EP2307130A1 - Pixel device for biological analysis, cmos biosensor and corresponding fabrication methods - Google Patents
Pixel device for biological analysis, cmos biosensor and corresponding fabrication methodsInfo
- Publication number
- EP2307130A1 EP2307130A1 EP09784225A EP09784225A EP2307130A1 EP 2307130 A1 EP2307130 A1 EP 2307130A1 EP 09784225 A EP09784225 A EP 09784225A EP 09784225 A EP09784225 A EP 09784225A EP 2307130 A1 EP2307130 A1 EP 2307130A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- pixel
- photosensitive layer
- hydrogel
- photosensitive
- biological analysis
- 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.)
- Withdrawn
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
- G01N21/6454—Individual samples arranged in a regular 2D-array, e.g. multiwell plates using an integrated detector array
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
- B01J2219/00527—Sheets
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/0054—Means for coding or tagging the apparatus or the reagents
- B01J2219/00572—Chemical means
- B01J2219/00576—Chemical means fluorophore
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00585—Parallel processes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00596—Solid-phase processes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
- B01J2219/00644—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being present in discrete locations, e.g. gel pads
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00659—Two-dimensional arrays
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00709—Type of synthesis
- B01J2219/00713—Electrochemical synthesis
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00718—Type of compounds synthesised
- B01J2219/0072—Organic compounds
- B01J2219/00725—Peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T156/00—Adhesive bonding and miscellaneous chemical manufacture
- Y10T156/10—Methods of surface bonding and/or assembly therefor
Definitions
- the present invention relates to a biological analysis device of the pixel type.
- the present invention also relates to a CMOS biosensor comprising a plurality of pixel-type biological analysis devices arranged in a matrix of pixels.
- the present invention also relates to a method of manufacturing a pixel-type biological analysis device and a method of manufacturing a CMOS biosensor comprising a plurality of pixel-type biological analysis devices arranged in a pixel array.
- the present invention generally relates to improvements to the biological analysis techniques described in US Pat. No. 6,325,977 or International Application WO 2006/082336.
- RNA, DNA oligonucleotides
- the oligonucleotides can be directly grafted onto the outer surface of the biosensors.
- biosensors made from such imagers are much less suitable for more fragile structures such as proteins, which, they easily lose their functionality, by changing their three-dimensional structure or denaturation, under the effect of temperature or dehydration.
- the intrinsic properties of the biomolecules of this family mean that their study in a standardized, multiplexed and miniaturized manner (in the format of a biosensor) remains at present, still delicate. However, they would make it possible to envisage applications in the field of the diagnostic test of diseases infectious, allergies, pollution, in the detection of drugs, or in the field of defense, etc.
- An embodiment of the invention relates to a pixel-type biological analysis device comprising a photosensitive layer, a capture mixture for capturing target proteins, the capture mixture being disposed on an outer surface of the photosensitive layer and comprising a probe protein grafted to a hydrogel, means for collecting photoelectrons in the photosensitive strain, and means for reading and processing an electrical quantity supplied by the collection means, for the supply of a characteristic value of a light intensity detected by the photosensitive layer.
- the outer surface of the photosensitive layer is of sufficient size to receive all of a predetermined dose of probe protein in the form of a drop.
- the outer surface of the photosensitive layer has an area of at least 25500 ⁇ 10 -12 m 2 .
- the electron collection means comprise a plurality of island-shaped collection zones spaced from one another in the photosensitive layer.
- the collection areas are spaced apart from one another in the photosensitive layer by a distance less than a predetermined maximum distance and set according to a recombination distance of photoelectrons emitted in the photosensitive layer.
- the photosensitive layer comprises a first portion, provided with a first portion of the collection means, able to detect an incident light intensity from the capture mixture and providing a first electrical quantity and a second portion, provided with a second part of the collection means, protected against the incident light intensity from the capture mixture and providing a second electrical magnitude
- the reading and processing means comprise combined treatment means of the first and second electrical quantities, for providing a characteristic value of a light intensity detected by the first portion of the photosensitive layer.
- the method comprises steps of: preparing the outer surface of the photosensitive layer intended to receive the capture mixture, comprising a step of silanizing the outer surface; oxidation of the hydrogel and grafting of the oxidized hydrogel on the silanized outer surface; carboxylation and activation of the grafted hydrogel, and grafting of the probe protein in the activated hydrogel.
- the step of preparation of the external surface by silanization comprises the following phases: cleaning of the external surface by successive rinsing with demineralised water, acetone and absolute ethanol in a hot water bath. ultrasound; alkaline oxidation of the outer surface by oxidation under the action of ozone plasma, followed by treatment with potassium hydroxide solution for several hours at room temperature; successive immersions in demineralized water and then in absolute ethanol under ultrasound and drying under an Argon flux; silanization of the outer surface with a solution of 3-aminopropyltriethoxysilane in ethanol for several hours; successive immersions in demineralized water and then in absolute ethanol under ultrasound and drying under an Argon flux; heating the device for several hours and then maintained under an inert atmosphere.
- the oxidation and grafting step of the hydrogel comprises the following phases: dissolution of the hydrogel in demineralised water, followed by treatment with a solution of sodium periodate whose volume is adjusted to obtain a mixture whose molar ratio between sodium periodate and a predetermined and repeated monomer unit of the hydrogel is of the order of 50%, protection of the reaction mixture obtained against light and stirring for several hours at room temperature ambient, to obtain an oxidized hydrogel, grafting the oxidized hydrogel on the silanized outer surface and stirring at room temperature and protected from light for several hours, treatment of the assembly obtained with an aqueous solution of sodium cyanoborohydride for several hours in order to reduce formed Schiff bases, then rinsing with deionized water, with ethanol and drying under an Argon flux.
- the carboxylation and activation step of the grafted hydrogel comprises the following phases: treatment of the grafted hydrogel with a solution of bromoacetic acid in sodium periodate for several hours at room temperature and under Argon, purging the reaction mixture obtained and rinsing with demineralized water, ethanol and drying under an Argon flux, activation in the form of esters of the carboxylate residues formed, then rinsing with water.
- the method comprises a preliminary step of treating the outer surface of the photosensitive layer intended to receive the capture mixture using a hydrophobic coating whose roughness is chosen so that at least one solution deposited on the photosensitive layer) in the preparation of the capture mixture does not spread beyond the photosensitive layer .
- the method comprises a step of dimensioning the outer surface of the photosensitive layer intended to receive the capture mixture so that it corresponds to the size of a predetermined dose of capture mixture that can be disposed on the outer surface in the form of a drop.
- An embodiment of the invention also relates to a biosensor comprising a plurality of pixel-type biological analysis devices as described above, arranged in a matrix of pixels, and a signal processing circuit provided at the output of the device. pixel matrix.
- each pixel of the pixel matrix comprises an analog signal converter in digital signals for the digital conversion of the characteristic value of a light intensity detected by the light-sensitive layer of this pixel, and the processing circuit of the pixels.
- signals provides at the output of the pixel array comprises a digital data transmission bus connected to the matrix pixel converters.
- each pixel is separated from an adjacent pixel by a shaped insulating seal to electrically isolate the photosensitive layer from the pixel of the photosensitive layer of the adjacent pixel.
- the biosensor has a contact surface with a fluid to be analyzed, this surface comprising a first central zone in which is disposed the pixel matrix, covered by a hydrophobic coating, a first peripheral zone surrounding the central zone, comprising electronic circuits and covered by a hydrophilic coating, a second peripheral zone surrounding the first peripheral zone, comprising electrical contact pads and covered by a hydrophobic coating, and a barrier of a biocompatible resin covering the second peripheral zone, fixed on the hydrophilic coating.
- the hydrophilic coating is silicon oxide.
- the hydrophobic coating is silicon nitride.
- FIG. 1 schematically represents the structure of an embodiment of a biological analysis device according to the invention
- FIG. 2 is the electrical diagram of a biosensor comprising a plurality of devices such as that represented in FIG. 1,
- FIGS. 3a, 3b, 3c are flowcharts showing successive steps of a manufacturing method of the device of FIG. 1,
- FIG. 4a represents an embodiment of electronic means of a biological analysis device according to the invention
- FIG. 4b represents another embodiment of electronic means of a biological analysis device according to the invention.
- FIG. 5 schematically represents a differential pixel structure that can be used in a biological analysis device according to the invention
- FIGS. 6a and 6b show adaptations of a differential pixel structure including an analog / digital converter
- FIG. 6c is the electrical diagram of a biosensor comprising a plurality of differential pixels such as that represented in FIG. 6a,
- FIGS. 7 and 8 show, in sectional views, embodiments of a photosensitive layer that can be used in a biological analysis device according to the invention
- FIGS. 9a, 9b, 9c, 9d show in more detail various embodiments of the differential pixel structure
- FIG. 10 represents a structure of adjacent pixels that can be used in a biosensor according to the invention.
- FIG. 11A, 11B show schematically, respectively from a top view and a sectional view, an embodiment of a biosensor according to the invention.
- FIG. 1 represents the general structure of a biological analysis device 10 and a biosensor according to one embodiment of the invention.
- the biological analysis device 10 is produced on a semiconductor substrate 25. It comprises a layer of a capture mixture 12 comprising a certain amount of a probe protein 14 grafted to a hydrogel 16, 18 and intended to capture a certain type of target protein.
- the hydrogel comprises a hydrophilic medium 16 incorporating water molecules 18, such as for example Dextran (registered trademark), nitrocellulose, hyaluronic acid or a derivative of these saccharide polymers or polyamide. More generally, this hydrogel is a natural or synthetic polymer capable, according to its formulation, of retaining up to 99% of water.
- Each molecule of the probe protein 14 is adapted to mate with a corresponding molecule of target protein likely to be in a substance to be analyzed.
- a labeled third molecule is added and associated with the target molecule, for example a secondary antibody, a quantity of light can be emitted, for example by chemiluminescence or fluorescence.
- the biological analysis device 10 furthermore comprises a photosensitive layer 20.
- This photosensitive layer comprises an external surface 22 on which the capture mixture layer 12 is arranged.
- This photosensitive layer 20 is adapted to absorb chemiluminescence or fluorescence-emitted photons. from the capture mixture 12 and to collect photoelectrons using at least one collection area not shown in Figure 1. This is for example a photodiode. More specifically, the outer surface 22 is treated, for example by silanization, to allow the deposition of the capture mixture 12 and in particular of the hydrogel 16, 18 on the photosensitive layer 20.
- the biological analysis device 10 comprises electronic means 24 for reading and processing an electrical quantity supplied by the collection means of the photosensitive layer 20, for the supply of a characteristic value of a light intensity detected by the photosensitive layer.
- These electronic means represented here schematically, are implanted on the semiconductor substrate 25 and are configured to carry out a transfer of the charges generated in the photosensitive layer 20.
- the biological analysis device 10 makes it possible to analyze a biological substance that may comprise a certain quantity of the target protein corresponding to the grafted probe protein 14, for example using a detection protocol for chemiluminescence.
- This chemiluminescence detection protocol comprises the following steps:
- the biological substance may or may not be treated with a lysing agent in order to chemically decompose some of its components: this step renders certain molecules of the biological substance accessible to the molecules of the probe protein 14 and thus analyzable; the mixture obtained is then deposited on the capture mixture layer 12 of the biological analysis device 10 comprising the probe protein 14, and then incubated for a variable duration depending on the treated species, a "secondary" antibody solution coupled to a marker chemiluminescence, for example the HorseRadish Peroxidase or HRP enzyme, is added and the whole is incubated for a few minutes: this antibody is coupled specifically and with high affinity to the pairs formed by the probe protein molecules and target protein; the concentrations and incubation times can, again, vary and are optimized according to the intended application in a manner known per se,
- a lysing agent in order to chemically decompose some of its components: this step renders certain molecules of the biological substance accessible to the molecules of the probe protein 14 and thus analyzable; the
- washing types time, composition, concentration, number
- the washing types are variable and can be adapted in a manner known per se according to the proteins manipulated during the detection protocol
- an activation of a chemiluminescence signal and a corresponding detection by the biological analysis device is carried out by adding a solution revealing the specific substrate of the HRP enzyme under the optimal conditions of concentration and pH for a chemiluminescence signal: this revealing solution can be obtained commercially or prepared, for example in the form of luminol; it is added over the entire surface of the analysis device 10 and the signal is immediately collected by the photosensitive layer 20,
- a computer post-processing may possibly be necessary in order to amplify the signal obtained
- the comparison of the luminous intensity value obtained by the biological analysis device with a predetermined calibration curve, or determined at the same time as the biological measurement makes it possible to estimate the quantity of target protein present in the the biological sample.
- the biological analysis device 10 can also be used to perform the analysis of a biological substance using a fluorescence detection protocol.
- a fluorescence detection protocol has the same steps as the chemiluminescence detection protocol except that the secondary antibody is coupled to a fluorophore instead of a chemiluminescence label and an excitatory light is provided to the instead of a revealing solution for the activation of a fluorescence signal.
- each device 1O 1J can be arranged in a matrix of pixels of a biosensor.
- Each device 1O 1J represents a line pixel i and column j of the matrix.
- Figure 2 only the first two rows and the first three columns of a pixel array are shown, but it is easy to generalize this representation to m rows and n columns to form a complete biosensor.
- the pixels 1O 1 1 , 1O 1 2 and 1O 1 3 are connected to a first-line bus I 1 and the pixels 1O 21 , 10 2.2 and 1O 23 to a second-line bus I 2 for the transmission of the data values. 'light intensity.
- the bus lines I 1 and I 2 are connected to a horizontal shift register 26.
- the pixels 1O 1-1 and 1O 21 are connected to a first column bus C 1 , the pixels 1O 1 2 and 1O 22 to a second column bus C 2 and the pixels 10 1 3 and 10 23 to a third column bus C 3 for the transmission of the light intensity values.
- the bus columns C 1 , C 2 and C 3 are in turn connected to a vertical shift register 28.
- the shift registers 26 and 28 are connected to a signal processing circuit 30 which may include in particular a digitizing circuit of the signals. signals.
- each pixel can be provided with its own converter of analog signals into digital signals. Examples of such structures will be described later with reference to Figures 6a, 6b and 6c.
- the capture mixture 12 is arranged in two steps on the biosensor. In a first step, the hydrogel 16 is deposited on the entire surface of the biosensor. In a second step, a specific protein probe 14 solution is deposited locally in the form of drop. Several different probe proteins can thus be deposited on the same hydrogel substrate, drop by drop, each drop comprising a specific probe protein.
- a drop of protein probe of a few nanoliters may be sufficient to allow a photodetection of target protein by chemiluminescence or fluorescence, which corresponds to a drop of about 150 microns (micrometers) in diameter. Below this size, the evaporation during the deposition of the protein probe solution becomes too great and no longer allows the maintenance of protein probes, or the amount of protein probe is not sufficient to then allow a photodetection by chemiluminescence (or fluorescence).
- a drop of specific probe protein covers a plurality of pixels of a biosensor because of their small surface area, generally ten, and the electrical signals obtained by the plurality of pixels of the biosensor must be combined by posterior processing to obtain a value effectively revealing the amount of target protein in the biological samples. It follows that this post-treatment is complex and can be tainted by errors.
- the probe protein drops in order to prevent chemiluminescent or fluorescent light intensity sources from interfering between two drops of neighboring specific probe protein, the probe protein drops must be spaced a sufficient distance apart. In general, this distance can represent up to three times the size of a drop of protein probe, ie about 450 microns. A non-negligible photosensitive surface is thus lost in the biosensor.
- each pixel is advantageously dedicated to the detection of a particular target protein and has a photosensitive surface which has dimensions larger than those of a conventional imager, adapted to the size of a particular image. such a drop.
- Each pixel has, for example, a width of 150 ⁇ m or more (for a square-shaped pixel) corresponding to a surface of the order of 25500 ⁇ m 2 or more (ie 25500.10 -12 m 2 or more).
- this minimum surface corresponds to the size of a drop of probe protein below which either the evaporation during the deposition becomes too great and no longer allows the maintenance of protein probes, ie the amount of probe protein does not increase. is not enough for then allow photodetection by chemiluminescence or fluorescence. Without these constraints, a pixel could reach much smaller dimensions, such as 20 microns x 20 microns, for example.
- a drop of specific probe protein 14 is disposed on the hydrogel substrate 16 above each biological analysis device, i.e. on each pixel 10, of the biosensor matrix so as to avoid any post-treatment as mentioned above.
- the chemiluminescence or fluorescence detection protocol can be easily adapted to the biosensor shown in FIG. 2, considering each pixel of this biosensor as a biological analysis device such as the analysis device 10 previously described: in this case, each pixel comprises its own type of probe protein and the biological substance, optionally chemically decomposed, is deposited on the entire photosensitive outer surface of the biosensor.
- the comparison of the light intensity values obtained for each pixel (and therefore for each type of probe protein of each pixel) with a predetermined calibration curve, or determined at the same time as the biological measurement makes it possible to quantify a significant amount of proteins distinct from the biological sample.
- a range of protein solutions of known increasing concentrations is systematically analyzed by the biosensor, under the same conditions as a real biological test.
- the value of the pixel for each of the analyzed concentrations is then plotted on a graph as a function of the protein concentration, or stored in memory on the biosensor.
- FIGS. 3a, 3b and 3c An embodiment of certain steps of a method for manufacturing a biological analysis device according to the invention will now be described with reference to FIGS. 3a, 3b and 3c. These steps are aimed at depositing a layer of the capture mixture 12 on the outer surface 22 of the photosensitive layer 20.
- CMOS electronic assembly comprising a photosensitive layer 20 and electronic means 24 for reading and processing an electrical quantity supplied by the photosensitive layer 20 (FIG. 1).
- the outer surface 22 of the photosensitive layer optionally comprises a hydrophobic coating, for example of the Si 3 N 4 type, which will be described later.
- the outer surface 22 of the photosensitive layer 20 of the CMOS electronic assembly is cleaned by successive rinsing with demineralised water, acetone and absolute ethanol in an ultrasonic bath .
- the outer surface 22 is oxidized under the action of an ozone plasma, for example for 15 minutes, and then treated with a 2.2M solution of KOH in an aqueous mixture, for example an H 2 mixture. O / EtOH (2: 3), for example for 3 hours at room temperature.
- the CMOS electronic assembly is then immersed successively in demineralized water and then in absolute ethanol under ultrasound before being dried under an Argon flux.
- silanol residues 32 formed on the outer surface 22 are silanized with a solution of APTES in 0.4M ethanol, for example for 12 to 20 hours, at room temperature and under an inert atmosphere ( Argon).
- CMOS electronic assembly is again washed several times with demineralized water in an ultrasonic bath, rinsed with absolute ethanol and dried under a stream of Argon. it is then heated, for example at 85 ° C., for several hours, for example 5 hours, then maintained under an inert atmosphere during a step S110, to finally obtain a silanized external surface 34.
- a hydrogel 16, 18, for example Dextran (0.5 g, molecular weight approximately 100 kiloDaltons), is dissolved in 50 ml of demineralised water and then treated with 0.1 M NaIO 4 solution. whose volume is adjusted in order to obtain a mixture of molar ratio between the moles of NaIO 4 and the moles of Dextran monomer glucose of the order of 50%, during a step S112.
- Dextran is a polymer whose base unit is a derivative of glucose: it is therefore a question of adjusting the volume of NaIO 4 0.1 M solution so that there are two times more molecules of the base motif of Dextran than of molecules of NaIO 4 . This results in the opening of every second base pattern in the Dextran, as illustrated at the output of step S112 in FIG. 3b.
- reaction mixture is protected from light and stirred vigorously for several hours at room temperature, for example 20 hours, to obtain oxidized Dextran 36.
- oxidized Dextran 36 Then, in a step S114, the silanized outer surface 34 is treated with the freshly prepared oxidized Dextran solution 36.
- the reaction is stirred at room temperature and protected from light for 48 hours for example.
- the reaction mixture is then purged in a step S116 and the CMOS electronic assembly treated with an aqueous solution of 0.1M NaBH 3 CN for, for example, 3 hours in order to reduce formed Schiff bases.
- the CMOS electronic assembly is rinsed intensively with deionized water and then with ethanol, and dried under an Argon flux, so as to obtain a grafted hydrogel 38.
- the grafted hydrogel 38 is then carboxylated and activated. To do this, during a step S118, it is treated with a solution of BrCH 2 COOH 0.1 M in 2M NaOH for for example 16 hours at room temperature and under Argon. Once this oxidation is complete, the reaction mixture is purged and the sensor rinsed with deionized water and ethanol, and then dried under an Argon flow.
- the carboxylate residues formed are then activated in esters during a step S120, for example using an aqueous solution of EDC (0.2 M) / NHS (50 mM), for example for 15 minutes at room temperature. room.
- EDC 0.2 M
- NHS 50 mM
- the CMOS electronic assembly is then thoroughly rinsed with water.
- steps S100 to S120 must be performed on all the pixels of this biosensor.
- step S122 is applied differently to each pixel, for the grafting of a drop of specific probe protein on the activated hydrogel substrate above this pixel.
- APTES 3-Aminopropyltriethoxysilane or (H2N (CH2) 3Si (OEt) 3)
- NalO4 Sodium periodate
- NaBH3CN Sodium cyanoborohydride
- BrCH2COOH Bromoacetic acid
- the photosensitive layer 20 comprises, for example, a doped region within the semiconductor substrate 25 (FIG.
- the energy of an incident photon extracts electrons from atoms in the photosensitive layer 20, thereby generating a charge and therefore a current.
- the photosensitive layer 20 of the biological analysis device 10 uses an electric field at a P-N junction to cause the separation of an ion and a photoelectron and to prevent recombination and loss of signal.
- these P-N junctions have a small loss current that the photosensitive layer 20 can not distinguish from a current that would actually be generated by incident light intensity, in this case chemiluminescence or fluorescence. This loss current is also present in the dark, so it is commonly called dark current.
- darkness is to be understood as a condition in which an incident light intensity is either absent or does not cause photo-generation of charges in the photosensitive layer 20 of the biological analysis device 10. This may be due to protecting the light-sensitive layer preventing incident light from passing, or maintaining the light-sensitive layer at a certain potential, such as an initialization potential, which prevents charge accumulation.
- This dark current is a limiting factor of the performance of the biological analysis device 10 or more generally of a CMOS biosensor.
- the dark current strongly depends on the temperature and is therefore difficult to compensate. It also varies considerably with any lack of uniformity in the doping gradients. Any improvement aimed at suppressing this dark current is particularly advantageous for the biological analysis device or the aforementioned biosensor, since the luminous intensity emitted by chemiluminescence or fluorescence is generally very low. In this type it is important that the biological analysis device or the biosensor is very sensitive.
- Fig. 4a is an electrical diagram showing an embodiment of the biological analysis device 10 having improved sensitivity.
- the biological analysis device 10 comprises a photosensitive layer 20 comprising a first photosensitive portion 42 and a second photosensitive portion 44.
- the photosensitive portions 42, 44 emit signals applied to the electronic reading and processing means 24.
- the signal supplied by the photosensitive portion 42 is applied to a negative input 46 of an operational amplifier 50 and the signal supplied by the photosensitive portion 44 is applied to a positive input 48 of the amplifier 50.
- Photosensitive portions are represented by photodiodes, but any other type of photosensitive sensor may be used.
- the first photosensitive portion 42 is able to detect an incident light intensity from the capture mixture 12 (FIG 1) by chemiluminescence or fluorescence effect, while the second photosensitive portion 44 is protected against the incident light intensity from the mixture capture 12 or any other source of light intensity.
- the second photosensitive portion 44 is protected against any source of light intensity, it is still described as "photosensitive” in that it has the same material and electrical characteristics as the first photosensitive portion 42. was not protected from light, it would generate, as the first photosensitive portion 42, a charge in response to any incident light.
- each of the first and second photosensitive portions may comprise photodiodes which, in one embodiment of the invention, may be of identical shape.
- An independent opaque protection is then formed above the second photosensitive portion 44.
- the first photosensitive portion 42 will be qualified as "illuminated” photosensitive portion, while the second photosensitive portion 44 will be qualified as a photosensitive portion placed in the dark.
- the biological analysis device 10 thus produced can then be described as a "differential pixel" as opposed to pixels that do not have these two photosensitive portions, one of which is, by construction, protected from incident light.
- the protection applied to the photosensitive portion placed in the dark 44 is obtained using mechanical means, for example by depositing a metal or other opaque substance above this photosensitive portion 44, or directly on its outer surface, either as a layer spaced from the outer surface of the photosensitive portion 44 with a suitable passivation layer.
- a metal layer or other spaced from the outer surface by a passivation layer is advantageous for reducing the coupling capacity between the metal shield and the photodiode of the photosensitive portion placed in the dark 44.
- this type of protection does not does not prevent the passage of a light intensity coming from the sides.
- the opaque layer can be removed and replaced by a mechanical barrier erected between the illuminated photosensitive portion 42 and the light-sensitive portion 44 in the dark, so that the capture mixture 12 is forced to remain above the illuminated photosensitive portion 42 without overflowing on the photosensitive portion placed in the dark 44. Therefore, the output of the light-sensitive portion placed in the dark 44 represents only a dark current in the sense that no chemiluminescence reaction or fluorescence occurring in the capture mixture 12 generates light intensity that can be captured by the photosensitive portion placed in the dark 44.
- the mechanical protection may take the form of an opaque housing of the biological analysis device 10 which may have a plurality of housing portions arranged to cover a suitable surface to define the photosensitive portion placed in the dark. .
- Each photosensitive portion 42, 44 of the photosensitive layer 20 is connected to a VRT initialization voltage via respective MOS switches 52 and 54.
- MOS switches 52 and 54 are shown in FIG. 4 as NMOS transistors but could be made from PMOS transistors or any other type of suitable switch.
- An initialization switch 56 is also provided in parallel with a reference capacitance Cfb 58 also arranged in parallel between the output and the negative input 46 of the operational amplifier 50. The initialization switch 56 may be selectively controlled to discharge the operational amplifier 50.
- an adjustable capacitor Cs bearing the reference 59 is added between the ground and the positive input 48 of the operational amplifier 50.
- the photosensitive portions 42 and 44 are modeled using their respective intrinsic capacities Cpd1 bearing the reference 42a and Cpd2 bearing the reference 44a as well as their respective current sources bearing the references 42b and 44b.
- the current source is Iph + Id1, where Iph is the current generated by chemiluminescence or fluorescence effect and Id 1 the dark current, through the photodiode.
- the current source comprises only Id2, that is to say the dark current that passes through it.
- Id1 (Cs + Cpd2) Id2 (Cpd1 + Cfb) (Equation 5)
- the effect of the dark current is canceled in the output voltage, if the dark current is the same in both the photosensitive portions 42 and 44 and if the sum of the adjustable capacity 59 and the intrinsic capacitance of the dark-light-sensitive portion 44 is equal to the sum of the feedback capacitance 58 of the operational amplifier 50 and the an intrinsic capacity of the illuminated photosensitive portion 42.
- the operational amplifier 50 may be designed to have a high input common mode rejection ratio so that the signals that appear on each output are ignored. This means that any noise on the mass is ignored and does not appear on the output.
- the dark current is very sensitive to the profile and precise levels of doping of a substrate in which it flows, whereas in fact there is usually a difference in these parameters between adjacent pixels of a matrix. pixels or even between two photosensitive portions of the same pixel.
- the adjustable capacitor Cs has in particular a terminal connected to the ground while the operational amplifier 50 requires that the two terminals of the capacitor Cfd are floating. Since these structures are different, it is difficult to match them. Equation 5 shows that, even if the currents Id 1 and Id 2 do not match, the effect of the dark current can always be canceled as long as the products Id1 (Cs + Cpd2) and Id2 (Cpd1 + Cfb) are equal. .
- FIG. 4b shows an embodiment of a biological analysis device 10 which comprises means for realizing the adjustable capacitance Cs and canceling the effect of the dark current even if the currents Id1 and Id2 are not equal.
- the difference between the two figures lies in the configuration of the adjustable capacitor Cs, which is not represented here as a single capacitor 59, but as a plurality of capacitors 59a, 59b, 59c arranged in parallel between the mass and the input positive 48 of the operational amplifier 50.
- a basic positive input capacitance CsO (capacitor 59a) is connected between the ground and the positive input 48 of the operational amplifier 50.
- Optional positive input capacitors Cs1 (capacitor 59b) and Cs2 (capacitor 59c) are selectively connectable in parallel, using switches D1 and D2.
- the basic feedback capacity CsO has a lower value than Cfb.
- Only three feedback capabilities have been shown in Figure 4b for purely illustrative and non-limiting, while a larger number may also be provided.
- Cs must be less than Cfb. This is achieved by leaving the switches D1 and D2 open, so that the adjustable capacitance Cs is provided by CsO only, which is less than Cfb.
- each capacitor of the plurality of capacitors arranged in parallel has the same common value, so that the adjustable capacitor Cs actually applied to the positive input of the operational amplifier 50 can be incremented by a constant value. .
- the number of capacitors in this plurality of capacitors arranged in parallel can be increased, so as to increase the resolution or amplitude of the variations that can be envisaged.
- the capacitances of the plurality of capacitors arranged in parallel may have different values so as to increase the flexibility of the assembly.
- a preferred embodiment is to introduce a factor of two between each successive optional capability of the parallel arrangement, thereby limiting the number of switches required at constant resolution.
- the CsO capacitor is chosen to correspond to 75% Cfb and the sum Cs1 + ... + CsN is chosen to correspond to 50% Cfb, so that the adjustable capacity Cs can effectively vary between 75% Cfb and 125% Cfb.
- the inequality of the dark currents Id1 and Id2 is often not known in advance and a calibration phase may be necessary to determine it. This calibration can be performed as a post-manufacturing test applied to the biological analysis device 10 and the configuration of the switches can be stored in memory. On the contrary, the calibration can also be performed on the fly, just before the device is put into operation.
- the output voltage VOUT can be measured by any appropriate technique, for example by means of an analog / digital converter or by using the device 10 in a lighting / frequency operation, in which the voltage VOUT is compared with a reference signal causing a reset of the device 10 when it is reached. The output voltage is then given by the frequency of the initialized pulses.
- Another factor on which one can play is the geometry of the biological analysis device 10 and the geometry of a set of biological analysis devices as pixels in a biosensor.
- An example of pixel geometry is shown in Figure 5 in a top view.
- a photosensitive portion placed in the dark 60 is disposed in an upper left portion of the pixel. It is adjacent to an illuminated photosensitive portion 62 disposed in a top right portion of the pixel.
- the electronic means 64 for reading and processing this pixel are arranged in a lower portion of the pixel located under the photosensitive portions 60 and 62.
- the size of RY1 depends on the precise composition of the electronic means 64, but it is preferable that it be equal to the dimensions DY1 and LY1, so that the pixel is generally square in shape.
- They can be arranged in line and in column, for example, by simple vertical or horizontal translation of the pixel shown in FIG.
- they can also be arranged so that two successive lines of pixels are vertically inverted relative to each other. and thus present their respective electronic means 64 or their respective sensitive portions 60, 62 in correspondence.
- the pixels may also be rectangular, the electronic means 64 being located under the photosensitive portion placed in the darkness 60, itself located under the illuminated photosensitive portion 62.
- the photosensitive portions may be triangular to form a photosensitive layer 20 of generally square shape, or more complex shapes while having equal areas.
- each differential pixel may be provided with its own converter of analog signals into digital signals. This is also possible when the pixel is not differential.
- the differential pixel 10 comprises, in the upper part, a photosensitive portion placed in the dark 60 and an illuminated photosensitive portion 62. These two photosensitive portions are connected to an amplifier circuit 24 ', for example such that the electronic means 24 detailed in figure 4a or 4b.
- This amplifier circuit outputs an analog voltage which is transmitted at the input of a converter 120 of analog signals to digital signals.
- the differential pixel 10 also comprises a digital decoding block 122 whose characteristics depend on the coordinates of the differential pixel 10 in the pixel array of the biosensor in question.
- This digital decoding block 122 monitors the state of an address bus 124 so that when a signal transiting on this address bus 124 corresponds to a predetermined value stored by the digital decoding block 122 and specific to the Differential pixel 10, block 122 transmits an activation signal Sa to converter 120 which in response provides the analog output voltage of the converted amplifier circuit to a digital value at a data transmission bus 126.
- FIG. 6b A second example, illustrated in FIG. 6b and showing two adjacent pixels, represents a variant of the example of FIG. 6a.
- the photosensitive portions 60 and 62 and the amplifier circuit 24 'of each differential pixel are not shown in this figure although present.
- the digital decoding block 122 is replaced by a shift register 128 in each pixel, ie a shift register 128a in the pixel 10a and a shift register 128b in the pixel 10b.
- Each shift register 128a, 128b receives an input signal SRI (Shift Register Input) and provides an output signal SRO (Shift Register Output).
- the output signal SRO of the shift register 128a forms the input signal SRI of the shift register 128b.
- the output signal SRO of the shift register 128b forms the input signal of a next pixel shift register while the input signal SRI of the shift register 128a is formed by an output signal of a previous pixel shift register.
- the output signal of each shift register 128a, 128b is also provided to the corresponding converter 120a, 120b of the pixel 10a, 10b so as to enable this converter to transmit its digital output value to the data bus 126 at the same time. desired moment.
- a clock signal CLK (Clock) is provided to the shift registers 128a, 128b to clock transmissions of digital pixel values to the data bus 126.
- each pixel of the pixel array is set to logic "0" (inactive) to block the output of the converters 120a, 120b,
- a logic state is set to "1" (active) at the input of a first pixel 10a so as to activate the clock signal for this pixel and thereby set the signal SRO of this pixel 10a to the state "1", for the converter 120a to transmit its digital output value to the data bus 126,
- the logic state of the first pixel 10a is then set to "0" (inactive) and the clock signal is again activated to activate the pixel 10b and deactivate the pixel 10a,
- the output signal SRO of the pixel 10a remains in the state "0" and the active state on the signal SRO propagates in the matrix to every clock stroke.
- FIG. 6b has the advantage of presenting identical electronic circuits on all the pixels of the biosensor, with fewer interconnections than in FIG. 6a, so that fewer connection pads are necessary to connect the biosensor to a external controller.
- the reading of the digital values of the converters offers less flexibility since it is predetermined by the wiring of the biosensor which can not, by definition, be modified during its operation. Random access rendered 9 000765
- multiple devices 1O 1J such ⁇ ue the device 10 of Figure 6a, may be arranged in an array of pixels I a biosensor.
- Each device 10 w represents a line pixel i and column I matrix.
- Figure 6c only four rows and four columns of a pixel array are shown, but it is easy to generalize this representation to m rows and n columns to form a complete biosensor.
- All pixels are connected to the same address bus 124 which determines the sequence in which the biosensor pixels can provide their digital output values. Likewise, all the pixels are connected to the same data transmission bus 126 which retrieves these digital output values.
- the differential pixel structure described with reference to FIGS. 4 to 6 is based on the assumption that the photosensitive portions are defined strictly by their boundaries, i.e., their external dimensions define the photoelectron collection zone. In this case, the requirement of correspondence between the first and second photosensitive portions means that the surfaces of these two photosensitive portions must be substantially equal.
- FIG. 7 A first embodiment of a biological analysis device according to the principle presented in this patent is illustrated in FIG. 7.
- a P-epitaxial layer 66 is formed on a substrate P 68.
- This substrate 68 is the semiconductor substrate 25 described. higher or a doped region thereof.
- a photoelectron collection zone 70 is designed as an island-shaped N-well in the P-epitaxial layer 66.
- the collection zone 70 collects e1 e8 photoelectrons generated by incident radiation 72, including radiation. chemiluminescence or fluorescence.
- Electronic means 74 for reading and processing include a well 76 in which an NMOS transistor 78 is housed. In a conventional pixel type biological analysis device, the N 70 well would extend over the entire available length between the electronic means 74 of successive pixels. But in the device of FIG. 7, the well N 70 is designed as an island, that is to say that it is surrounded by P-epitaxial material not connected to the ground and very little doped in comparison with well P 76.
- the smaller size of the N 70 well means that the capacity of the photosensitive portion is relatively small. But the collection efficiency is not compromised. Indeed, the vast majority of photoelectrons, such as the photoelectrons e1,..., E6 shown in FIG. 7, diffuse into the P-epitaxial layer 66 and are finally collected by the well N 70. The electron e7 can statistically to move indifferently either to the well N 70, or to the well P 76. As for the electron e8, it will certainly focus on the well P 76 and will be lost.
- FIG. 8 A second embodiment of a biological analysis device according to the principle presented in US Pat. No. 6,998,659 is illustrated in FIG. 8.
- the electronic reading and processing means 80 comprise a thin layer 84 of P + material. extending over a large part of the device, so that the surface of the P-epitaxial layer 66 around the well N 70 is covered by this thin layer, with the exception of a narrow zone in the vicinity of the well N 70.
- the thin layer 84 extends from the well P 82 of the electronic means 80 and is therefore connected to the latter.
- the well P 82 is generally connected to the ground and therefore the thin layer 84 too.
- the thin layer 84 is also shallower and, at a lower potential than the N 70 well, the photoelectrons are more likely to go to the well N 70 and to be collected by it than by the thin layer 84 or well P 82.
- the electron e7 in Figure 8 is more likely to go to the well N 70, while in Figure 7 it is as likely to go to the well N 70 only to the well P 76.
- the size of a collection area such as the N 70 well is dependent on the manufacturing technology used, but can generally be as low as 1 ⁇ m x 1 ⁇ m. This minimum size is not recommended, however, because in these dimensions, the manufacturing hazards generate significant differences in size between the collection areas which generates significant differences in their characteristics. Since, in a differential pixel, the photosensitive portion placed in the dark must necessarily correspond precisely to the illuminated photosensitive portion, too small collection areas are undesirable. On the other hand, an increase in the size of the Nits increases its area and perimeter, leading to an increase in darkness and associated noise.
- a good compromise size for a collection area such as the N well is a size between 5 microns and 15 microns.
- a differential pixel 86 has a first illuminated photosensitive portion 88 and a second photosensitive portion placed in the dark 90.
- the dimensions of the first and second photosensitive portions 88 and 90 it is not necessary for the dimensions of the first and second photosensitive portions 88 and 90 to be identical, it is sufficient that the dimensions of the collection areas 92 and 94 are. It is therefore possible to envisage great flexibility in the geometry of the differential pixels.
- the recombination distance it is necessary to take into account the distance that a photoelectron can travel in a substrate before recombining. This distance is called the recombination distance. It is determined firstly by the doping level of the substrate and then by the defects of the substrate. The higher the doping, the lower the recombination distance. This recombination distance therefore imposes a maximum distance between a collection zone and the edges of the photosensitive portion in which it is located so that all the photoelectrons can be collected before being recombined.
- the limited size of the collection zones 92 and 94 therefore imposes a limited size of the photosensitive portions of the differential pixel 86.
- the recombination distance is generally between 30 and 50 ⁇ m.
- the photosensitive portion 88 can not reach these dimensions.
- a solution shown in FIG. 9b consists of increasing the size of the collection zones 92 and 94. It is thus possible to reach pixel dimensions of the order of 150 ⁇ m. However, as already indicated above, increasing the size of the collection areas 92 and 94 increases the effects of the dark current and its associated noise. To obtain a pixel of larger dimensions, it is also possible to provide a plurality of island-shaped N wells forming collection zones in the same pixel-type biological analysis device, spaced apart from one another by a distance less than the recombination distance.
- a differential pixel 86 comprises two photosensitive portions 88 and 90 each having four collection areas 92 and 94 respectively.
- the geometry of the two photosensitive portions 88 and 90 is not necessarily the same.
- the collection zones 92 are not necessarily arranged in the same way in the illuminated photosensitive portion 88 as the collection zones 94 in the photosensitive portion placed in the dark 90.
- the four collection zones 92, on the one hand, and the four collection zones 94, on the other hand have the same dimensions: they are for example 5 or 10 ⁇ m.
- the four collection zones 92 are spaced from each other by 75 ⁇ m. It is thus possible to obtain a differential pixel 86 whose illuminated photosensitive portion 88 is a square of 150 ⁇ m on a side and whose light-sensitive portion placed in the dark 90 is a rectangle of 150 ⁇ m by 20 ⁇ m.
- the differential pixel 86 comprises two photosensitive portions 88 and 90 each comprising nine collection zones 92 and 94 respectively.
- the dimensions of the collection zones 92 and 94 are for example 5 ⁇ m.
- the nine collection zones 92 are spaced apart from each other by 50 ⁇ m, for example. It is thus possible to obtain a differential pixel 86 whose illuminated photosensitive portion 88 is a square of 150 ⁇ m on a side and whose light-sensitive portion placed in the dark 90 is a rectangle of 150 ⁇ m by 20 ⁇ m.
- pixels comprising more collection areas (16 or more).
- the pixels of a biosensor according to the invention can be sized to match the size of a drop of probe protein. This advantageous dimensioning allows a simpler processing of the signals obtained.
- two drops of protein probe neighbors do not interfere by chemiluminescence or fluorescence, it is usually necessary impose a minimum distance between two neighboring drops, usually several hundred micrometers.
- the first pixel 86a comprises an illuminated photosensitive portion 88a, here of square shape, for example 150 ⁇ m side. It further comprises a photosensitive portion placed in the dark 90a of rectangular shape, 150 ⁇ m in length and 20 ⁇ m in width.
- the second pixel 86b comprises an illuminated photosensitive portion 88b of 150 ⁇ m square shape. It further comprises a photosensitive portion placed in the dark 90b of rectangular shape, 150 ⁇ m in length and 20 ⁇ m in width.
- the first pixel 86a is separated from the second pixel 86b by an insulating joint 92 shaped to electrically isolate the illuminated photosensitive portion 88a of this pixel from the illuminated photosensitive portion 88b of the second pixel 86b and to mechanically isolate the two illuminated light-sensitive portions, so that a source of chemiluminescence or fluorescence located in one of the two illuminated light-sensitive portions is not detectable by the other illuminated light-sensitive portion.
- a photoelectron e1 emitted in the illuminated photosensitive portion 88a, bordering thereof, and in particular being able to point towards the illuminated photosensitive portion 88b, would be absorbed by the insulating joint 92 in the event that it actually moves towards the photosensitive portion illuminated 88b.
- a photoelectron e2 emitted in the illuminated photosensitive portion 88b, bordering the latter, and in particular being able to point towards the illuminated photosensitive portion 88a would be absorbed by the insulating joint 92 in the event that it actually moves towards the photosensitive portion. illuminated 88a.
- the insulating joint may be a few micrometers thick, which is much smaller than the size of a pixel or a drop of protein probe.
- the presence of such an insulating joint 92 makes it possible to bring the pixels of a biosensor according to the invention considerably closer together and thus to considerably increase the number of specific probe protein drops that can be disposed on a predetermined biosensor surface.
- the molecules or photoelectrons trapped by the insulating gasket 92 are lost for detection. However, if we consider the diffusion rate of the photon-emitting molecules by chemiluminescence (or fluorescence) and the thickness of the insulating joint 92, it is estimated at about 2% maximum, in the least favorable configuration, the loss. of photoelectrons or photons generated sut ⁇ in pixel but not detected by it.
- an additional insulating seal may also be interposed between the photosensitive portions (88a, 90a, and 88b, 90b) of each pixel 86a, 86b and their respective electronic reading and processing means.
- the electronic means are not of the same nature as the photosensitive portions and may be a parasitic electron source that contribute to the effects of the dark current of the photosensitive portions.
- the electronic means have a higher potential than that of the photosensitive portions thus attracting the photoelectrons diffusing in the photosensitive portions. These phenomena are likely to deteriorate the detection of proteins by chemiluminescence or fluorescence.
- This additional insulating gasket comprises, for example, a grounded P + / P- well and an N + / N- positive potential well.
- the surface of a biosensor according to the invention is shown in FIG. 11A in plan view.
- the biosensor is arranged on a semiconductor wafer and comprises a central photosensitive zone 114 intended to be brought into contact with a biological substance, the zone 114 forming here a matrix of pixels of the type described above, and a peripheral zone 116 which surrounds the zone.
- central photosensitive 114, the zone 116 comprising various electrical circuits (eg digital readout circuits, control logic circuit, etc.) and electrical connections between these elements (wiring).
- the biosensor comprises electrical contact pads 117 intended, for example, to allow the wiring of the biosensor ("bonding pads”) to an interconnection support.
- the biosensor is subjected to a humid medium, in particular biological solutions or chemical reagents, or even acidic / basic or oxidizing / reducing solutions.
- a humid medium in particular biological solutions or chemical reagents, or even acidic / basic or oxidizing / reducing solutions.
- the zone 116 is thus likely to be in contact with the humic medium, which could damage the electrical parts that it comprises.
- An improvement aimed here is to protect the zone 116 of the iumide media intended to be in contact with the central photosensitive zone 114, and moreover to promote the deposition of the capture mixture as described above.
- This Derfectionnism includes the forecast of the following dispositions or stages, when I manufacture the biosensor:
- This hydrophilic coating is for example silicon oxide (SiO 2 ) and forms a kind of "ring", for example with a width of about 700 microns, around the central photosensitive zone 114;
- the remainder of the surface of the biosensor is covered with the low-roughness hydrophobic coating, ie the peripheral zone 118, the zones 114 and 118 being able to be treated simultaneously.
- This coating allows a clear demarcation between the peripheral zone 116 to be protected and the central photosensitive zone 114.
- the peripheral zone 116 may be covered with a thick layer of biocompatible and non-conductive resin 119, forming a kind of barrier which completely encircles the central zone, as illustrated in FIG. 11B by a sectional view.
- This resin barrier isolates from the wet environment the zone 118 receiving the contact pads 117 while also protecting the wiring zone 116, which it completely covers. Its height may be several millimeters and is thus much greater than the thickness of the biosensor, for example 30 to 100 ⁇ m.
- the protective resin 119 tends, before curing by polymerization, to automatically distribute itself around the central photosensitive zone 114, without encroaching on it or Even in this way, the hydrophobic surface treatment has a repellent effect on the resin and limits its spreading until it is polymerized.
- the hydrophobic coating on the central photosensitive zone 114 also makes it possible to ensure that solutions deposited on the zone 114 during the Biological preparation steps do not extend beyond the pixel matrix by chemically attacking elements belonging to the zone 116 and lying on the inner edge thereof.
- Such elements located on the inner edge of the zone 116 for example aluminum conductors, could indeed be imperfectly covered by the resin 119 and thus be imperfectly protected.
- the process for forming the hydrogel as described above comprises the step S104 for depositing a potassium hydroxide (KOH) solution that is capable of chemically attacking fragile elements at the edge of the zone. 116.
- KOH potassium hydroxide
- the solution is deposited on the pixel matrix in a determined dosage.
- the hydrophobic coating prevents the dose of potassium hydroxide solution from spreading beyond the central photosensitive zone 114 under the effect of surface tension forces.
- a roughness of the order of 50 nm was measured on a coating of silicon nitride (Si3N4) using conventional measurement techniques of the roughness depth.
- the silicon nitride coating was deposited using a conventional manufacturing method implemented in the applicant's production units and achieved the desired result by limiting the spread of the potassium hydroxide solution.
- Those skilled in the art may however determine other coatings or surface treatment processes for rendering hydrophobic the surface of the central photosensitive zone 114.
- the combination of these features allows optimal arrangement and adhesion of the protective resin 119 both by the chemical characteristics of the treated surfaces and by the arrangement of the biosensor components and their spacing.
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Abstract
Description
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FR0803639A FR2933198B1 (en) | 2008-06-27 | 2008-06-27 | METHOD FOR MANUFACTURING A BIOSENSOR ON A SEMICONDUCTOR SUBSTRATE |
FR0803627A FR2933197A1 (en) | 2008-06-27 | 2008-06-27 | Pixel type bioassay device useful in a biosensor, comprises a photosensitive layer, a unit for collecting photoelectrons in the photosensitive layer, a unit for reading and processing electric quantity, and a capture mixture |
PCT/FR2009/000765 WO2010004115A1 (en) | 2008-06-27 | 2009-06-24 | Pixel device for biological analysis, cmos biosensor and corresponding fabrication methods |
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EP09784226A Withdrawn EP2300147A1 (en) | 2008-06-27 | 2009-06-24 | Method for the fabrication of a biosensor on a semiconductor substrate |
EP09784225A Withdrawn EP2307130A1 (en) | 2008-06-27 | 2009-06-24 | Pixel device for biological analysis, cmos biosensor and corresponding fabrication methods |
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EP2908576B1 (en) * | 2011-04-04 | 2019-06-05 | Kyocera Corporation | Mobile communication method and radio terminal |
US20130170415A1 (en) | 2011-04-04 | 2013-07-04 | Kyocera Corporation | Mobile communication method and radio terminal |
JP6135333B2 (en) * | 2013-06-28 | 2017-05-31 | ソニー株式会社 | Power supply switching circuit, electronic device, and control method for power supply switching circuit |
JP5627808B2 (en) * | 2014-02-06 | 2014-11-19 | 京セラ株式会社 | Wireless terminal and apparatus |
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2010
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US20110140208A1 (en) | 2011-06-16 |
US20110158853A1 (en) | 2011-06-30 |
EP2300147A1 (en) | 2011-03-30 |
WO2010004115A1 (en) | 2010-01-14 |
WO2010004116A1 (en) | 2010-01-14 |
US8858885B2 (en) | 2014-10-14 |
US8440470B2 (en) | 2013-05-14 |
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