FR2933197A1 - 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 - Google Patents

Abstract

The pixel type bioassay device comprises a photosensitive layer (20), a unit for collecting photoelectrons in the photosensitive layer, a unit for reading and processing electric quantity supplied by the collecting unit for supplying a characteristic value of light intensity detected by the photosensitive layer, and a capture mixture (12) comprising a protein probe (14) grafted to a hydrogel (16, 18). The capture mixture is arranged on an external surface (22) of the photosensitive layer for capturing the predetermined target proteins. The pixel type bioassay device comprises a photosensitive layer (20), a unit for collecting photoelectrons in the photosensitive layer, a unit for reading and processing electric quantity supplied by the collecting unit for supplying a characteristic value of light intensity detected by the photosensitive layer, and a capture mixture (12) comprising a protein probe (14) grafted to a hydrogel (16, 18). The capture mixture is arranged on an external surface of the photosensitive layer for capturing the predetermined target proteins. The outer surface of the photosensitive layer has a sufficient size for receiving a predetermined dose of probe protein in drop form, and a surface of 25500x 10 -> 1> 2>m 2>. The unit for collecting electrons comprises collection areas present in islets and spaced from each other in the photosensitive layer. The collection areas are spaced at a distance less than a maximum predetermined distance, and fixed according to a distance of recombination of photoelectrons emitted in the photosensitive layer. The photosensitive layer comprises a first portion fitted with a first part of the collecting unit to detect an incident light intensity from the capture mixture and providing a first electrical quantity (Iph + Idl), and a second portion fitted with a second part of the collecting unit protected against the incident light intensity from the capture mixture and providing a second electrical quantity (Id2). The unit for reading and processing comprises a treatment unit combined with first and second electrical quantities for supplying a characteristic value of light intensity detected by the first portion of the photosensitive layer. Independent claims are included for: (1) a process for manufacturing a bioassay device; and (2) a biosensor.

Description

PIXEL-LIKE BIOLOGICAL ANALYSIS DEVICE, CMOS BIOSENSOR AND METHODS OF MAKING SAME

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. These techniques envisage the use of pixel-type biological analysis devices comprising. a photosensitive layer and means for collecting photoelectrons in the photosensitive layer, and means for reading and processing an electrical quantity supplied by the collection means for supplying a characteristic value of a detected light intensity. by the photosensitive layer. In these documents, commercial CCD or CMOS imagers are used for the study of solid organic molecular structures, that is to say structurally stable and resistant to even extreme experimental conditions, such as for example oligonucleotides (RNA, DNA ...). In these applications,

the oligonucleotides can be directly grafted onto the outer surface of the biosensors. But 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 infectious diseases, allergies, pollution, in the detection of drugs, or in the field of the defense, etc. It may thus be desirable to provide a biological analysis device capable of using more fragile structures than the oligonucleotides envisaged in the documents of the state of the art. One embodiment of the invention thus relates to a pixel-type biological analysis device comprising a photosensitive layer, means for collecting photoelectrons in the photosensitive layer, and means for reading and processing an electrical quantity supplied by the collection means for providing a characteristic value of a light intensity detected by the photosensitive layer. The analysis device comprises a capture mixture comprising a hydrogel-grafted probe protein, the capture mixture being disposed on an outer surface of the photosensitive layer for capturing predetermined target proteins. According to one embodiment, 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. According to one embodiment, the outer surface of the photosensitive layer has an area of at least 25500.10-12 m2. According to one embodiment, the electron collection means comprise a plurality of island-shaped collection zones spaced from one another in the photosensitive layer.

According to one embodiment, 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. According to one embodiment, 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, and 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.

According to one embodiment, the method comprises steps of: preparing the outer surface of the photosensitive layer for receiving the capture mixture, comprising a step of silanization of 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. According to one embodiment, 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 demineralised water and then in absolute ethanol under ultrasound and drying under argon flow silanization of the outer surface by means of 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. According to one embodiment, 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 the 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 while

several hours to reduce formed Schiff bases, followed by rinsing with deionized water, ethanol and drying under Argon flux. According to one embodiment, 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. According to one embodiment, the method comprises a preliminary step of treating the outer surface of the photosensitive layer intended to receive the capture mixture with the aid of a hydrophobic coating whose roughness is chosen so that at least one The solution deposited on the photosensitive layer during the preparation of the capture mixture does not spread beyond the photosensitive layer. According to one embodiment, the method comprises a step of dimensioning the outer surface of the photosensitive layer for receiving 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.

According to one embodiment, 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.

According to one embodiment, 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. According to one embodiment, 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. According to one embodiment, the hydrophilic coating is silicon oxide. According to one embodiment, the hydrophobic coating is silicon nitride. Embodiments of the invention will be described in greater detail in the description which follows, given in a nonlimiting manner in relation to the appended figures in which: FIG. 1 schematically represents the structure of an embodiment of a Biological analysis device according to the invention, - Figure 2 is the electrical diagram of a biosensor 5 having a plurality of devices such as that shown in Figure 1; - Figures 3a, 3b, 3c are flow diagrams 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 embodiment of electronic means of a biological analysis device 15 according to the invention; - FIG. 5 schematically represents a differential pixel structure that can be used in a device. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 6a and 6b show adaptations of a differential pixel structure including an analog / digital converter; FIG. 6c is an electrical diagram of a biosensor comprising a plurality of pixels; As shown in FIG. 6a, FIGS. 7 and 8 show sectional views of embodiments of a photosensitive layer that can be used in a biological analysis device according to the invention, FIGS. 9b, 9c, 9d show in more detail various embodiments of the differential pixel structure, - Figure 10 shows an adjacent pixel structure usable in a biosensor according to the invention, and

- Figures 11A, 11B show schematically, respectively from a top view and a sectional view, an embodiment of a biosensor according to the invention. In the following description, various specific details are given for a better understanding of embodiments of the invention. The described embodiments may be implemented by omitting one or more of these details, or by using other methods, equipment, materials, etc.

In some cases, materials or operations that are well known in themselves are not described in detail to avoid obscuring certain aspects of the described embodiments. Referring to an "embodiment" in the description means that a particular feature or structure described in connection with this embodiment is included in this embodiment. Thus, the use of the terms "in one embodiment" or "in one embodiment" at various points of the description does not necessarily refer to the same embodiment. On the other hand, the particular features relating to each embodiment may be suitably combined to form one or more other embodiments. Finally, the analyzes, comments, deductions and conclusions set out in the following with respect to the prior art are deemed to be an integral part of the present 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. In case of pairing of the two molecules, when 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 further comprises a photosensitive layer 20. This photosensitive layer comprises an outer 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 zone not shown in FIG. 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.

Finally, 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 detected light intensity. 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 be treated or not with a lysing agent in order to chemically break down some of its components: this step makes certain molecules of the biological substance accessible to the molecules of the probe protein 14 and therefore analysable; the mixture obtained is then deposited on the capture mixture layer 12 of the biological analysis device 10 comprising the probe protein 14, then incubated for a variable duration depending on the treated species, - a "secondary" antibody solution coupled to a a chemiluminescence marker, 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 incubation concentrations and times can, again, vary and are optimized according to the intended application in a manner known per se, - several washes are then carried out in order to eliminate any trace of secondary antibody adsorbed in a non-specific manner to the surface of the biological analysis device 10 and can generate a false

detection; the washing types (time, composition, concentration, number) are variable and can be adapted in a manner known per se according to the proteins manipulated during the detection protocol, - activation of a chemiluminescence signal and corresponding detection by the device Biological analysis is carried out by adding a solution revealing the specific substrate of the HRP enzyme under optimal conditions of concentration and pH for a chemiluminescence signal: this revealing solution can be obtained commercially or prepared by example in the form of luminol; it is added over the entire surface of the biological 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, - finally, the comparison of the luminous intensity value obtained by the biological analysis device 10 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 sample of biological substance . The biological analysis device 10 can also be used to perform the analysis of a biological substance using a fluorescence detection protocol. Such 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.

As shown in FIG. 2, several devices 10i, j, such as the device 10 of FIG. 1, can be arranged in a matrix of pixels of a biosensor. Each device 10i, i represents a line pixel i and column j of the matrix. In 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 101,1, 101,2 and 101,3 are connected to a first line bus 11 and the pixels 102,1, 102,2 and 102,3 to a bus of second line 12 for the transmission of the values of light intensity. The bus lines 11 and 12 are connected to a horizontal shift register 26. The pixels 101,1 and 102,1 are connected to a first-column bus c1r the pixels 101,2 and 102,2 to a bus second column c2 and pixels 101,3 and 102,3 to a third column bus c3 for the transmission of the light intensity values. The bus columns c1, c2 and c3 are connected to a vertical shift register 28. The shift registers 26 and 28 are connected to a signal processing circuit 30 may include in particular a digitizing circuit signals. To produce a biosensor arranged in a matrix of pixels, structures other than that which has just been described with reference to FIG. 2 are conceivable. In particular, 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. Typically, the capture mixture 12 is arranged in two stages on the biosensor. Initially

time, 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. It has been experimented that a drop of probe protein of a few nanoliters may be sufficient to allow photodetection of target protein by chemiluminescence or fluorescence, which corresponds to a drop of about 150 microns (microns) 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). In the prior art, given the size of the pixels of a conventional biosensor, much less than 150 μm, a drop of specific probe protein covers a plurality of pixels of a biosensor because of their small surface area, generally a ten, and the electrical signals obtained by the plurality of pixels of the biosensor must be combined by a posteriori treatment to obtain a value effectively revealing the amount of target protein in the biological sample. It follows that this post-treatment is complex and can be tainted by errors.

In addition, 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 protein probe drop, about 450 μm. A non-negligible photosensitive surface is thus lost in the biosensor. According to a preferred embodiment of the invention, 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 μm or more (for a square-shaped pixel) corresponding to a surface of the order of 25500 pm 2 or more (ie 25500 × 10 -12 m2 or more). As indicated above, 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 sufficient to subsequently allow photodetection by chemiluminescence or fluorescence. Without these constraints, a pixel could reach much smaller dimensions, such as 20 pm x 20 pm for example. Thus, a drop of specific probe protein 14 is placed on the hydrogel substrate 16 above each biological analysis device, that is to say on each pixel 10i, j 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 has its own type of probe protein and the possible biological substance

chemically decomposed is deposited on the entire external photosensitive surface of the biosensor. At the end of the protocol, 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.

More precisely, for the calibration of the proteins to be quantified, 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. During the actual biological test, it is then sufficient to compare the obtained pixel value with the graph previously plotted, or with the value previously stored, to obtain an estimate of the concentration of the target protein in the biological sample tested. Deposition of the capture mixture 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.

As shown in FIG. 3a, during a first step 5100, there is provided a 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 layer

photosensitive optionally comprises a hydrophobic coating, for example of Si3N4 type which will be described later. Then, during a step 5102, the outer surface 22 of the photosensitive layer 20 of the CMOS electronic assembly is cleaned by successive rinsing with demineralized water, acetone and absolute ethanol in an ultrasonic bath . During a step S104, 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 O / EtOH (2: 3), for example for 3 hours at room temperature.

During a step S106, 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. During a step S108, 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). Then the 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, and then maintained under an inert atmosphere during a step S110, to finally obtain a silanized external surface 34. As represented in FIG. 3b, a hydrogel 16, 18, for example Dextran (0.5 g, molecular weight approximately 100 kiloDalton), is dissolved in 50 ml of demineralised water, and then treated with 0.1M NaIO 4 solution

whose volume is adjusted 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%, in a step S112. Note in fact in Figure 3b that Dextran is a polymer whose base unit is a derivative of glucose: it is therefore to adjust the volume of 0.1M NaIO4 solution so that we have twice more molecules of the basic motif of Dextran than of molecules of NaIO4. This results in the opening of every second base pattern in the Dextran, as illustrated at the output of step S112 in FIG. 3b. The reaction mixture is protected from light and stirred vigorously for several hours at room temperature, for example 20 hours, so as to obtain oxidized Dextran 36. Then, during a step 5114, the silanized external surface 34 is treated by the oxidized dextran solution 36 freshly prepared. 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 0.1M NaBH3CN solution for, for example, 3 hours in order to reduce formed Schiff bases. After aspiration of the suspension, 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. As shown in FIG. Grafted hydrogel 38 is then carboxylated and activated. For this purpose, during a step S118, it is treated with a solution of 0.1M BrCH2COOH 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 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. The CMOS electronic assembly is then thoroughly rinsed with water. Finally, in a last step S122, an appropriate solution of the protein of interest (probe protein) is grafted into the activated hydrogel. In order to produce a complete biosensor such as that shown in FIG. 2, steps S100 to 5120 must be performed on all of the pixels of this biosensor. Finally, the last step 5122 is applied differently to each pixel, for grafting a drop of specific probe protein onto the activated hydrogel substrate above that pixel. The usual abbreviations used above will be noted. (CH3) 2O: Acetone C2H5OH or EtOH: Ethanol O3: Ozone KOH: Potassium hydroxide NaOH: Sodium hydroxide H2O: Water Ar: Argon "APTES" 3-Aminopropyltriethoxysilane or (H2N (CH2) 3Si (OEt) 3) NaIO4: Sodium periodate NaBH3CN: Sodium cyanoborohydride BrCH2COOH: Bromoacetic acid EDC 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride NHS: N-Hydroxysuccinimide.

We will now describe improvements of a biological analysis device and a biosensor according to the invention or a method of manufacturing such a biosensor. These improvements can be implemented separately or in combination, at the choice of the person skilled in the art depending on the intended result and the economic or technical constraints that may need to be taken into account for the production of biosensors according to the invention.

Prediction of a Differential Pixel Structure In the biological analysis device 10 described above, the photosensitive layer 20 comprises for example a doped region within the semiconductor substrate 25 (FIG 1). 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. However, 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 by chemiluminescence or fluorescence. This loss current is also present in the dark, so it is commonly called dark current. The term 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 20 at a certain

potential, for example an initialization potential, which prevents the accumulation of charges. 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 of application, 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. In this exemplary embodiment, the signal provided by the photosensitive portion 42 is applied to a negative input 46 of an operational amplifier 50 and the signal provided by the photosensitive portion 44 is applied to a positive input 48 of the amplifier 50.

The 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.

chemiluminescence or fluorescence, while the second photosensitive portion 44 is protected against the incident light intensity from the capture mixture 12 or any other source of light intensity. Although the second photosensitive portion 44 is protected against any source of light intensity, it is still qualified photosensitive in that it has the same material and electrical characteristics as the first photosensitive portion 42. Thus, if it was not protected from light, it would generate, as the first photosensitive portion 42, a charge in response to any incident light. For example, 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. For the sake of clarity in the following description, the first photosensitive portion 42 will be qualified illuminated photosensitive portion, while the second photosensitive portion 44 will be described as 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 by means of a

appropriate 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. On the other hand, this type of protection does not does not prevent the passage of a light intensity coming from the sides. Alternatively, 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 taking place in the capture mixture 12 generates light intensity that can be sensed by the photosensitive portion placed in the dark 44. Alternatively, the mechanical protection can take the form of an opaque housing of the device biological analysis 10 which may comprise several housing portions arranged so as to cover a suitable surface for defining the darkened photosensitive portion 44. Each photosensitive portion 42, 44 of the photosensitive layer 20 is connected to a VRT initialization voltage via respective MOS switches 52 and 54. These MOS switches 52 and 54 are shown in FIG. 4 as NMOS transistors but could be made from PMOS transistors or any other suitable type of switch. An initialization switch 56 is also

provided in parallel with a feedback capacitance Cfb bearing the reference 58 also arranged in parallel between the output and the negative input 46 of the operational amplifier 50. The initialization switch 56 can be selectively controlled to discharge the amplifier It is important to ensure that the illuminated light-sensitive portion 42 and the dark-light-sensitive portion 44 are as similar as possible so as to generate the most similar dark currents possible. The constraints imposing this, as well as the different options for their arrangement will be detailed below. For this, an adjustable capacitor Cs bearing the reference 59 is added between the ground and the positive input 48 of the operational amplifier 50. In FIG. 4a, the photosensitive portions 42 and 44 are modeled using their capacities. Cpd1 with the reference 42a and 20 Cpd2 with the reference 44a and their respective current sources bearing the references 42b and 44b. For the illuminated photosensitive portion 42, the current source is Iph + Idl, where Iph is the chemiluminescent or fluorescent effect current and Id1 is the dark current, through the photodiode. For the photosensitive portion placed in the dark 44, the current source comprises only Id2, that is to say the dark current that passes through it. At the positive input 48 of the operational amplifier 50, the current and voltage values imply the following equation: (Cpd2 + Cs) (6VINP / 5t) + Id2 = 0 (Equation 1) 24

At the negative input 46 of the operational amplifier 50, the current and voltage values imply the following equation: Cpol 5VINN / 6t + Cfb (5VOUT / 6t-5VINN / 6t) - (Iph + Idl) = 0 (equation 2)

By rearranging the previous equation, we obtain the following equation: (Iph + Id1) + (Cpol + Cfb) 5VINN / 6t - Cfb 5VOUT / 6t = 0 (equation 3)

At the output of the operational amplifier 50, there is, in the general case, the following relation:

5VOUT / 5t = (IPH (Cs + Cpd2) + Idl (Cs + Cpd2) - Id2 (Cpol + Cfb)) / (Cfb (Cs + Cpd2)) (Equation 4)

From equation 4 it follows that in the general case the effect of the dark current on the output voltage can be suppressed if the following relation is satisfied:

Idl (Cs + Cpd2) = Id2 (Cpol + Cfb) (Equation 5)

In particular, equation 5 can be satisfied, and the effect of the dark current canceled, if:

Idl = Id2 and (Cs + Cpd2) = (Cpol + Cfb) (Equation 6)

In other words, it is deduced from equations 5 and 6 that the effect of the dark current is canceled when the product enters the dark current in the

illuminated photosensitive material 42 and the sum of the adjustable capacitance 59 and the intrinsic capacitance of the light-sensitive portion 44 placed in darkness is equal to the product between the dark current in the light-sensitive portion 44 and the sum of the feedback capacitance 58 of the operational amplifier 50 and the intrinsic capacitance of the illuminated photosensitive portion 42. In particular, the effect of the dark current is canceled in the output voltage, if the dark current is the even in the two photosensitive portions 42 and 44 and if the sum of the adjustable capacity 59 and the intrinsic capacity of the dark-light-sensitive portion 44 is equal to the sum of the feedback capacitance 58 of the operational amplifier 50 and the intrinsic capacity of the illuminated photosensitive portion 42. In addition, the operational amplifier 50 may be designed to have a High input common mode rejection 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. On this basis, there are several options for designing a biological analysis device 10 corresponding to that of FIG. 4. The first is to impose identical photosensitive portions 42 and 44 and to adjust the adjustable capacity 59 so as to verify the equality (Cs + Cpd2) = (Cpdl + Cfb). Assuming that Cpd1 = Cpd2, this amounts to adjusting the adjustable capacity 59 Cs to the feedback capability 58 Cfb. Several techniques for matching photosensitive portions will be discussed below. It can be noted that the dark current is very sensitive to the profile and precise levels of doping.

of a substrate in which it circulates, when in fact there is usually a difference on these parameters between neighboring pixels of a matrix of pixels or even between two photosensitive portions of the same pixel.

Another complication comes from the structural differences between the adjustable capacity Cs, on the one hand, and the feedback capability Cfb, on the other hand. 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 Id1 and Id2 do not match, the effect of the dark current can still be canceled as long as the products Id1 (Cs + Cpd2) and Id2 (Cpol + 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. In this figure, the elements identical to those shown in Figure 4a have the same references.

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 connectable selectively in parallel,

using switches Dl and D2. The basic feedback capacity CsO has a lower value than Cfb. Of course, only three feedback capabilities have been shown in Figure 4b for purely illustrative and non-limiting, while a larger number may also be provided. Thus, in one embodiment, the values of Cs0, Cs1 and Cs2 are chosen so that Cs1 = Cs2 and CsO + Cs1 = Cfb.

Therefore, if the dark current Idl is slightly higher than the dark current Id2, then to maintain the equality provided by Equation 5, 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. If the two dark currents Id1 and Id2 are identical, then the switch D1 is opened and the switch D2 is closed. As a result, the resulting adjustable capacitor Cs across the operational amplifier 50 is Cs = Cs0 + Cs1 = Cfb. If the dark current Idl is slightly lower than the dark current Id2, then to maintain the equality provided by Equation 5, Cs must be greater than Cfb. This is achieved by closing the switches D1 and D2 so that the adjustable capacitance Cs is provided by Cs0 + Cs1 + Cs2. This method can also be applied in the case where, even when the dark currents of the two photosensitive portions 42 and 44 are identical, it is desired to tolerate a small difference between the capacitance values Cfb and CsO. In one embodiment, each of the plurality of capacitors arranged in parallel has the same common value, so that the adjustable capacitance

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. Alternatively, 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. By thus providing a basic positive input capacitance CsO and capacitances of 1, 2, 4, 8 and 16 times an increment Cs1, an effective adjustable capacitor Cs can be obtained whose value can vary between the basic capacitance CsO and CsO + 31 CsI with an increment of Cs1. More generally, when N optional capacitors are used in parallel, to enable the electronic means 24 to adapt to manufacturing variations, generally of the order of +/- the capacitance CsO is chosen from in such a way as to correspond to 75% Cfb and the sum Cs1 + ... + CsN is chosen so as 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 Idl 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. As has been seen previously, it is preferable for the suppression of the effect of the dark current that the first and second photosensitive portions 42 and 44 correspond as best as possible. But in reality, even if identical photosensitive portions are manufactured, the dark current may vary slightly from one to the other due to factors involved in their manufacture or operation. In practice, the respective dark currents of the two photosensitive portions will often be different. Indeed, strict equality can hardly be achieved. A realistic goal is therefore to try to minimize this difference.

The minimization of this difference is the object of the device shown in FIG. 4b. However, the resolution of this system is limited by the number and the value of the capacitances provided in the capacitive assembly disposed in parallel with the positive input of the operational amplifier 50. Another factor that can be played is the geometry of the capacitor. biological analysis device 10 and the geometry of a set of biological analysis devices as pixels in a biosensor. A

An example of pixel geometry is shown in FIG. 5 by a view from above. In this figure, 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 part of the pixel located under the photosensitive portions 60 and 62. For a good match of the photodiodes, the dimensions of the photosensitive portions 60 and 62 may be equal (DX1 = LX1 and DY1 = LY1) in which case RX1 = DX1 + LX1. In addition, these photosensitive portions may be square (DX1 = DY1). 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.

On the basis of this pixel configuration, several pixels can then be arranged in different ways online and / or in a column to form a complete biosensor. They can be arranged in line and in column, for example, by simple vertical or horizontal translation of the pixel shown in FIG. 6. Alternatively, 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 respective photosensitive portions 60, 62 in correspondence. Moreover, instead of being square, the pixels can also be rectangular, the electronic means 64 being located under the portion

in the dark 60, itself located under the illuminated photosensitive portion 62. Furthermore, also the photosensitive portions may be triangular to form a photosensitive layer 20 of generally square shape, or more complex shapes while having surfaces equal. Several variants can thus be imagined by those skilled in the art and will not be detailed further.

To produce a biosensor arranged in a matrix of differential pixels, the structure that has been described with reference to FIG. 2 is conceivable. But others are also possible. In particular, 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. A first example is illustrated in Figure 6a. In this figure, 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, the block 122 transmits an activation signal Sa to the converter 120 which in response provides the analog output voltage of the converted amplifier circuit to a digital value to a data transmission bus 126. A second example, illustrated in FIG. 6b and showing two adjacent pixels, represents a variant of the example of FIG. 6a. To simplify the illustration, the photosensitive portions 60 and 62 and the amplifier circuit 24 'of each differential pixel are not shown in this figure although present. In this variant, 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 (English Shift Register Output). The output signal SRO of the shift register 128a forms the input signal SRI of the shift register 128b. Similarly, 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. In addition, a clock signal CLK (Clock English) is provided to the shift registers 128a, 128b to clock the transmissions of values

The operation of this variant comprises the following steps: firstly, each pixel of the pixel array is set to logic state 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 in state 1, for the converter 120a to transmit its digital output value to the data bus 126, the logical 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 disable the pixel 10a, - in the following process of reading the numerical values of the converters of the pixel matrix, the output signal SRO of the pixel 10a remains in state 0 and the state active on the signal SRO spreads in the a matrix at each clock stroke. The variant of 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. On the other hand, 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. The random access made possible by the exemplary embodiment of FIG. 6b is therefore advantageous compared with the variant of FIG. 6b.

As shown in FIG. 6c, several devices 10i, such as the device 10 of FIG. 6a, can be arranged in a matrix of pixels of a biosensor. Each device 10i: represents a pixel of row i and column j of the matrix. In 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. Prediction of a plurality of photoelectron collection zones 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 area. 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. However, a possible improvement in the structure of the photosensitive layer 20 of the biological analysis device 10, and in particular in the structure of the photoelectron collection means, makes it possible to design photosensitive portions of different surfaces while ensuring the correspondence of the photosensitive currents. generated darkness.

The principle to illustrate this structural improvement is described in the patent published under number US 6,998,659. 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 photoelectrons el, ..., e8 generated by incident radiation. 72, in particular a chemiluminescence or fluorescence radiation. 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 the well P 76. The smaller size of the well N 70 means that the capacity of the photosensitive portion is relatively low. But the collection efficiency is not compromised. Indeed, the great majority of photoelectrons, such as the photoelectrons el, ..., 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 move either indifferently towards the N 70 well, or towards the

wells P 76. As for the electron e8, it will certainly move towards the well P 76 and will be lost. 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. In this embodiment, 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. For example, 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 μm x 1 μm. 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 are not desirable. On the other hand, an increase in the size of the N-well increases its area and perimeter, leading to an increase in the dark current and the associated noise.

Today, a good compromise size for a collection area such as the N well, given the aforementioned constraints, is a size between 5 pm and 15 pm. An implementation of the improvement presented with reference to Figures 7 and 8 is illustrated in Figure 9a. In this figure, a differential pixel 86 has a first illuminated photosensitive portion 88 and a second photosensitive portion placed in the dark 90. As previously indicated, 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.

On the other hand, 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. With a conventional substrate having a resistivity of 100 Ohms per

centimeter, the recombination distance is generally between 30 and 50 pm. However, it has been seen previously that it is advantageous to provide pixels whose photosensitive surface has dimensions adapted to the size of a probe protein drop of a few nanoliters for detection by chemiluminescence or fluorescence, that is to say that is, dimensions of the order of 150 μm. With a collection zone 92 whose size is between 5 and 15 μm, the photosensitive portion 88 can not reach these dimensions. To obtain a differential pixel 86 of larger dimensions, a solution shown in FIG. 9b consists in increasing the size of the collection zones 92 and 94. It is thus possible to reach pixel dimensions of the order of 150 μm. 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. In Figure 9c, a differential pixel 86 comprises two photosensitive portions 88 and 90 each having four collection areas 92 and 94 respectively. As illustrated in this figure, the geometry of the two photosensitive portions 88 and 90 is not necessarily the same. In addition, the collection areas 92 are not necessarily arranged in the same way in the illuminated photosensitive portion 88 as the collection areas 94 in the dark photosensitive portion 90.

In contrast, the four collection areas 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 μm. Moreover, the four collection zones 92 are spaced from each other by 75 μm. It is thus possible to obtain a differential pixel 86 whose illuminated photosensitive portion 88 is a 150 μm square and whose light-sensitive portion 90 is a rectangle of 150 μm by 20 μm. In FIG. 9d, 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 μm. Moreover, the nine collection zones 92 are spaced apart from each other by 50 μm for example. It is thus possible to obtain a differential pixel 86 whose illuminated photosensitive portion 88 is a 150 μm square and whose light-sensitive portion 90 is a rectangle of 150 μm by 20 μm. It is possible to generalize to pixels (not shown) comprising more collection areas (16 or more). It will be noted that, since the geometries of the photosensitive portions 88 and 90 are not necessarily the same, the presence of a plurality of collection zones in FIGS. 9c and 9d makes it possible to envisage a geometry in which the photosensitive portion placed in the darkness 90 surrounds the illuminated photosensitive portion 88. Prediction of an insulating joint between two adjacent pixels It has been described how the pixels of a biosensor according to the invention can be sized to correspond to the size of a drop of protein probe. This advantageous dimensioning allows a simpler processing of the signals obtained. However, so that two drops of protein probe neighbors do not interfere by

chemiluminescence or fluorescence, it is generally necessary to impose a minimum distance between two neighboring drops, generally several hundred micrometers.

Another improvement that can be made to a biosensor according to the invention, shown in FIG. 10, makes it possible to considerably reduce the distance required between two neighboring drops. This figure represents two neighboring pixels 86a and 86b, sized to each receive a drop of specific probe protein. The first pixel 86a comprises an illuminated photosensitive portion 88a, here of square shape, for example 150 μm side. It further comprises a photosensitive portion placed in the dark 90a of rectangular shape, 150 pm long and 20 pm wide. The second pixel 86b comprises an illuminated photosensitive portion 88b of square shape of 150 μm side. It further comprises a photosensitive portion placed in the dark 90b of rectangular shape, 150 pm long and 20 pm wide. The first pixel 86a is separated from the second pixel 86b by an insulating seal 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. Thus, a photoelectron el emitted in the illuminated photosensitive portion 88a, on the edge 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 illuminated photosensitive portion 88b. Conversely, 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. Similarly, a molecule excited on the first pixel 86a diffusing a light intensity towards the second pixel 86b would see this light intensity absorbed by the insulating joint, and vice versa. To ensure good mutual protection of the two neighboring pixels, 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. Thus, the presence of such an insulating seal 92 makes it possible to bring the pixels of a biosensor according to the invention substantially closer together and thus to considerably increase the number of specific probe protein drops which can be arranged on a predetermined surface of the biosensor. The molecules or photoelectrons trapped by the insulating gasket 92 are lost for detection. However, considering the diffusion rate of the photon-emitting molecules by chemiluminescence (or fluorescence) and the thickness of the insulating joint 92, it is estimated that at least 2, in the least favorable configuration, the loss of photoelectrons or photons generated on a pixel but not detected by it.

In addition, 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. Indeed, the electronic means are not of the same nature as the photosensitive portions and

may be a source of parasitic electrons that contribute to the effects of the dark current of the photosensitive portions. In addition, 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. Thus, the presence of the additional insulating seal makes it possible to avoid this migration of undesirable electrons. This additional insulating gasket comprises, for example, a grounded P + / P- well and an N + / N- positive potential well. Treatment of the surface of the biosensor 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 has a central photosensitive zone 114 intended to be placed in 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 central photosensitive zone 114, the zone 116 comprising various electrical circuits (for example digital readout circuits, control logic circuit, etc.) and electrical connections between these elements (wiring). Finally, the biosensor comprises electrical contact pads 117 intended, for example, to allow the wiring of the biosensor ("bonding pads") to an interconnection support. During its manufacture or its use, the biosensor is subjected to 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 wetlands intended to be in contact with the central photosensitive zone 114, and also to promote the deposition of the capture mixture as described above. This improvement comprises the provision of the following arrangements or steps, during the manufacture of the biosensor: - moving the contact pads 117 away from the central photosensitive zone 114, for example at a distance of 850 m micrometers thereof, by arranging them in a peripheral zone 118 which surrounds the peripheral zone 116. This distance avoids their contact with the liquids during the chemical treatments of the sensor or during the course of the biological test; - Cover the peripheral zone 116 with a hydrophilic coating with high roughness. This hydrophilic coating is, for example, silicon oxide (SiC 2) and forms a kind of ring ", for example approximately 700 μm wide, around the central photosensitive zone 114 - covering the central photosensitive zone 114 with a hydrophobic coating of low roughness, for example silicon nitride (Si 3 N 4). Preferably, the remainder of the surface of the biosensor is covered with the hydrophobic coating having a low roughness, namely the peripheral zone 118, the zones 114 and This coating allows a clear demarcation between the peripheral zone 116 to be protected and the central photosensitive zone 114. Once these steps have been carried out, the peripheral zone 116 may be covered with a thick layer of biocompatible and non-biocompatible resin 119. conductive, forming a kind of barrier that completely encircles the central area, as shown in Figure 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 μm. Due to the hydrophilic variation between the zones 116 and 114, 118, the protective resin 119 tends, before curing by polymerization, to automatically distribute itself around the central photosensitive zone 114, without encroaching on it or In other words, the hydrophobic surface treatment has a "repellent" effect on the resin and limits its spreading, until this is polymerized. The hydrophobic coating on the central photosensitive zone 114 further 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 to the area 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. In particular, 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. 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

of the central photosensitive zone 114, under the effect of the surface tension forces. As a numerical example, 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 even surface treatment processes making it possible to render the surface of the central photosensitive zone 11 hydrophobic. Thus, the combination of these characteristics allows optimal arrangement and adhesion of the surface. Protective resin 119 both by the chemical characteristics of the treated surfaces and by the arrangement of the biosensor components and their spacing. It will be clear to those skilled in the art that a biological analysis device and a biosensor according to the invention, to which the above-mentioned improvements are optionally added, notably allow the use of CMOS imaging technology for chemiluminescence analysis. or by fluorescence of biological substances comprising fragile target proteins. It will also be apparent to those skilled in the art that the various embodiments as well as the various improvements described above can be combined to achieve other embodiments. Moreover, various modifications may be made by those skilled in the art to the embodiments described above, in the light of the teaching that has just been disclosed.

In the following claims, the terms used are not to be construed as limiting the claims to the embodiments set forth in this description, but should be interpreted to include all the equivalents that the claims are intended to cover because of their formulation and whose forecasting is within the reach of those skilled in the art by applying his general knowledge to the implementation of the teaching that has just been disclosed to him. 47

Claims (18)

REVENDICATIONS1. A pixel-type biological analysis device (10) comprising: - a photosensitive layer (20), - photoelectron collection means (70, 92) in the photosensitive layer, and - reading and imaging means (24). treatment of an electrical quantity supplied by the collection means, for the supply of a characteristic value of a light intensity detected by the photosensitive layer, characterized in that it further comprises a capture mixture (12) comprising a probe protein (14) grafted to a hydrogel (16, 18), the capture mixture being disposed on an outer surface (22) of the photosensitive layer for capturing predetermined target proteins. The biological assay device of claim 1, wherein the outer surface (22) of the photosensitive layer is of sufficient size to receive a full predetermined dose of probe protein (14) in the form of a drop. The biological assay device of claim 2, wherein the outer surface (22) of the photosensitive layer has an area of at least 25500 × 10-12 m 2. The biological analysis device according to one of claims 2 and 3, wherein the electron collection means comprises a plurality of island-shaped collection regions (70; 92) spaced apart from one another in the photosensitive layer. (20). A biological analysis device according to claim 4, wherein the collection areas (70; 92) are spaced apart from one another in the photosensitive layer (20) by a distance less than a predetermined maximum distance and set according to a recombination distance of photoelectrons emitted in the photosensitive layer. 6. biological analysis device according to one of claims 1 to 5, wherein the photosensitive layer comprises: - a first portion (42) provided with a first portion of the collection means, adapted to detect a light intensity incident from the capture mixture (12) and providing a first electrical magnitude (Iph + Idl) and - a second portion (44), provided with a second portion of the collection means, protected against incident light intensity from the capture mixture (12) and providing a second electrical magnitude (Id2), and wherein the reading and processing means (24) comprises means (50) for combined treatment of the first and second electrical quantities for the supply of a characteristic value of a light intensity detected by the first portion (42) of the photosensitive layer (20). 7. A method of manufacturing a biological analysis device according to one of claims 1 to 6, characterized in that it comprises steps of: - preparation (S100, S102, S104, S106, S108, S110) of the outer surface (22) of the photosensitive layer (20) for receiving the capture mixture, comprising a step of silanizing the outer surface, - oxidation (S112) of the hydrogel and grafting (S114, S116) of the oxidized hydrogel on the silanized outer surface, - carboxylation (5118) and activation (5120) of the grafted hydrogel, and - grafting (S122) of the probe protein into the activated hydrogel. 8. Process according to claim 7, in which the step of preparing the external surface by silanisation comprises the following stages: cleaning (S102) of the external surface by successive rinsing with demineralised water, with acetone and with absolute ethanol in an ultrasonic bath, - alkaline oxidation (5104) of the outer surface by oxidation under the action of ozone plasma, and then treatment with a potassium hydroxide solution for several hours at room temperature - successive immersions (S106) in demineralized water and then in absolute ethanol under ultrasound and drying under argon flow, - silanization (108) of the external surface by means of a solution of 3-aminopropyltriethoxysilane in ethanol for several hours, successive immersions (5108) in deionized water and then in absolute ethanol under ultrasound and drying under an Argon flux, - heating the device (S110) for several hours s kept in an inert atmosphere. 30 9. Method according to one of claims 7 and 8, wherein the step of oxidation and grafting of the hydrogel comprises the following phases: - dissolution (S112) of the hydrogel in demineralized water, then treatment with a sodium periodate solution whose volume is adjusted to obtain a mixture whose molar ratio between the sodium periodate and a predetermined and repeated monomer unit of the hydrogel is of the order of 50%; protection (S112) of the reaction mixture obtained against the light and stirring for several hours at room temperature, to obtain an oxidized hydrogel, - grafting (S114) of the oxidized hydrogel on the silanized outer surface and stirring at ambient temperature and at room temperature. protected from light for several hours, - treatment (S116) 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 alisee, ethanol and drying under an Argon flux. 10. Method according to one of claims 7 to 9, wherein the step of carboxylation and activation of the grafted hydrogel comprises the following phases: - treatment (S118) of the hydrogel grafted with an acid solution bromoacetic acid in sodium periodate for several hours at room temperature and under argon, purge (S118) of the resulting reaction mixture and rinsing with deionized water, ethanol and drying under an Argon flux, - activation (S120) in the form of esters of the carboxylate residues formed, followed by rinsing with water. 11. Method according to one of claims 7 to 10, comprising a preliminary step (S100) of treatment of the outer surface (22) of the photosensitive layer for receiving the capture mixture (12) using a hydrophobic coating whose roughness is chosen so that at least one solution deposited on the photosensitive layer during the preparation of the capture mixture does not spread beyond the photosensitive layer. 12. Method according to one of claims 7 to 11, comprising a step of dimensioning the outer surface (22) of the photosensitive layer for receiving the capture mixture (12) so that it corresponds to the size a predetermined dose of capture mixture that can be disposed on the outer surface in the form of a drop. 13. Biosensor comprising: - a plurality of pixel-type biological analysis devices (10i) according to one of claims 1 to 6, 15 arranged in a matrix of pixels, and - a circuit (30) for processing the signals provided. at the output of the pixel matrix. The biosensor according to claim 13, wherein: - each pixel (10i, i) of the pixel array comprises a converter (120) of analog signals to digital signals for digital conversion of the characteristic value of a light intensity detected by the photosensitive layer of this pixel, and the signal processing circuit at the output of the pixel array comprises a digital data transmission bus (126) connected to the matrix pixel converters. The biosensor according to claim 13 or 14, wherein each pixel (86a, 86b) is separated from an adjacent pixel (86a, 86b) by an insulating seal (92) shaped to electrically isolate the photosensitive layer (88a, 90a, 88b, 90b) of the pixel of the photosensitive layer of the adjacent pixel. 16. Biosensor according to one of claims 13 to 5 having a contact surface with a fluid to be analyzed, this surface comprising: - a first central zone (114) in which is disposed the pixel matrix, covered by a hydrophobic coating A first peripheral zone (116) surrounding the central zone, comprising electronic circuits and covered by a hydrophilic coating, - a second peripheral zone (118) surrounding the first peripheral zone (116), comprising electrical contact pads 15 (117) and coated with a hydrophobic coating, and - a barrier of a biocompatible resin (119) covering the second peripheral zone (118), fixed on the hydrophilic coating. 20 17. Biosensor according to one of claims 16 and 17, wherein the hydrophilic coating is silicon oxide. 25 18. Biosensor according to one of claims 16 and 17, wherein the hydrophobic coating is silicon nitride.