode 16 in a one-to-one correspondence.
  • one working terminal 22 may be electrically connected to a plurality of annular working electrodes 16 so that the annular working electrodes 16 of the plurality are connected in parallel.
  • the wells 17 corresponding to the annular working electrodes 16 connected in parallel preferably form an islet 170.
  • the transducer 1 may comprise more than one islet 170 of wells 17. Within each islet 170, all the wells 17 are electrically connected to a same working terminal 22; each working terminal 22 being electrically connected to only one islet 170.
  • a combination of individually connected annular working electrodes 16 and in parallel annular working electrodes 16 may be provided. This means that a first set of a plurality of working terminals 22 are electrically connected to only one annular working electrode 16 in a one-to-one correspondence, and a second set of a plurality of working terminals 22 are electrically connected to a plurality of annular working electrodes 16.
  • the transducer 1 may further comprise a planar reference electrode 20, preferably made of a lower layer of titanium, an intermediate layer of platinum and an upper layer of polypyrrole or silver/silver chloride.
  • the thickness of the lower layer of titanium may be 15 to 25 nm, preferably 17.5 to 22.5 nm, more preferably 19 to 21 nm; for example about 20 nm.
  • the thickness of the intermediate layer of platinum may be 100 to 140 nm, preferably 110 to 130 nm, more preferably 115 to 125 nm; for example about 120 nm.
  • the upper layer of polypyrrole may be replaced by an upper layer of silver (preferably in AgCl form), however, an upper layer of polypyrrole is preferred to avoid the dissolution of silver chloride salt during experiments and so the direct exposition of biological entities to silver which displays strong antibacterial properties.
  • the reference electrode 20 preferably has a strip shape.
  • the strip shape may form part of a regular geometrical figure such as a circle, triangle or square, preferably a circle. More preferably, the wells 17 are all included inside the geometrical figure. In case the wells 17 or the islets are arranged in concentric circles, the strip shape of the reference electrode 20 is preferably part of a circle concentric with the circles formed by the wells 17 or islets 170.
  • the transducer 1 typically comprises a reference terminal 23 electrically connected to the reference electrode 20.
  • the transducer 1 may further comprise a planar counter-electrode 21, preferably made of a lower layer of titanium and an upper layer of platinum.
  • the thickness of the lower layer of titanium may be 15 to 25 nm, preferably 17.5 to 22.5 nm, more preferably 19 to 21 nm; for example about 20 nm.
  • the thickness of the upper layer of platinum may be 100 to 140 nm, preferably 110 to 130 nm, more preferably 115 to 125 nm; for example about 120 nm.
  • the counter-electrode 21 preferably has a strip shape.
  • the strip shape may form part of a regular geometrical figure such as a circle, triangle or square, preferably a circle. More preferably, the wells are all included inside the geometrical figure.
  • wells 17 are all included inside the geometrical figure of the reference electrode 20 and the whole is included inside the geometrical figure of the counter-electrode 21.
  • the strip shape of the counter-electrode 21 is preferably part of a circle concentric with the circles formed by the wells 17 or islets 170.
  • the transducer typically 1 comprises a counter terminal 24 electrically connected to the counter-electrode 21.
  • the transducer 1 may be manufactured into a, preferably disposable, chip.
  • the chip may be coupled to flexible working cables 25 to form a chip and cables assembly.
  • the flexible working cables 25 typically comprise a first end connector electrically to be connected to the working terminals 22 and a second end connector.
  • the transducer 1 may be electrically connected to devices through the second end connector.
  • the chip and cables assembly may further comprise a flexible reference cable 26 with a first end connector electrically to be connected to the reference terminal 23 and a second end connector.
  • the chip and cables assembly may further comprise a flexible counter cable 27 with a first end connector electrically connected to the counter terminal 24 and a second end connector.
  • the cables 25, 26, 27 are intended to be connected to devices such as a potentiostat.
  • the transducer chip may have a 22 mm x 22 mm square shape. This makes it possible to couple it to existing fluidic chambers.
  • the optoelectrochemical system 10 comprises a chip and cables assembly as described above, a platform 3, and a fluidic chamber 4.
  • the platform 3, chip and cables assembly, and fluidic chamber 4 are configured to be stacked in this order.
  • the platform 3 may have different shapes, but preferably a circular shape. It comprises a receiving part 31 for receiving the chip and cables assembly and the fluidic chamber 4, typically a recess having the same peripheral form as the peripheral form of the fluidic chamber 4, preferably exhibiting a depth of the same size as the thickness of the fluidic chamber 4 so that the upper surface of the fluidic chamber 4 is flush with the upper surface of the platform 3 around the receiving part 31.
  • the platform 3 may be made of a material providing good heat conductivity such as anodized aluminum.
  • the platform 3 may further comprise a magnet 33 in the vicinity of the receiving part 31.
  • the platform 3 further exhibits a through opening 32 in the receiving part 31 optical observations through the opening 32 are possible.
  • the platform 3 is preferably a microscope platform.
  • the fluidic chamber 4 comprises a substantially central chamber 41 for receiving fluid therein and formed through the thickness of the fluidic chamber 4.
  • the size of the chamber 41 is adapted to the size of the transducer 1 and preferably bigger to ensure that all the wells 17 of the transducer chip are immerged by the fluid when the chamber 41 is filled with it.
  • the chamber 41 may exhibit a straight cylindrical shape with a base of different shape, such as a circular shape, a squared shape, a diamond shape.
  • the axis of the cylindrical shape is perpendicular to the mean plane of the fluidic chamber 4.
  • the fluidic chamber 4 is configured to be placed on top of the transducer chip so that the chamber 41 is opened at the bottom to the wells 17 of the transducer chip.
  • the fluidic chamber 4 further may comprise on its bottom face a bottom recess 42, the shape of which is the same as the peripheral shape of the chip and cables assembly, preferably the bottom recess 42 exhibits a depth of the same size as the thickness of the chip and cables assembly so that the bottom surface of the chip and cables assembly is flush with the bottom surface of the fluidic chamber 4 surrounding the bottom recess 42.
  • the chamber 41 entirely opens downwards into the bottom recess 42.
  • the fluidic chamber further 4 may comprise a sample input port 44 for the provision of a sample comprising a biological entity, preferably in the form of a suspension.
  • the sample input port 44 is typically fluidically connected to the chamber 41, preferably through a linear sample path.
  • the linear sample path preferably leads into one of the angles.
  • the fluidic chamber 4 may further comprise a drug input port 45 for the provision of drugs into the chamber.
  • the drug input port is typically fluidically connected to the chamber, preferably through a linear drug path.
  • the linear drug path preferably leads into one of the angles, most preferably into the same angle as the linear sample path.
  • the fluidic chamber 4 may further comprise an output port 46 for evacuating the fluid contained in the chamber 41.
  • the output port 46 is typically fluidically connected to the chamber 41, preferably through a linear output path.
  • the linear output path preferably leads into one of the angles different from the out into which the linear sample path opens.
  • the fluidic chamber 4 may be made of PDMS, SU8, parylene, or more preferably in polycarbonate.
  • the optoelectrochemical system 10 may further comprise thermistors 5.
  • the thermistors 5 are configured to regulate the temperature of the fluidic chamber 41. It is preferably removably embedded in the platform 3.
  • the platform 3 may comprise a thermistor receiving part 34, typically a recess having the same peripheral form as the thermistor 5, preferably exhibiting a depth of the same size as the thickness of the thermistor 5 so that the upper surface of the thermistor 5 is flush with the upper surface of the platform 3 around the thermistor receiving part 34.
  • the thermistor 5 makes long-term and temperature controlled experiments possible.
  • the optoelectrochemical system 10 may further comprise a cover 6 to be placed on top of the fluidic chamber 4.
  • the cover 6 may comprise only one glass cover 61 optionally coated with ⁇ which is particularly useful for electrophoresis.
  • the cover 6 preferably comprises a glass cover 61 and a magnetic cover 62.
  • the glass cover 61 is to be disposed on top of the fluidic chamber 4 to cover entirely the chamber 41 thereof.
  • the fluidic chamber 4 may present on its top surface a top recess 43 with the same peripheral shape as that of the glass cover 61 and a depth equal to the thickness of the glass cover 61 so that the top surface of the glass cover 61 is flush with the top surface of the fluidic chamber 4 surrounding the top recess 43.
  • the chamber 41 entirely opens upwards into the top recess 43.
  • the magnetic cover 62 is configured to be coupled to the magnets 33 of the platform 3 to maintain the whole assembly described above in place.
  • the magnetic cover 62 further presents a through opening 621 to be aligned at least partially with the through opening 32 of the platform 3.
  • the through opening 621of the magnetic cover 62 also opens entirely onto the portion of the transducer chip presenting wells 17.
  • the optoelectrochemical system 10 may further comprise a microscope on which the platform is placed for observation, and optionally a camera coupled to the microscope and configured to capture images seen through the camera.
  • the transducer may be manufactured by successive depositions of insulator and conductive materials on a transparent substrate using for example techniques derived from the microelectronics field.
  • cathode sputtering on the transparent substrate may be used.
  • PECVD plasma enhanced chemical vapor deposition
  • a working material layer between two interfacial layers may be deposited onto the first insulator layer through physical vapor deposition (PVD) and lift-off technique.
  • PVD physical vapor deposition
  • PVD and lift-off technique may be used.
  • a passivation layer is deposited through inductively-coupled plasma enhanced chemical vapor deposition (ICPECVD) and the active surface of the reference electrode and the counter- electrode are defined by lift-off.
  • this stack of layers is ion etched (reactive ion etching - RIE) through a mask to obtain the wells with integrated annular working electrodes as described in [7].
  • This final RIE steps results in the formation of the wells and the definition of the annular working electrodes at mid-depth and electrode made of ITO at the well bottoms.
  • the electrical contacts of the annular working electrodes may be opened by wet etching using buffered oxide etchant.
  • Silver may be electrochemically deposited onto the intermediate layer of platinum of the reference electrode by chrono-amperometry in aqueous solution.
  • polypyrrole may be used instead.
  • chrono-amperometry [9] may be used. The process may be automatically interrupted when the coulometric charge reaches a certain value. Thus, the thickness of the deposited layer can be precisely controlled and excessive constriction of the well can be avoided.
  • chrono-amperometry [11] may be used.
  • microcontact printing may be used [12].
  • a 370 nm layer of ITO is deposited through cathode sputtering.
  • a first 2.5 ⁇ layer of Si0 2 is deposited through PECVD on the layer of ITO.
  • a 200 nm layer of platinum is deposited between two interfacial 20 nm layer of titanium through PVD and lift-off technique.
  • a second 2.5 ⁇ layer of Si0 2 is deposited through PECVD.
  • a 20 nm layer of titanium and 120 nm layer of platinum are deposited through PVD and lift-off technique.
  • a 100 nm passivation layer of S1 3 N 4 is deposited by inductively coupled inductively-coupled plasma enhanced chemical vapor deposition and the active surface of the reference electrode and the counter-electrode are defined by lift-off. Wells are then formed through RIE. Silver is electrochemically deposited onto the intermediate layer of platinum of the reference electrode by chrono-amperometry in aqueous solution containing 0.3 M of silver nitrate previously deaerated. The final AgCl layer is formed by linear voltammetry in 1 M KC1 solution.
  • Black platinum is then electrodeposited at the annular working electrodes surface through chrono-amperometry at -0.06 V vs Ag/AgCl in aqueous solution containing 31 mM of hydrogen hexachloroplatinate and 1 mM of lead acetate.
  • the process is automatically interrupted when the coulometric charge reaches 0.05 ⁇ / ⁇ 2 .
  • pyrrole monomer is diluted in aqueous solution at 20 mM, in presence of anti-VDAC at 25 ⁇ g/ml. Electropolymerization of the pyrrol polymer on the bottom surface of the well is carried out by chronoamperometry at +2.0 V vs Pt for 250 ms.
  • polydimethylsiloxane PDMS
  • FDTS perfluorodecyltrichlorosilane
  • both the PDMS stamp and the transducer chip undergo a plasma 0 2 treatment to generate negative charges at their surfaces.
  • the PDMS stamp is incubated for two minutes in PBS containing 1 mg/mL of PLL-g-PEG.
  • the stamp is then dried under a flux of nitrogen and the PLL-g-PEG layer is transferred onto the transducer chip by applying a soft pressure for two minutes.
  • the functionalization step is validated by the contact angle measurement of a water drop deposited onto the surface, which should be equal to about 35°.

Abstract

The present invention relates to a transducer (1) comprising: a transparent substrate (11); a transparent microelectrode (13) on top of the transparent substrate (11); a first insulator layer (14) on top of the transparent microelectrode (13) and a second insulator layer (15) on top of the first insulator layer (14) with wells (17) defined therethrough; the transducer (1) further comprises: a layer of annular working electrodes (16) sandwiched between the first and second insulator layers (14, 15) so that each well (17) is provided with an annular working electrode; adhesion enhancing agent (18) at the bottom of the wells (17) on the transparent microelectrode (13); and antibiofouling agent (19) at the free surface of the second insulator layer (15). The invention also relates to a chip and cable assembly comprising this transducer and an optoelectrochemical system comprising this chip and cables assembly.

Description

Transducer for the monitoring of metabolic status of a biological entity
Technical field
The present invention relates to the technical field of transducers and more particularly of biochemical transducers. More particularly, the present invention relates to the technical field of transducers for the monitoring of metabolic status of biological entities such as organs, cells and organelles.
Prior art
Recent studies have demonstrated a direct relationship between the metabolic status of a sub-population of leukemic cells and its capacities to overcome chemotherapy [1]. In particularly, it has been shown that in acute myeloid leukemia (AML), leukemic cells which exhibit higher basal level of oxidative metabolism, reactive oxygen species (ROS) content, mitochondrial oxygen consumption rate (OCR), fatty acid oxidation dependency and oxidative phosphorylation (OXPHOS) gene signature are relatively insensitive to chemotherapy whereas glycolytic cells respond better in vivo. These resistant leukemic cells are considered to be the cause of the frequent relapses observed during treatment of AML and the cause of the patient death.
Human mitochondria are typically 1 μιη spherical organelles responsible for 90% of the oxygen consumption of the cell and directly involved in mechanisms resulting in production of H202, a member of the ROS family participating in cell signaling and oxidative stress [2, 3]. The number of mitochondria contained in a single cell can vary from zero to a few thousand depending on its function. Innovative tools that make it possible to decipher cellular heterogeneity of cancer cells by single cell analysis are in constant and fast development [4]. Mitochondria heterogeneity also exists within a single cell [5, 6]. Therefore, devices enabling single mitochondrion analysis would make it possible to observe biological processes at a new scale, to go deeper in the understanding of the mechanisms underlying chemoresistance in leukemia and cancer, and also those involved in many diseases linked to mitochondrial dysfunction.
Summary of the invention
Therefore, one aim of the invention is to provide biological process observation at a different scale to those currently available. To this aim, the present invention provides a transducer
comprising:
a transparent substrate;
a transparent microelectrode on top of the transparent substrate;
a first insulator layer on top of the transparent microelectrode;
a second insulator layer on top of the first insulator layer;
the first and second insulator layers being perforated so that wells are defined therethrough from a free surface of the second insulator layer to the surface of the transparent microelectrode;
the transducer further comprises a layer of annular working electrodes sandwiched between the first and second insulator layers so that each well is provided with an annular working electrode;
the transducer further comprises:
adhesion enhancing agent at the bottom of the wells on the transparent microelectrode ; and
antibiofouling agent at the free surface of the second insulator layer.
This transducer makes it possible to monitor the metabolic status of a single biological entity such as a cell or an organelle. In particular, it enables to confine the metabolic biomarkers such as oxygen, hydrogen peroxide, protons, etc., in a small volume of a few picoliters and thus maximizing the transducer' s sensitivity.
In particular, it is thus possible to monitor and/or record the metabolic status of mitochondria isolated from leukemic cells and more particularly for the simultaneous monitoring of oxygen (02) consumption and hydrogen peroxide (H202) production of the mitochondria. The transducer according to the invention makes it possible to both monitor and/or record the combined metabolic status of a plurality (for example 10,000) of mitochondria (multiple mitochondria analysis) or the isolated metabolic status of each of the mitochondria of the plurality thereof (single mitochondrion analysis). The monitoring and/or recording can be made in real-time thus providing both quantitative and kinetic data.
Further, since the biological entity is attached at the bottom of the well, the confinement due to the geometric structure of the wells maximize the collection ratio of the transducer since the diffusion layers relative to the annular working electrode polarization almost totally close the microwell. In other words, the confining system acts as an electrochemical filter so that any chemical species entering or exiting the wells are detected by the annular working electrodes. Other optional features are summarized as follows.
The well may have a base with a diameter of 2 to 10 μιη. Alternatively, the well may have a base with a diameter of 10 to 50 μιη.
The material of the transparent microelectrode may be chosen from the group consisting of transparent conducting oxides, preferably indium tin oxide.
The material of the first and second insulator may be independently chosen from the group consisting of biocompatible insulators or silicon dioxide free from electrochemical activity.
The thickness of the first and/or second insulator layers may be 2 to 40 μιη, preferably the first and second insulator layers have same thickness.
The annular working electrodes may exhibit protrusions on the surface of the well, preferably of black platinum or another porous electro-active material. Additionally or alternatively the annular working electrodes may be ring nano-electrodes.
The adhesion enhancing agent may comprise antibodies trapped in conductive polymers, preferably polyp yrrole and PEDOT.
The anti-biofouling agent may comprise poly(L-lysine)-gra/i-poly(ethylene-glycol),
Figure imgf000005_0001
or hyperhydrophobic materials.
The transducer may further comprise working terminals electrically connected to the annular working electrodes. In such case, at least part of the working terminals may be individually electrically connected to only one annular working electrode. Additionally or alternatively, one working terminal may be electrically connected to a plurality of annular working electrodes so that the annular working electrodes of the plurality are connected in parallel.
The transducer may further comprise a reference electrode, preferably made of a lower layer of platinum and an upper layer of polypyrrole or silver/silver chloride, and optionally a reference terminal electrically connected to the reference electrode.
The transducer may further comprise a counter-electrode, preferably made of a lower layer of titanium and an upper layer of platinum, and optionally a counter terminal electrically connected to the counter-electrode.
The invention also provides a chip and cable assembly comprising the transducer as described above, further comprising flexible working cables with a first end connector electrically connected to or to be connected to the working terminals and a second end connector. In the case where the transducer further comprises a reference electrode, preferably made of a lower layer of platinum and an upper layer of polypyrrole or silver/silver chloride, and optionally a reference terminal electrically connected to the reference electrode, the chip and cable assembly may further comprise a flexible reference cable with a first end connector electrically connected or to be connected to the reference terminal and a second end connector.
In the case where the transducer further comprises a counter-electrode, preferably made of a lower layer of titanium and an upper layer of platinum, and optionally a counter terminal electrically connected to the counter-electrode, the chip and cable assembly may further comprise a flexible counter cable with a first end connector electrically connected or to be connected to the counter terminal and a second end connector.
The invention also provides an optoelectrochemical system comprising a chip and cables assembly as described above, a fluidic chamber, and a platform with a receiving part for receiving the chip and cables assembly and the fluidic chamber, and optionally at least one of thermistors configured to regulate the temperature of the fluid contained in the fluidic chamber, a cover to be placed on top of the fluidic chamber and a microscope on which the platform is placed for observation, and optionally a camera coupled to the microscope and configured to capture images seen through the camera.
Drawings
Other aims, features and advantages of the invention will become apparent upon reading the following non-limiting description with reference to the drawings, among which:
- figure 1 is a schematic cross-section view of a well of a transducer according to the invention;
- figure 2 is a schematic top view of a transducer according to the invention; and - figure 3 is an exploded view of a optoelectrochemical system according to the invention.
Description
Transducer and transducer chip
A transducer according to the invention will be described hereafter with reference to figures 1 to 3.
The transducer 1 comprises a transparent substrate 11, a transparent microelectrode 13, a first insulator layer 14, a second insulator layer 15, layer of annular working electrodes 16, an adhesion enhancing agent 18 and an antibiofouling agent 19. The word "transparent" refers to the ability of the material to let at least a portion of light going therethrough. Preferably, "transparent" refers to the ability of the material to let at least 90 % of a wavelength of interest go through it. Most preferably, "transparent" refers to the ability of the material to let at least 90 % of a plurality of wavelengths of interest, such as visible light, to go through it.
The use of a transparent substrate 11 makes it possible to couple the transducer 1 with an optical system (described hereafter), typically to evaluate the fill rate of the wells during experimentation and to perform simultaneously electrochemical and optical measurements. The transparent substrate 11 may be made of any material that is transparent such as glass, optical plastic. The transparent substrate 11 may advantageously exhibit a thickness of 100 to 600 μιη, preferably of 120 to 170 μιη to improve focus through the transparent substrate.
The transparent microelectrode 13 is positioned on top of the transparent substrate 11, i.e. in contact with the transparent substrate 11. The transparent microelectrode 13 may be chosen from the group consisting of transparent conducting oxides, e.g. indium tin oxide (ITO). The transparent microelectrode 13 may exhibit a thickness of 320 to 420 nm, preferably 345 to 395 nm, still preferably 360 to 380 nm, for example about 370 nm.
The first insulator layer 14 is positioned on top of the transparent microelectrode 13, i.e. in contact with the transparent microelectrode 13. The first insulator layer 14 may be made of a material chosen from the group consisting of biocompatible insulators (such as polymers like PDMS, SU8, parylene, polyimide, dry-films...) or silicon dioxide (Si02) free from electrochemical activity. Preferably, the first insulator layer 14 is made of Si02, particularly because of its higher dielectric properties, lower electrochemical activities and higher biocompatibility. The first insulator layer 14 may exhibit a thickness of 2 to 40 μιη depending on the type of biological entity. For example, the thickness of the first insulator layer 14 is advantageously 2 to 3 μιη, preferably 2.25 to 2.75 μιη, more preferably 2.4 to 2.6 μιη; for example about 2.5 μιη, for organelles. The thickness of the first insulator layer 14 is advantageously 10 to 40 μιη, preferably 20 to 35 μιη, more preferably 24 to 31 μιη; for example about 25 μιη or 30 μιη, for cells.
The second insulator layer 15 is positioned on top of the first insulator layer 14. The second insulator layer 15 may be made of a material chosen from the group consisting of biocompatible insulators (such as polymers like PDMS, SU8, parylene, polyimide, dry- films...) or silicon dioxide (Si02) free from electrochemical activity. Preferably, the second insulator layer 15 is made of Si02. The second insulator layer 15 may exhibit a thickness of 2 to 40 μιη depending on the type of biological entity. For example, the thickness of the second insulator layer 15 is advantageously 2 to 3 μηι, preferably 2.25 to 2.75 μηι, more preferably 2.4 to 2.6 μιη; for example about 2.5 μιη, for organelles. The thickness of the second insulator layer 15 is advantageously 10 to 40 μιη, preferably 20 to 35 μιη, more preferably 24 to 31 μιη; for example about 25 μιη or 30 μιη, for cells.
Preferably, the first and second insulator layers 14, 15 have same thickness.
The first and second insulator layers 14, 15 are perforated so that wells 17 are defined therethrough from a free surface of the second insulator layer 15 to the surface of the transparent microelectrode 13.
The wells 17 may have a base with a diameter of 10 to 50 μιη, especially a diameter of 20 to 30 μιη (preferably of 21, 22, 23, 24, 25, 26, 24, 28, 29 μιη) when the transducer 1 is intended for analysis of cells, and a diameter of 2 to 10 μιη (preferably of 3, 4, 5, 6, 7, 8, 9 μιη) when the transducer 1 is intended for analysis of organelles. More generally, when used for the analysis of a particular biological entity, the diameter of the well 17 is preferably at least twice as big as the size of the biological entity, and at most 4 times as big as the size of the biological entity. Preferably, the diameter of the well is 3 times as big as the average size of the biological entity.
The number of wells 17 may range from 1 to 100,000 or even more. Preferably, the number of wells 17 ranges from 100 to 10,000. The wells 17 are preferably separated from one another of a center- to-center distance of 15 to 35 times the diameter of the well (each value with 0.5 increment is contemplated as lower and upper values of ranges), preferably 17.5 to 32.5 times the diameter of the well. The wells 17 may be arranged in a regular pattern such as triangular and squared patterns. The wells 17 may also be arranged in concentric circles with one well 17 at the center, the radius of a bigger circle being equal to the radius of the adjacent smaller circle increased by a constant value, such as the radius of the smallest circle. Preferably, six wells 17 are disposed on the smallest circle and two adjacent wells 17, 17' on a same circle are separated by a distance equal to the distance separating two adjacent wells 17, 17' on the smallest circle.
Each well 17 may have a right cylindrical shape or a right frustoconical shape with its smaller base at the transparent microelectrode side. Other shapes are also contemplated such as parallelepipeds. The base of the cylindrical or frustoconical shape is preferably circular but other shapes may be contemplated such as polygons.
These shape definitions encompass the situation where there is a slight discrepancy in diameter and/or longitudinal axis shift between the first and second insulator layers 14, 15 on their sides at which they are opposed to each other. The slight discrepancy is lower than 25 , preferably lower than 20 %, 15 %, 10 %, 5 %, of the average diameter at the opposed sides of the first and second insulator layers 14, 15. Usually, the diameter of the well 17 at the side of the first insulator layer 14 opposed to the side of the second insulator 15 is smaller. The slight shift in longitudinal axis is lower than 25 %, preferably lower than 20 %, 15 %, 10 %, 5 %, of the average diameter at the opposed sides of the first and second insulator layers.
In case the right cross section of the wells 17 is not circular, the diameter is to be understood as the diameter of the circumscribed circle.
The wells 17 may be distributed into islets 170. Each islet 170 may comprise 1 to 100,000 wells 17, but at least one islet has more than two wells. Within each islet 170, the wells 17 are preferably separated from one another of a center-to-center distance of 15 to 35 times the diameter of the well (each value with 0.5 increment is contemplated as lower and upper values of ranges), preferably 17.5 to 32.5 times the diameter of the well. The wells 17 may be arranged in a regular pattern such as triangular and squared patterns. The wells 17 may also be arranged in concentric circles with one well 17 at the center, the radius of a bigger circle being equal to the radius of the adjacent smaller circle increased by the radius of the smallest circle. Preferably, six wells 17 are disposed on the smallest circle and two adjacent wells 17, 17' on a same circle are separated by a distance equal to the distance separating two adjacent wells 17, 17' on the smallest circle.
The shorter distance separating a well 17 of an islet 170 from a well 17 of an adjacent islet 170' is preferably at least 10 times longer than the greater distance separating two adjacent wells 17, 17' within an islet.
The islets 170 are preferably also distributed in a regular pattern, such as triangular and squared patterns. The islets may also be arranged in concentric circles with one islet 170 at the center, the radius of a bigger circle being equal to the radius of the adjacent smaller circle increased by the radius of the smallest circle. Preferably, six islets 170 are disposed on the smallest circle and two adjacent islets on a same circle are separated by a distance equal to the distance separating two adjacent islets on the smallest circle.
The layer of annular working electrodes 16 is sandwiched between the first and second insulator layers 14, 15 so that each well 17 is provided with an annular working electrode 16. Each of the annular working electrodes 16 is coaxial with its corresponding well 17. A slight shift of the alignment lower than 25 % of the diameter of the annular working electrode 16 may be observed. In such case, it is considered within the present disclosure that the annular working electrodes 16 are coaxial with its corresponding well 17. The annular working electrodes 16 preferably comprise a working material chosen from the group consisting of platinum, gold, carbon or diamond; preferably platinum. The working material preferably has a thickness of 150 to 250 nm, preferably 175 to 225 nm, more preferably 190 to 210 nm; for example about 200 nm.
The annular working electrodes 16 preferably further comprise an interfacial layer on both sides of the working material to ensure adhesion of the working material to the first and second insulator layers 14, 15. The interfacial layer is preferably in a material chosen from the group consisting of titanium, tantalum, chromium..., preferably titanium. Preferably, the thickness of each interfacial layer is 15 to 25 nm, preferably 17.5 to 22.5 nm, more preferably 19 to 21 nm; for example about 20 nm.
Each annular working electrode 16 protrudes from the lateral surface of its corresponding well diminishing the diameter of the well at this level of at least 1 , and/or at most 10 % of the average diameter at the opposed sides of the first and second insulator layers, any digit increment being contemplated as lower and upper values of preferably ranges.
The protrusions 161 of the annular working electrode 16 may be made of black platinum or another porous electroactive material. Black platinum is known to enhance the catalytic activity of platinum regarding Η202 and other ROS. The protrusions typically present a cauliflower- like shape which increases the surface/volume ratio.
To allow the rigorous normalization of the measurements, the biological entities must be located only inside the wells. Several washes are generally performed to evacuate excess biological entities outside the wells. However, those washes are also susceptible to cause the biological entities to escape from the wells. The adhesion enhancing agent 18 prevents that.
The adhesion enhancing agent 18 is disposed at the bottom of the wells on the transparent microelectrode. The adhesion enhancing agent 18 may comprise antibodies 181 trapped in polyp yrrole 182 or other conductive polymers such as poly(3,4- ethylenedioxythiophene (PEDOT). The anti-body 181 is preferably an antibody directed against proteins of the biological entity; for example, in the case the biological entity is mitochondria, against proteins of mitochondrial outer membrane (MOM) e.g. the voltage- dependent anion channel (VDAC), which is the most abundant protein of MOM, when the transducer is intended for analyzing mitochondria.
Biological entities have the tendency to sediment onto surfaces and form a biofilm. During a typical experiment the normalization is possible through optical observation through the rear face of the transparent substrate 11. If some biological entities are located at the inter- wells surface, they will not be taken into consideration so that normalization and measurements will be distorted. The anti-biofouling agent helps to prevent the biological entity to be analyzed from adhering to the free surface of the second insulator layer 15, especially to ensure that no biological entity remains at the free surface after washing of the transducer. It also enables the use of a milder method for washing the transducer and thus reducing the risk of detachment of the biological entity present in the wells.
The anti-biofouling agent 19 is disposed at the free surface of the second insulator layer 15. The anti-biofouling agent 19 may comprise poly(L-lysine)-^ra i-poly(ethylene- glycol) (PLL-g-PEG),
Figure imgf000011_0001
(PLL-g-dextran), or hyperhydrophobic materials such as Teflon® for example.
The transducer 1 may further comprise working terminals 22 electrically connected to the annular working electrodes 16. At least part of the working terminals 22 may be electrically connected to only one annular working electrode 16 in a one-to-one correspondence.
Alternatively, one working terminal 22 may be electrically connected to a plurality of annular working electrodes 16 so that the annular working electrodes 16 of the plurality are connected in parallel. In this case, the wells 17 corresponding to the annular working electrodes 16 connected in parallel preferably form an islet 170. Thus, the transducer 1 may comprise more than one islet 170 of wells 17. Within each islet 170, all the wells 17 are electrically connected to a same working terminal 22; each working terminal 22 being electrically connected to only one islet 170.
A combination of individually connected annular working electrodes 16 and in parallel annular working electrodes 16 may be provided. This means that a first set of a plurality of working terminals 22 are electrically connected to only one annular working electrode 16 in a one-to-one correspondence, and a second set of a plurality of working terminals 22 are electrically connected to a plurality of annular working electrodes 16.
The transducer 1 may further comprise a planar reference electrode 20, preferably made of a lower layer of titanium, an intermediate layer of platinum and an upper layer of polypyrrole or silver/silver chloride. The thickness of the lower layer of titanium may be 15 to 25 nm, preferably 17.5 to 22.5 nm, more preferably 19 to 21 nm; for example about 20 nm. The thickness of the intermediate layer of platinum may be 100 to 140 nm, preferably 110 to 130 nm, more preferably 115 to 125 nm; for example about 120 nm. The upper layer of polypyrrole may be replaced by an upper layer of silver (preferably in AgCl form), however, an upper layer of polypyrrole is preferred to avoid the dissolution of silver chloride salt during experiments and so the direct exposition of biological entities to silver which displays strong antibacterial properties.
The reference electrode 20 preferably has a strip shape. The strip shape may form part of a regular geometrical figure such as a circle, triangle or square, preferably a circle. More preferably, the wells 17 are all included inside the geometrical figure. In case the wells 17 or the islets are arranged in concentric circles, the strip shape of the reference electrode 20 is preferably part of a circle concentric with the circles formed by the wells 17 or islets 170.
In such case, the transducer 1 typically comprises a reference terminal 23 electrically connected to the reference electrode 20.
The transducer 1 may further comprise a planar counter-electrode 21, preferably made of a lower layer of titanium and an upper layer of platinum. The thickness of the lower layer of titanium may be 15 to 25 nm, preferably 17.5 to 22.5 nm, more preferably 19 to 21 nm; for example about 20 nm. The thickness of the upper layer of platinum may be 100 to 140 nm, preferably 110 to 130 nm, more preferably 115 to 125 nm; for example about 120 nm.
The counter-electrode 21 preferably has a strip shape. The strip shape may form part of a regular geometrical figure such as a circle, triangle or square, preferably a circle. More preferably, the wells are all included inside the geometrical figure. Preferably, wells 17 are all included inside the geometrical figure of the reference electrode 20 and the whole is included inside the geometrical figure of the counter-electrode 21. In case the wells 17 or the islets 170 are arranged in concentric circles, the strip shape of the counter-electrode 21 is preferably part of a circle concentric with the circles formed by the wells 17 or islets 170.
In such case, the transducer typically 1 comprises a counter terminal 24 electrically connected to the counter-electrode 21.
Thus, it is possible to provide an all-integrated three-electrode electrochemical transducer.
The transducer 1 may be manufactured into a, preferably disposable, chip.
The chip may be coupled to flexible working cables 25 to form a chip and cables assembly. The flexible working cables 25 typically comprise a first end connector electrically to be connected to the working terminals 22 and a second end connector. Thus, the transducer 1 may be electrically connected to devices through the second end connector.
The chip and cables assembly may further comprise a flexible reference cable 26 with a first end connector electrically to be connected to the reference terminal 23 and a second end connector. The chip and cables assembly may further comprise a flexible counter cable 27 with a first end connector electrically connected to the counter terminal 24 and a second end connector.
The cables 25, 26, 27 are intended to be connected to devices such as a potentiostat. The transducer chip may have a 22 mm x 22 mm square shape. This makes it possible to couple it to existing fluidic chambers.
Optoelectrochemical system
An optoelectrochemical system 10 according to the invention is described hereafter with reference to figure 3.
The optoelectrochemical system 10 comprises a chip and cables assembly as described above, a platform 3, and a fluidic chamber 4. The platform 3, chip and cables assembly, and fluidic chamber 4 are configured to be stacked in this order.
The platform 3 may have different shapes, but preferably a circular shape. It comprises a receiving part 31 for receiving the chip and cables assembly and the fluidic chamber 4, typically a recess having the same peripheral form as the peripheral form of the fluidic chamber 4, preferably exhibiting a depth of the same size as the thickness of the fluidic chamber 4 so that the upper surface of the fluidic chamber 4 is flush with the upper surface of the platform 3 around the receiving part 31. The platform 3 may be made of a material providing good heat conductivity such as anodized aluminum. The platform 3 may further comprise a magnet 33 in the vicinity of the receiving part 31. The platform 3 further exhibits a through opening 32 in the receiving part 31 optical observations through the opening 32 are possible. The platform 3 is preferably a microscope platform.
The fluidic chamber 4 comprises a substantially central chamber 41 for receiving fluid therein and formed through the thickness of the fluidic chamber 4. The size of the chamber 41 is adapted to the size of the transducer 1 and preferably bigger to ensure that all the wells 17 of the transducer chip are immerged by the fluid when the chamber 41 is filled with it. The chamber 41 may exhibit a straight cylindrical shape with a base of different shape, such as a circular shape, a squared shape, a diamond shape. The axis of the cylindrical shape is perpendicular to the mean plane of the fluidic chamber 4. The fluidic chamber 4 is configured to be placed on top of the transducer chip so that the chamber 41 is opened at the bottom to the wells 17 of the transducer chip.
The fluidic chamber 4 further may comprise on its bottom face a bottom recess 42, the shape of which is the same as the peripheral shape of the chip and cables assembly, preferably the bottom recess 42 exhibits a depth of the same size as the thickness of the chip and cables assembly so that the bottom surface of the chip and cables assembly is flush with the bottom surface of the fluidic chamber 4 surrounding the bottom recess 42. The chamber 41 entirely opens downwards into the bottom recess 42.
The fluidic chamber further 4 may comprise a sample input port 44 for the provision of a sample comprising a biological entity, preferably in the form of a suspension. The sample input port 44 is typically fluidically connected to the chamber 41, preferably through a linear sample path. When the shape of the chamber 44 comprises angles, the linear sample path preferably leads into one of the angles.
The fluidic chamber 4 may further comprise a drug input port 45 for the provision of drugs into the chamber. The drug input port is typically fluidically connected to the chamber, preferably through a linear drug path. When the shape of the chamber comprises angles, the linear drug path preferably leads into one of the angles, most preferably into the same angle as the linear sample path.
The fluidic chamber 4 may further comprise an output port 46 for evacuating the fluid contained in the chamber 41. The output port 46 is typically fluidically connected to the chamber 41, preferably through a linear output path. When the shape of the chamber 44 comprises angles, the linear output path preferably leads into one of the angles different from the out into which the linear sample path opens.
The fluidic chamber 4 may be made of PDMS, SU8, parylene, or more preferably in polycarbonate.
The optoelectrochemical system 10 may further comprise thermistors 5. The thermistors 5 are configured to regulate the temperature of the fluidic chamber 41. It is preferably removably embedded in the platform 3. To this aim, the platform 3 may comprise a thermistor receiving part 34, typically a recess having the same peripheral form as the thermistor 5, preferably exhibiting a depth of the same size as the thickness of the thermistor 5 so that the upper surface of the thermistor 5 is flush with the upper surface of the platform 3 around the thermistor receiving part 34. The thermistor 5 makes long-term and temperature controlled experiments possible.
The optoelectrochemical system 10 may further comprise a cover 6 to be placed on top of the fluidic chamber 4. The cover 6 may comprise only one glass cover 61 optionally coated with ΓΓΟ which is particularly useful for electrophoresis. However, the cover 6 preferably comprises a glass cover 61 and a magnetic cover 62. The glass cover 61 is to be disposed on top of the fluidic chamber 4 to cover entirely the chamber 41 thereof. In order to receive the glass cover 61, the fluidic chamber 4 may present on its top surface a top recess 43 with the same peripheral shape as that of the glass cover 61 and a depth equal to the thickness of the glass cover 61 so that the top surface of the glass cover 61 is flush with the top surface of the fluidic chamber 4 surrounding the top recess 43. The chamber 41 entirely opens upwards into the top recess 43. The magnetic cover 62 is configured to be coupled to the magnets 33 of the platform 3 to maintain the whole assembly described above in place. The magnetic cover 62 further presents a through opening 621 to be aligned at least partially with the through opening 32 of the platform 3. The through opening 621of the magnetic cover 62 also opens entirely onto the portion of the transducer chip presenting wells 17.
The optoelectrochemical system 10 may further comprise a microscope on which the platform is placed for observation, and optionally a camera coupled to the microscope and configured to capture images seen through the camera.
Method
The transducer may be manufactured by successive depositions of insulator and conductive materials on a transparent substrate using for example techniques derived from the microelectronics field.
To form the transparent microelectrode, cathode sputtering on the transparent substrate may be used. To form the first insulator layer, plasma enhanced chemical vapor deposition (PECVD) on the transparent microelectrode may be used. To form the layer of annular working electrodes, a working material layer between two interfacial layers may be deposited onto the first insulator layer through physical vapor deposition (PVD) and lift-off technique. To form the second insulator layer, PECVD on the entire top surface may be used. To form the reference electrode and the counter-electrode, PVD and lift-off technique may be used. A passivation layer is deposited through inductively-coupled plasma enhanced chemical vapor deposition (ICPECVD) and the active surface of the reference electrode and the counter- electrode are defined by lift-off.
Then, this stack of layers is ion etched (reactive ion etching - RIE) through a mask to obtain the wells with integrated annular working electrodes as described in [7]. This final RIE steps results in the formation of the wells and the definition of the annular working electrodes at mid-depth and electrode made of ITO at the well bottoms. The electrical contacts of the annular working electrodes may be opened by wet etching using buffered oxide etchant.
Silver (notably AgCl) may be electrochemically deposited onto the intermediate layer of platinum of the reference electrode by chrono-amperometry in aqueous solution. Alternatively, polypyrrole may be used instead. To form the protrusions of the annular working electrodes, chrono-amperometry [9] may be used. The process may be automatically interrupted when the coulometric charge reaches a certain value. Thus, the thickness of the deposited layer can be precisely controlled and excessive constriction of the well can be avoided.
To functionalize the bottom of the wells, chrono-amperometry [11] may be used. To functionalize the free surface of the transducer, microcontact printing may be used [12].
Example
On a glass chamber, a 370 nm layer of ITO is deposited through cathode sputtering. A first 2.5 μιη layer of Si02 is deposited through PECVD on the layer of ITO. A 200 nm layer of platinum is deposited between two interfacial 20 nm layer of titanium through PVD and lift-off technique. A second 2.5 μιη layer of Si02 is deposited through PECVD. Then, a 20 nm layer of titanium and 120 nm layer of platinum are deposited through PVD and lift-off technique. A 100 nm passivation layer of S13N4 is deposited by inductively coupled inductively-coupled plasma enhanced chemical vapor deposition and the active surface of the reference electrode and the counter-electrode are defined by lift-off. Wells are then formed through RIE. Silver is electrochemically deposited onto the intermediate layer of platinum of the reference electrode by chrono-amperometry in aqueous solution containing 0.3 M of silver nitrate previously deaerated. The final AgCl layer is formed by linear voltammetry in 1 M KC1 solution.
Black platinum is then electrodeposited at the annular working electrodes surface through chrono-amperometry at -0.06 V vs Ag/AgCl in aqueous solution containing 31 mM of hydrogen hexachloroplatinate and 1 mM of lead acetate. The process is automatically interrupted when the coulometric charge reaches 0.05 μθ/μΐή2.
To functionalize the bottom of the wells, pyrrole monomer is diluted in aqueous solution at 20 mM, in presence of anti-VDAC at 25 μg/ml. Electropolymerization of the pyrrol polymer on the bottom surface of the well is carried out by chronoamperometry at +2.0 V vs Pt for 250 ms.
To functionalize the free surface of the transducer, polydimethylsiloxane (PDMS) is polymerized onto a flat silicon wafer previously treated with perfluorodecyltrichlorosilane (FDTS) to facilate the demolding. Then, both the PDMS stamp and the transducer chip undergo a plasma 02 treatment to generate negative charges at their surfaces. The PDMS stamp is incubated for two minutes in PBS containing 1 mg/mL of PLL-g-PEG. The stamp is then dried under a flux of nitrogen and the PLL-g-PEG layer is transferred onto the transducer chip by applying a soft pressure for two minutes. Finally, the functionalization step is validated by the contact angle measurement of a water drop deposited onto the surface, which should be equal to about 35°.

Claims

Claims
1. A transducer (1) comprising:
a transparent substrate (11);
a transparent microelectrode (13) on top of the transparent substrate (11);
a first insulator layer (14) on top of the transparent microelectrode (13);
a second insulator layer (15) on top of the first insulator layer (14);
the first and second insulator layers (14, 15) being perforated so that wells (17) are defined therethrough from a free surface of the second insulator layer (15) to the surface of the transparent microelectrode (13);
the transducer (1) further comprises a layer of annular working electrodes (16) sandwiched between the first and second insulator layers (14, 15) so that each well (17) is provided with an annular working electrode;
the transducer (1) further comprises:
adhesion enhancing agent (18) at the bottom of the wells (17) on the transparent microelectrode (13); and
antibiofouling agent (19) at the free surface of the second insulator layer (15).
2. The transducer (1) of claim 1, wherein the well has a base with a diameter of 2 to 10 μιη.
3. The transducer (1) of claim 1, wherein the well has a base with a diameter of 10 to 50 μιη.
4. The transducer (1) of any claim 1 to 3, wherein the material of the transparent microelectrode (13) is chosen from the group consisting of transparent conducting oxides, preferably indium tin oxide (ITO).
5. The transducer (1) of any claim 1 to 4, wherein the material of the first and second insulator are independently chosen from the group consisting of biocompatible insulators or silicon dioxide (Si02) free from electrochemical activity.
6. The transducer (1) of any claim 1 to 5, wherein the thickness of the first and/or second insulator layers (14, 15) is 2 to 40 μιη, preferably the first and second insulator layers (14, 15) have same thickness.
7. The transducer (1) of any claim 1 to 6, wherein the annular working electrodes (16) exhibit protrusions on the surface of the well, preferably of black platinum or another porous electro- active material.
8. The transducer (1) of any claim 1 to 7, wherein the annular working electrodes (16) are ring nano-electrodes.
9. The transducer (1) of any claim 1 to 8, wherein the adhesion enhancing agent (128) comprises antibodies (181) trapped in conductive polymers, preferably polypyrrole and PEDOT.
10. The transducer (1) of any claim 1 to 9, wherein the anti-biofouling agent (19) comprises poly(L-lysine)-gra/i-poly(ethylene-glycol), poly(L-lysine)-gra t--dextran, or hyperhydrophobic materials.
11. The transducer (1) of any claim 1 to 10, further comprising working terminals (22) electrically connected to the annular working electrodes (16).
12. The transducer (1) of claim 11, wherein at least part of the working terminals (22) are individually electrically connected to only one annular working electrode (16).
13. The transducer (1) of claim 11 or claim 12, wherein one working terminal (22) is electrically connected to a plurality of annular working electrodes (16) so that the annular working electrodes (16) of the plurality are connected in parallel.
14. The transducer (1) of any claim 1 to 13, further comprising a reference electrode (20), preferably made of a lower layer of platinum and an upper layer of polypyrrole or silver/silver chloride, and optionally a reference terminal (23) electrically connected to the reference electrode (20).
15. The transducer (1) of any claim 1 to 14, further comprising a counter-electrode (21), preferably made of a lower layer of titanium and an upper layer of platinum, and optionally a counter terminal (24) electrically connected to the counter-electrode (21).
16. A chip and cable assembly comprising the transducer (1) of claim 11 to 13, further comprising flexible working cables (25) with a first end connector electrically connected to or to be connected to the working terminals (22) and a second end connector.
17. A chip and cable assembly comprising a transducer of claim 14, further comprising a flexible reference cable (26) with a first end connector electrically connected or to be connected to the reference terminal (23) and a second end connector.
18. A chip and cable assembly comprising a transducer of claim 15, further comprising a flexible counter cable (27) with a first end connector electrically connected or to be connected to the counter terminal (24) and a second end connector.
19. An optoelectrochemical system (10) comprising a chip and cables assembly according to any claim 16 to 18, a fluidic chamber (4), and a platform (3) with a receiving part (31) for receiving the chip and cables assembly and the fluidic chamber (4).
20. The optoelectrochemical system (10) of claim 19, further comprising thermistors (5) configured to regulate the temperature of the fluid contained in the fluidic chamber 41.
21. The optoelectrochemical system (10) of claim 19 or 20, further comprising a cover (6) to be placed on top of the fluidic chamber (4).
22. The optoelectrochemical system (10) of any claim 1 to 21, further comprising a microscope on which the platform is placed for observation, and optionally a camera coupled to the microscope and configured to capture images seen through the camera.
PCT/EP2018/068362 2017-07-07 2018-07-06 Transducer for the monitoring of metabolic status of a biological entity WO2019008136A1 (en)

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