Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6825—Nucleic acid detection involving sensors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/403—Cells and electrode assemblies
  • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
  • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
  • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

• KUMAR et al. entitled “Silanized nucleid acids: a gênerai platform for DNA immobilization ”published in Nucleid Acid Research 2000, volume 28, n ° 14, pages i to vi.
• Two methods of fixing the probe molecules to the surface can be used in the context of the present invention. The first consists of a direct synthesis on solid, as described for example in the article by S.P.A. Fodor and Collaborateurs entitled “Light-directed, spatially addressable parallel chemical synthesis” published in Science 251, pages 767 to 773 (1991). The second is the fixation of molecules from a dilution.
• the present invention thus relates to a method of electronic detection of at least one specific interaction between probe molecules attached at least to an active area of a sensor and target biomolecules, characterized in that said sensor consists of a network of transistors field effect (Ti, T 2 , ...) each of which has a source region (5), a drain region (D) as well as a gate region which constitutes an active area (3) on which said specific interaction must be detected, and characterized in that it comprises the following steps: a) bringing at least one said active area (3) into contact with probe molecules of a given type fixed on said active area. b) bringing at least some of the probe molecules into contact with target biomolecules capable of interaction with said probe molecules and effecting said interaction in a reaction buffer having a first salt concentration.
• any molecule in particular biomolecules (DNA, RNA, proteins, etc.), or else chemically synthesized oligonucleotide DNA, or even nucleic peptide acids (“PNA”), is considered as probe molecule, which can be grafted onto a said active surface and which can be specifically recognized by a target biomolecule.
• biomolecules DNA, RNA, proteins, etc.
• PNA nucleic peptide acids
• the differential measurement of step c) is carried out on the same probe molecules, before the interaction of step b).
• the differential measurement of step c) is carried out between two groups of probe molecules of the same type for example (DNA), having or not the same sequences, fixed on separate active areas, one groups having been subjected to the interaction of step b), and the other not.
• the differential measurement in step c) is carried out with the measurement buffer on two groups of probe molecules arranged on separate active zones, these two groups of probe molecules, for example of the same type, but having or not the same sequences, and having been subjected to different specific interactions.
• the probe molecules and / or the target biomolecules are for example molecules of DNA, RNA, "PNA", proteins, or even small molecules such as vitamins.
• the reaction buffer and the measurement buffer are for example KCI.
• the salt concentration of the measurement buffer is for example greater than 0.002 mM and less than 20 mM, and it is for example greater than 0.005 mM and less than 20 mM, and in particular between 0.005 Mm and 15 mM, or even between 0.01 mM and 15 mM.
• the concentration of the reaction ramp is for example between 20 mM and 1 M (molar concentration).
• the method according to the invention is compatible with conventional detection of molecular interaction by fluorescence.
• the method can be characterized in that said measurement of at least one point of the characteristic implements the application of a given voltage (U D s) between the drain and the source of at least one transistor, as well as the application in a first case of a given voltage (UGS) between the door and the source of said transistor or, in a second case of a drain current (l D ) given to said transistor.
• U D s a given voltage
• UGS drain current
• the measurement of the characteristic point consists of the measurement of the drain current Id.
• the voltage UGS is measured.
• a circulation is carried out through at least one fluid microchannel of at least a solution which constitutes a reference or which contains target molecules for bringing it into contact with at least one active area of a field effect transistor.
• a reference solution for example salt buffer
• a solution containing target molecules successively in contact with one or more active zones, or else (in parallel) with one or more different active zones.
• two solutions of target molecules can be brought into contact with one or more different active zones in parallel.
• FIG. 1 shows two field effect transistors of a detection chip comprising a plurality of such transistors organized according to a mono or two-dimensional network of transistors;
• FIG. 2 shows a top view of a detail of a detection chip and the arrangement of the active areas each corresponding to a field effect transistor
• FIG. 3 illustrates the electrical connections for draining transmissions from the mono or two-dimensional network.
• FIG. 4 shows a device for depositing the solution on selected active areas
• FIGS. 7a and 7b illustrate the implementation of microfluidic channels
• Figures 1 to 3 illustrate a sensor having an array of FET field effect transistors on a silicon substrate.
• a transistor Ti or T 2 shown in section in FIG. 1 is provided with a source region S and a drain region D which each have an electrical contact and which are surmounted by an insulating layer 1 and 2 respectively, for example a thermal oxide of Si0 2 .
• the active region 3 between the source S and the drain D forms the gate region G of the transistor and has an insulating layer 4 of reduced thickness, for example a layer of thermal SiO 2 . It is also possible not to have oxide on this active region.
• the active surface is then delimited by a portion 4 'of the substrate which is exposed.
• Probe molecules for example single stranded DNA molecules, are attached by a known method to at least some of the active surfaces 4 or 4 '.
• the active surfaces 4 or 4 ' For DNA, use is preferably made of depletion n-channel field effect transistors (for which the charge carriers are the electrons, more mobile hence an increase in sensitivity) with a negative gate polarization (c that is, the electrolyte is negatively polarized with respect to the semiconductor), the DNA being negatively charged (for an electrolyte of neutral pH).
• the current l D depends on the fixation of the probe molecules, for example DNA molecules on the active surface 4 or 4 '. It is possible to carry out a detection at source-gate voltage USG e at constant source-drain voltage USD, by measuring the drain current Isa or even, as in Examples 1 and 2 below, with drain current Isd and at voltage source-drain U S D constants, by measuring the source-gate voltage
• the different structures are at a short lateral distance from each other and their active surfaces are in contact with the same measurement solution.
• a typical lateral dimension in current microelectronics is less than ⁇ m.
• the lateral dimension is 5-10 ⁇ m for direct synthesis on the solid and 50-100 ⁇ m in the case of fixing of the molecules from dilution.
• several pads with different types of immobilized probe molecules are in contact with the same solution, in particular for measurement, and at least one transistor structure is located below each pad. The implementation of several transistors per pad is possible in view of the dimensions mentioned above and allows redundancy in detection.
• An E electrode (Ag / AgCI, for example) is used to fix the potential of the measurement solution 6 (electrolyte) relative to the silicon structure it covers and to fix the working point of the sensors (transistors) .
• the potential of the electrolyte 6 can in certain cases be equal to zero.
• the measurement solution 6 which bathes the sensors contains ions at a concentration which gives sufficient conductivity and which does not give rise to excessive screening at the level of the active surfaces. Its pH is preferably neutral.
• the proposed technique facilitates detection using different approaches
• A- Characterizing the layers by electronic measurements Electronic measurement allows a rough characterization of the (electrostatic) states of the molecular layers deposited on the transistors. Electronic measurements are made between the different surface preparation steps. Each chemical treatment or molecule deposition step induces a shift in the measured voltage and the differences between transistors reflect a possible inhomogeneity. All of these offsets then give a characterization of the system with multiple layers deposited.
• the measurements can be repeated without carrying out further checks.
• the deposition of the probes, the recognition reaction and the electronic measurement can be carried out in different buffers. This allows these steps to be optimized largely independently.
• the example below shows in particular a hybridization with a salt concentration of 50 mM coupled with electronic detection with a salt concentration of 0.01 mM.
• the measurement electronics would be more complicated than that implemented in the context of the present invention for measurement in DC direct current based on the FET field effect transistors.
• Active structures FET are therefore more suitable for miniaturization. By using active surfaces of 2 microns by 20 microns, we are not limited by the noise of the FET transistors.
• the method for detecting molecular recognitions can be based on a comparison approach, in particular a differential approach.
• the measurement is for example carried out using several transistor structures.
• the measurement can be differential with respect to the different types of grafted molecules and possibly include several transistors per type of molecule. It allows to compare signals before / after the interaction reaction which reveals molecular recognition (and / or evolution during this reaction).
• the reference measurement can be carried out in the measurement buffer having the second concentration, but also in another buffer, for example in said reaction buffer having a first salt concentration.
• the method according to the invention overcomes the difficulties associated with the sensitivity of an individual sensor to pH and ionic strength and those associated with variability from one individual transistor to another (this includes the structure of the transistor and the quality of the fixation of the probes).
• a method implements the following steps: a) homogeneous treatments of the entire insulating surface to prepare the attachment of the probe molecules; b) local grafting of different types of probe molecules onto at least some of the individual active surfaces; c) possibly homogeneous rinses; d) electronic measurement: add the measurement electrolyte, immerse the electrode and measure the transistors (for example one or more points of the characteristic as a function of U SD and U S G), and compare the results obtained according to the transistors ; e) possibly homogeneous rinses; f) addition of the solution of target molecules in the presence of electrolyte and recognition reaction; g) possibly homogeneous rinses; h) electronic measurement, as (d).
• Certain transistors which have not been put in the presence of probe molecules can serve as a control. Their characteristics are measured after adding the measurement electrolyte which, for example, bathes all of the transistors.
• the grafting of the probe molecules is carried out by depositing micro-droplets with a diameter of approximately 100 ⁇ onto the active surfaces of the transistors using metallic micro-feathers which are commercially available, or else a commercial microdeposition system (for example Nanoplotter NP1 from the Ge Sim Company).
• the series resistors R c associated with these connections have values which depend on the index 1 ... n of the drain.
• a chip 15 incorporating the network of n transistors is placed on a support 14.
• Another plate 20 comprising a table 21 ensuring a displacement in the three directions X, Y, and Z is used to move an arm 22 carrying a micro-feather or a pipette 23 to ensure the deposition of the micro-droplets on at least some of the n transistors.
• a lens 17 and / or a camera coupled to a screen 19 make it possible to observe the deposition of the micro-droplets and to control the operations.
• an overall treatment of the surface of the Si / SiO 2 structure is carried out by incubation for 1-2 minutes in sulfochromic acid and rinsing under a stream of deionized water then incubation for 3-5 minutes in a solution of NaOH (60 ⁇ l 16N NaOH, 420 ⁇ l of ethanol and 220 ⁇ l of water), and finally rinsing under a stream of deionized water.
• the sample is dried 15 minutes in a humid atmosphere and then 5 minutes at 50 ° C.
• Poly-L-lysine is positive in the measurement electrolyte (neutral pH) due to the ionized amino groups.
• the decrease in current observed on poly-L-lysine deposits is compatible with the adsorption of a positive charge on the surface.
• the difference in surface potential ⁇ U S G corresponding to the measurement before / after deposition is measured.
• the two-dimensional characteristic for example ID (USG, USD)
• ID USG, USD
• the intrinsic characteristics of the 96 transistors are determined by digitally correcting the measured characteristics as a function of the resistances R c of the drain lines in series.
• the modification of the state of the SiO 2 interface induces a change in the intrinsic characteristic which corresponds to an offset ⁇ U S G to USD and constant drain current L D.
• This offset makes it possible to directly obtain a measurement independent of the working point of the transistor, unlike the change in current ⁇ ID presented in FIG. 5a.
• the value ⁇ USG allows as a first approximation to quantify the change in the interface Sj ⁇ 2 / liquid induced by the local deposit.
• USG is varied so as to keep l D constant.
• FIGS. 5a to 5c show differential measurements carried out before and after deposition of poly-L-lysine (FIG. 5a), carried out as a function of the concentration of KCI (FIG. 5b), and carried out as a function of the concentration of poly-L- lysine deposited.
• the differences ⁇ ID between two measurements carried out before a local deposit but separated by rinsing with water are represented by circles.
• the differences ⁇ ID corresponding to measurements made before and after a local deposition of poly-L-lysine are represented by stars. After local deposition, the sample is left for 15 minutes at room temperature in a humid environment, before drying at 50 ° C. for 5 minutes.
• the dilution Co of the poly-L-lysine is 0.01% w / v final "W ⁇ /" (P8920, Sigma) in a 0.1x PBS buffer at pH 7.
• the differences between a reference measurement (performed before local deposition and with a KCI concentration of 0.01 mM) and two series of measurements (performed after local deposition of poly-L-lysine and with different KCI concentrations) are represented by circles and stars.
• a local deposition of poly-L-lysine was carried out in two distinct zones with the same dilution Co as in the case of FIG. 5a.
• the concentration of KCI in the measurement buffer is varied between 0.01 mM and 100 mM, passing through the values 0.1 mM, 1 mM and 10 mM. Between the two series of measurements, the surface is rinsed with water. There is an appreciable sensitivity of the detection of poly-L-lysine for KCI concentrations between 0.01 mM and 1 mM, and the height of the peaks gradually decreases beyond these values.
• 5c shows the variations ⁇ USG of the voltage U SG as a function of the concentration of deposited polymer (poly-L-lysine), namely 2Co, C 0 , Co / 2, C 0/4 , Co / 8 in a buffer 0.1 x PBS pH 7, C 0 having the value indicated for the measurements in FIG. 5a.
• FIGS 6a and 6b show electronic DNA detection.
• the stars represent the measurement after initial surface treatment with soda.
• the circles represent the measurement after incubation of poly-L-lysine over the entire network.
• the network of FET transistors is incubated for 30 minutes in a dilution of poly-L-lysine (Co concentration). Then, without drying beforehand, it is rinsed with water and then air-dried.
• the incubation leads to shifts in the USG voltage of a value of 97 ⁇ 50 mV (statistics on 67 prepared surfaces) which reduce the variations between transistors in the electronic signal. These shifts are compatible with those observed with the values measured in relation to FIG. 5c on local deposits at the same concentration.
• the squares represent the measurements after local deposit of oligonucleotides (5 'modified Cy-5 20 mer, concentration 50 ⁇ M in deionized water) around transistors n ° 30, 60 and 90.
• oligonucleotides 5 'modified Cy-5 20 mer, concentration 50 ⁇ M in deionized water
• FIG. 6a the microfluorescence image of the three abovementioned DNA points is represented.
• FIG. 6b shows the electronic and fluorescence detection of modified Cy5 oligonucleotides.
• the squares show the intensity of the fluorescence measured on the dried FET transistors, once the electronic measurement has been carried out with the electrolyte. It will be noted that the same electronic detection is obtained with oligonucleotides of the same type, but not modified.
• Figures 7a and 7b show an integrated circuit having transistors T arranged along a line (or several lines).
• Two microfluidic channels (for example parallel) Ci and C 2 of a substrate 30 make it possible to put one or more field effect transistors T in contact with a reference solution or a solution containing target molecules which circulates in a channel Ci and / or C 2 .
• the material of a substrate 30 which comprises the microfluidic (or capillary) channels can be a PDMS (polydimethylsiloxane) or other polymer, a glass, silicon, etc.
• the detection of at least one specific interaction between probe biomolecules and target biomolecules is advantageously carried out by implementing a measurement buffer whose salt concentration (for example KCI) is lower than that of the reaction buffer.
• a measurement buffer whose salt concentration (for example KCI) is lower than that of the reaction buffer.
• the biomolecules concerned can for example be DNA, RNA, proteins and vitamins.
• the specific interactions may, for example, be DNA-DNA, DNA-RNA, DNA-protein, RNA-protein, protein-protein, or even vitamin-protein interactions.
• DNA can be chemically synthesized oligonucleotide DNA. It is also possible to perform interactions with a nucleic acid peptide "PNA".
• PLL poly-L-lysine solution, P8920 (Sigma), 0.01% w / v in 0.1x PBS buffer.
• Oligonucleotide ARS3 5 'CCG CGA ACT GAC TCT CCG CC
• Oligonucleotide ARS5 5' CAG GCG GCA GGG CTG ACG TT
• Cy3-ARS3sens oligonucleotide (complementary to ARS3 and with fluorophore Cy3)
• Measuring buffer KCI 0.01 mM (see figure 8a).
• This measurement is followed by rinsing with water and drying.
• the purpose of this second measurement is to verify the stability of the measurement at this stage.
• a micropipette 0.2 microliters of a solution containing the oligonucleotide ars5 is deposited on the left side of the FET network. On the right side of the network, 0.2 microliters of a solution containing the oligonucleotide Ars3 is deposited. In both cases, the dilutions contain 1 micromole of oligonucleotide in a 50 mM KCI buffer. Incubation is carried out for 15 minutes, in a humid atmosphere, to avoid drying.
• a measurement buffer is used: KCI 0.01 mM.
• the "Sondel -PL2" curve shows an offset of 25 mV for the two deposition regions left (deposition of Ars3 on transistors 1 to 13) and right (deposition of Ars5 on transistors 21 to 31). With this value, we have a sufficient concentration of probe biomolecules and are not yet too close to saturation.
• the electrolyte is then pumped and replaced with 1 ml of 50 mM KCI, without drying.
• Hybridization 1 (with Cy5-Ars5sens).
• step 8 When measuring "Sonde2" (step 8) there was 1 ml of KCI on the FET transistors.
• the final dilution in oligonucleotides is then of the order of 100 nM. It is stirred by pumping and this solution is redeposited twice. After stirring, the recognition reaction is carried out for 5 minutes protected from light (to avoid bleaching or "bleaching" of the fluorophores).
• Probe3 is approximately +3.9 mV on the Ars3 region and approximately +2.3 mV on the Ars5 region (here the probe biomolecules and the target biomolecules have complementary sequences). This offset is approximately -1.6 mV over the central region without deposit of DNA probes (transistors 14 to 20).
• the measurement electrode is immersed in the 0.01 mM KCI buffer and the electronic measurement is made.
• the 0.01 mM KCI electrolyte is pumped and replaced with 1 ml of 50 mM KCI. Afterwards, 100 ⁇ l of Cy3Ars3sens at 1 ⁇ M are added directly (without rinsing). The final dilution in oligonucleotides is then of the order of
• the electrode is immersed in the KCI buffer at 50 mM and the electronic measurement is carried out.
• the result of these measurements is represented by the Hyb2 - Sonde3 curve in Figure 8d. It is observed in FIG. 8d, that the difference Delta USG between the measurements referenced
• Hyb1 and Sonde3 is more positive on the Ars3 region (on the left) than on the Ars5 region (on the right, where the probes and targets have complementary sequences for hybridization 1). This trend is reversed for the difference between Hyb2 and Sonde2, in agreement with the fact that for hybridization 2 the probe and target biomolecules have complementary sequences on the Ars3 region.
• rinse pumping of the electrolyte and addition of 1 ml of 0.01 mM KCI, stirring, pumping over, repetition 5 times.
• the electrode is immersed in the 0.01 mM KCI buffer and the electronic measurement is made.
• the result is represented by the Hyb22 - Probe 1 curve in Figure 8f.
• Steps 1 to 5 are identical to those of Example 1. 6- Deposit of the DNA probes.
• substantially 0.2 ⁇ l of a solution containing the oligonucleotide ars5 is deposited on the left side of the network of FET transistors.
• 0.2 ⁇ l of a solution containing the oligonucleotide Ars3 is deposited on the right side of this network.
• a mixture of Ars5 and Ars3 is deposited in the center.
• the dilutions contain 1 ⁇ M (1 micromole) of oligonucleotide in a buffer of deionized water.
• the probes dry a few seconds after deposition at ordinary temperature and humidity. No rinsing.
• the probe 1-PL2 curve in FIG. 9a shows the Delta USG differences between measurements made after the deposition of the probes and the PL2 measurement carried out before this deposition (with the same measurement buffer).
• oligonucleotides 1 ⁇ M.
• the final dilution in oligonucleotides is then about 100 nM. It is stirred by pumping and this solution is redeposited twice. After stirring, the recognition reaction is carried out for 5 minutes protected from light.
• the electrode is immersed in the 20 mM KCI buffer and the electronic measurement is carried out.
• RNA probe molecules are grafted which carry different base sequences at different locations on the surface of the chip. Each DNA probe is chosen to interact specifically with a type of RNA molecule (characterized by its sequence). The relative number of hybrid RNA molecules on two different probe regions gives the relative abundance of two types of RNA molecules (and with that the relative level of expression of the two corresponding genes).
• the interest is to follow a large number of genes in parallel, for development studies, for a genetic characterization of pathologies, for a molecular analysis of the effect of drugs, etc.
• Another example concerns the detection of mutations on DNA chips.
• different oligonucleotides typically 12 seas
• the basic sequences and the hybridization conditions are optimized so that the presence of a mutation (even a point mutation) induces a measurable difference in the hybridization rates of the various probes.
• the target molecule sample here often consists of double-stranded DNA molecules obtained by "PCR" from a small amount of patient's genomic DNA.
• the detection of a specific interactivity can be carried out on one or more gate regions of the network of FET field effect transistors.
• the advantage of using several door regions makes it possible to highlight inhomogeneities.

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