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• • 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules
• • 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
• • 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
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
  • B01L2300/0654—Lenses; Optical fibres
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
  • B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
  • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
  • G01N2030/8827—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving nucleic acids
• • 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
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis

Definitions

• cfDNA free DNA circulating outside the cell
• cfDNA is in the form of double-stranded DNA with an average size of 150-180 bp corresponding to the winding of DNA around the nucleosome. Its lifespan is less than two hours, before it is filtered and eliminated from the bloodstream by the spleen, liver and kidneys.
• the device described in the documents PCT/FR2016/051774 and WO/2017/009566 makes it possible to easily and economically determine the characterization of the size profile of the cfDNA.
• the device operates in two stages of concentration and separation carried out in line.
• the DNA is concentrated via a system of capillaries formed by the junction of a small capillary and another of larger section.
• the researchers laminarly flow a solution containing DNA into the large capillary and use an electric field to slow the migration. Because of the shear brought by laminar flow, this counter-electrophoresis causes a transverse force to appear, dependent on the size of the DNA, which pushes the DNA towards the walls.
• FIG 1 is a diagram of such a device 40.
• This device 40 can be installed in a CE (acronym for "Capillary electrophoresis” or capillary electrophoresis) Agilent (registered trademark), in place of a standard capillary.
• CE acronym for "Capillary electrophoresis” or capillary electrophoresis
• Agilent registered trademark
• the total length of the device is approximately 30 cm, the minimum length that can be installed in an Agilent CE, which allows pressure and voltage to be applied to the terminals of the capillary device.
• Agilent CE which allows pressure and voltage to be applied to the terminals of the capillary device.
• This capillary 47 has a larger diameter than the capillary 45 so that its electrical resistance and its hydraulic resistance are small compared to the combined resistances of the injection nozzle 43 and the separation capillary 45. If the separation capillary 45 went up to outlet 42, the voltage and the pressure to be applied to retain small DNA fragments would be beyond what the instrument allows.
• a slightly larger capillary diameter can be suitable for very large DNA fragments (200 kb), or the way of assembling the capillaries can be modified to do DNA fractionation, for example to favor the quantity of DNA that can be stored at a junction, to the detriment of the analysis resolution.
• Figure 2 shows the typical sequence of DNA analysis with this device 40.
• the distal capillary 47 is not shown in this figure.
• the sample is injected into the device, by applying pressure for a given duration.
• the injected sample is pushed by pressure into the middle of the injection chamber 44.
• the DNA retained at the concentration junction 48 is separated according to size by a gradual reduction in the electric field applied between the inlet 41 and the outlet 42, the pressure generally being kept constant.
• the DNA fragments are detected by fluorescence when they pass in front of the detector 46.
• the viscosity and the resistivity of the solution in the capillary are respectively 40 mPa.s and 12.4 Q.m at 25°C.
• the value of the electric field given above is valid as long as the conductivity of the sample is close to, or less than, that of the analysis buffer (approximately 12.4 Q.m at 25°C). This is generally the case when the DNA is purified beforehand, and taken up in a buffer that is not very conductive.
• the electric field in the separation capillary 45 which must retain the DNA in the injection chamber 44, is therefore only 7% of the value of the electric field in the tip of the device.
• an electric field of 21400 V/cm would then be required in the tip 43, which is not achievable in practice because of heating by the Joule effect that this would cause, this heating in turn causing degassing of the electrolyte in the capillary, hence the formation of air bubbles cutting the electrical continuity of the electrolyte, virtually canceling any electric field downstream, in the separation capillary .
• BIABooster Online DNA Concentration and Size Profiling with a Limit of Detection of 10 fg/pL and Application to High-Sensitivity Characterization of Circulating Cell-Free DNA”, Anal. Chem. 2018, 90, 3766-3774; And
• the present invention aims to remedy all or part of these drawbacks.
• the present invention relates to a process for desalting and concentrating a sample of nucleic acids that is more conductive than an analysis buffer, a process which comprises at least one iteration of an alternation :
• the inventors have discovered that, when the electric field is insufficient to retain the small DNA fragments at the concentration junction, these fragments leak into the separation capillary. But the phenomena implemented always push them towards the wall, even if this pressing against the wall is insufficient for the counter-electrophoresis to be stronger than the fluid flow. It follows that the migration speed of these DNA fragments is lower than the average speed of the flow. On the other hand, the ions forming the salts are too small for there to appear a transverse force pushing them towards the wall, and they advance at the average speed of the flow, more or less their speed of electrophoresis according to their positive charge or negative.
• the concentration operation is stopped before the smallest fragments arrive at the end of the separation capillary 45 and a pressure is applied, alone or in the presence of a potential difference, in the opposite direction to the initial pressure to return the volume of the separation capillary to the injection chamber. Then, a new concentration step is carried out and, possibly, this alternation of return and concentration phases is repeated.
• the salts are thus gradually evacuated from the device, while the DNA molecules are essentially preserved.
• the method further comprises a step of measuring the amperage flowing in the capillary during at least one flow step in the first flow direction and a step of selecting a number of iterations of the alternation according to the measured amperage. Thanks to these arrangements, the conductivity of the sample, representative of its salinity, is estimated by means of the measurement of the current passing through the sample and, depending on this salinity, a number of alternations of flows are chosen in one direction and then the other to reduce this salinity to an adequate level.
• nucleic acids For example, in a biological sample containing proteins, some of these proteins are positively charged and then bind to the walls of the capillary, as well as to nucleic acids, which are negatively charged. This causes the nucleic acids to stick to the walls, a sticking that is harmful for the analysis.
• a high pH buffer By choosing a high pH buffer, the majority of proteins are negatively charged, and the sticking of nucleic acids to the walls is reduced, or even disappears. But, at high pH, a separation of nucleic acids is not as resolute as at neutral or slightly acidic pH. With a change of buffer, the ability to separate nucleic acids is restored.
• the present invention relates to a method for analyzing a sample of nucleic acids, which comprises the steps of the method of desalting and concentrating a sample which is the subject of the invention and, after the last iteration of the alternation, a step of separation by laminar flow of the sample in the capillary in the first flow direction, during which the sample is subjected to an electrical potential difference less than or equal to the first potential difference, of which the action on the nucleic acid molecules is opposite to the first direction of flow and causes partial retention of nucleic acid molecules in the capillary.
• the nucleic acids are thus partially retained, which improves their separation according to their size.
• the analysis method comprises, during or after the separation step, a step for measuring a temporal profile of fluorescence of the fluorescent molecules of nucleic acids and a step for converting the temporal profile of fluorescence into a concentration profile of nucleic acid molecules of different lengths, by implementing a fluorescence profile of a standard sample, the concentration of which is known for each length of fluorescent nucleic acid molecule.
• the present invention relates to a device configured to implement a method of desalting and concentration object of the invention or a method of analysis object of the invention of a sample of nucleic acids more conductive than an analysis buffer, the device comprising:
• the capillary is formed of a microfluidic channel.
• FIG. 1 schematically represents a capillary device of the prior art
• figure 2 represents a typical operating sequence of a DNA analysis with the device illustrated in figure 1
• figure 3 represents a calculated electric field, over time, at one mm downstream of the concentration junction of the device illustrated in figure 1 operating as illustrated in figure 2, when the sample has a conductivity 18 times higher than that of the analysis buffer
• figure 4 represents an analysis result for an unsalted DNA sample
• figure 5 represents an analysis result for more or less salty standard DNA samples
• FIG 6 reports the areas of the peaks in Figure 5, as a function of NaCl concentration in the sample
• Figure 7 shows the amperage flowing through the capillary during the concentration step of the assays shown in
• all or part of the means and variants of the devices disclosed in international application WO/2017/009566 are configured to implement the method object of the invention.
• the structural characteristics of the devices disclosed for example with respect to the dimensions of the capillary provided with a local restriction, can be transposed to the method which is the subject of the invention.
• the capillary has a diameter, upstream of the local restriction, of at least 100 ⁇ m and preferably of at least 300 ⁇ m.
• the device has a plurality of local restrictions.
• such local restrictions have similar dimensions.
• the device has:
• the narrow channel has a width of at least 2 ⁇ m and preferably at least 10 ⁇ m.
• Examples 1 and 2 described with reference to Figures 1 to 9, specify the experimental results of a device 40 of the prior art operating according to the sequence illustrated in Figure 2, sequence also called "0 return".
• Example 1 “0 return” method in device 40, unsalted sample.
• the number of returns preferably depends on the salinity of the samples.
• two types of analysis are defined, one for unsalted or slightly salted samples, the other for salted samples. So :
• the 1K standard sample contains DNA fragments of the following sizes: 100 bp, 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 1000 bp, and 1500 bp.
• the 0 return method for device 40 is described in Table 2 below.
• FIG. 4 represents an analysis result 80 for a standard sample of unsalted DNA: trace of the fluorescence of a standard unsalted sample according to a DNA 1 K analysis, “0 return” method.
• Each fluorescence peak 81 corresponds to the DNA size indicated on the graph in base pairs (bp).
• Example 2 “0 return” method in device 40, salted samples.
• the “0 return” method for device 40 is the same as in Example 1.
• Figure 5 represents the 90 result for more or less salty standard DNA samples.
• This analysis with the “0 return” method of samples containing: 0 mM NaCI is represented in curve 91, 10 mM NaCI, curve 92, 15 mM NaCI, curve 93, 20 mM NaCI, curve 94, 50 mM of NaCI, curve 95, 100 mM NaCI, curve 96, and 130 mM NaCI, curve 97.
• the fluorescence values have been shifted on the vertical fluorescence axis by adding a constant to facilitate the visualization of the curves.
• Figure 6 reports the areas 100 of the peaks as a function of the concentration of NaCI in the sample, for the peaks 10Obp, curve 101, 150bp, curve 102, 200bp, curve 103, 400bp, curve 104 and 1000bp, curve 105 as a function of the salt concentration in the DNA1 K buffer with the “0 return” method.
• FIG 8 represents the 120 analysis with the "0 return” method of a sample of purified circulating DNA containing: 0 mM of NaCI, curve 121, 10 mM of NaCI, curve 122, 15 mM of NaCI, curve 123, 20 mM of NaCI, curve 124, 50 mM NaCI, curve 125, 100 mM NaCI, curve 126 and 130 mM NaCI, curve 127.
• Figure 9 represents the evolution 130 of the area 131 of the first peak, on the left, and of the area 132 of the second peak, on the right, as a function of the NaCI concentration with the “0 return” method. The values are taken from figure 8. It is observed that, with the “0 return” method, the area of the second peak is stable whatever the concentration of salts in the sample. On the other hand, the area of the first peak is stable up to 10 to 15 mM, then decreases sharply. With 130 mM of salts in the sample, only 16% of the first peak 131 is retained during the concentration step.
• Examples 3 to 9 illustrate results obtained by the implementation of the present invention.
• experiments performed with samples containing DNA are described.
• the present invention is not limited to this type of nucleic acid but extends, on the contrary, to other nucleic acids, for example single-stranded RNA or DNA.
• Example 3 illustrated opposite Figures 11 to 15, by inserting two returns during the concentration step, it became possible to analyze small DNA samples containing up to 15 mM of salts, i.e. 50% more content than the 0 return method allows. Above 15 mM of salts, the small fragments are no longer correctly retained.
• FIG 13 the temporal positioning of the two durations of returns 161 and 162 in the chronogram of the method. During these returns, since no electrical voltage is applied, the electrical current is zero.
• the fluorescence profile is converted into a concentration profile thanks to the fluorescence profile of the standard sample, the concentration of which is known for each length of DNA fragment .
• the slight decrease in intensity for the smaller DNA fragments is therefore compensated by the calibration based on the profile of the standard sample.
• concentration at high pH it is preferable to carry out the concentration at high pH, so that the vast majority of proteins are negatively charged, and that the bonding of DNA to the walls via proteins disappears.
• concentration at high pH takes place with returns, which makes it possible to evacuate the salts and the remains of proteins from the sample.
• a new phase of concentration takes place, this time at neutral or slightly acidic pH, more favorable to separation.
• This concentration phase allows the sample buffer to be changed.
• a third step takes place the separation, identical to that which takes place in the other methods described in examples 1 and 2.
• Example 6 thus shows how a standard DNA migrates in the same way in a plasma and in a non-salted buffer.
• Example 7 we confirm the reliability of the method that is the subject of the invention by comparing circulating DNA size profiles obtained either by the method of Example 3 (two returns) after purification of the DNA, or by the process of example 5 directly on plasma (seven returns process).
• the technology also works in a microfluidic chip format, and the present invention can be perfectly implemented for analyzes of salted DNA samples, or analyzes of DNA directly in biological fluids, using microfluidic chips.
• Example 9 describes such a microfluidic chip.
• FIG. 10 represents a typical operating sequence of a DNA analysis with the method object of the invention implemented with two returns and the device 40.
• the distal capillary 47 is not represented in this figure.
• the concentrated DNA is represented in black and the salts in hatching.
• the sample is injected into the device, by applying pressure for a given duration.
• the injected sample is pushed by pressure into the middle of the injection chamber 44.
• the migration speed of the smallest DNA fragments is lower than the average speed of the flow.
• the ions forming the salts are too small for there to appear a transverse force pushing them towards the wall, and they advance at the average speed of the flow, more or less their speed of electrophoresis according to their positive charge or negative.
• the sample is caused to flow in a second direction of flow opposite to the first direction of flow by applying a second pressure in the opposite direction to the pressure exerted on the solution of the sample.
• Part of the sample solution which had entered the separation capillary 45 flows back into the injection chamber 44, entraining DNA fragments which thus return to the injection chamber 44.
• the operation 65 is called a "return".
• the concentration operation is stopped before the smallest fragments arrive at the end of the separation capillary and a second pressure is applied in the opposite direction to the first pressure to bring the volume of the separation capillary 45 back into the chamber. injection 44.
• the DNA is again concentrated at concentration junction 48 of injection chamber 44, by application of the first pressure causing flow in the first direction of flow and of the first electrical potential difference whose action on the nucleic acid molecules is opposite to the first direction of flow and causes the retention of nucleic acid molecules in the capillary.
• the separation capillary 45 fills with the sample solution, except for the largest DNA fragments which remain retained at the concentration junction 48. A new concentration step is thus carried out.
• the DNA is again concentrated at concentration junction 48 of injection chamber 44, by application of the first pressure causing flow in the first direction of flow and of the first electrical potential difference whose action on the nucleic acid molecules is opposite to the first direction of flow and causes the retention of the nucleic acid molecules in the capillary.
• the separation capillary 45 fills with the sample solution.
• the DNA is separated according to size by a gradual drop in the electric field applied between the inlet 41 and the outlet 42, the pressure generally being kept constant.
• the viscosity and the resistivity of the solution in the capillary are respectively 40 mPa.s and 12.4 O.m at 25°C.
• the sample is subjected to a second electrical potential difference whose action on the nucleic acid molecules is opposite in the first direction of flow.
• the second potential difference can therefore be identical to the first potential difference.
• Example 3 Procedure with two returns for more or less salty standard DNA samples.
• a method implementing two returns in a device 40 is described below.
• Graph 220 of figure 19 shows the evolution of the area 221 of the first peak and of the area 222 of the second peak as a function of the NaCI concentration with the six-return process. The values are taken from figure 18.
• Plasma samples are pretreated by proteinase K digestion in the presence of detergent, an operation intended to free the nucleic acids from the vesicles and nucleoprotein complexes in which they are most often trapped.
• This proteinase K digestion step is commonly performed as the first step in DNA purification methods from a biological sample. We will simply make sure to put enough proteinase K (the activity of the enzyme depends on the supplier and the reference chosen), and to choose a non-ionic detergent (an ionic detergent causes electroosmosis and an electric current too high for the method).
• 100 pL of plasma were pretreated with proteinase K; only 15 ⁇ L of pretreated plasma was placed in the Agilent CE injection vial. And about 1 pL of pretreated plasma was injected by the CE Agilent into the device 40 and analyzed by the 7-return method.
• the size profile in Figure 20 is a typical circulating DNA profile, with a main peak around 165 bp, and a second, smaller peak around 305 bp.
• the concentration curve 270 is represented as a function of the size of the DNA fragments of the standard sample added to a plasma sample, using as calibration the migration of the pure standard sample. ; in other words, curve 262 is interpreted using curve 261 to calibrate DNA peak sizes.
• Example 7 comparison of circulating DNA size profiles with or without prior purification of the DNA.
• the concentration of circulating DNA in plasma between 75 and 1650 bp was measured at 7.1 pg/pL by the seven-return method.
• the size profile is visually close; in particular, the asymmetry of the secondary peak and the ratio between the secondary peak and the main peak are similar in the two methods.
• the seven-return process therefore makes it possible to determine the size profile of circulating DNA from only a few microliters of plasma, and without having to purify the DNA beforehand.
• Example 8 performing a return for large DNAs
• the 10K standard sample contains DNA fragments of the following sizes: 1kbp, 2kbp, 3kbp, 4kbp, 5kbp, 6kbp, 8kbp, 10kbp, and 20kbp.
• 1 kbp is equal to 1000 bp, 1000 base pairs.
• Examples 1 to 8 involve capillary devices 40, shown schematically in FIG.
• the method that is the subject of the present invention also applies to microfluidic channels.
• the present invention can therefore be implemented in microfluidic chips having a geometric shape configured so that small DNA fragments leak slowly in the event of high sample conductivity.
• the flow zone that is to say the injection chamber 341 and the capillary 342 corresponding to the narrow channel have a depth (measured perpendicular to Figure 31) of two micrometers.
• the shooting zone 343 is partially represented on the right of FIG. 31 . In this shooting zone, the narrow channel 342 downstream of the constriction has a width, measured from top to bottom in FIG. 31, of 15 ⁇ m.
• the depth here 2 ⁇ m
• the width here 15 ⁇ m
• the depth can be increased, for example, up to 50 ⁇ m
• the width here 15 ⁇ m
• FIG. 32 illustrates, in the form of a flowchart, the steps of a particular embodiment of the desalting and concentration process 370 which is the subject of the invention and of the analysis process 350 which is the subject of the invention.
• the method for desalting and concentrating 370 a sample of nucleic acids that is more conductive than an analysis buffer comprises, first of all, a step 352 of injecting the sample consisting of the nucleic acids and the first buffer in the device. During a step 353, the sample is transferred into the injection chamber 44. During a step 354, a laminar flow of the sample is carried out in a capillary provided with a local restriction of its section , in a first direction of flow. During this flow, the sample is subjected to a first electrical potential difference whose action on the nucleic acid molecules is opposite to the first direction of flow and causes the retention of nucleic acid molecules in the capillary, or at least their braking by pLAS effect in the separation capillary 45.
• a measurement is made of the amperage flowing in the capillary during step 354.
• a selection is made of a number of iterations of a alternation of steps 354 and 358, depending on the amperage measured.
• the selected number of iterations of the alternation is an increasing function of the amperage of a peak of this measured amperage or of a peak of a derivative of this measured amperage.
• the number of returns is not determined by a salinity measurement of the sample, it is predetermined, for example with a value given in one of examples 3 to 8.
• the number of returns, and therefore of iterations is predetermined according to the minimum size of nucleic acids to be concentrated.
• a favorable salinity is defined according to a minimum threshold fixed by the size of the nucleic acids to be concentrated.
• the sizes of the nucleic acids retained are conditioned in particular by the flow rate, the voltage applied and the nature of the analysis buffer such as its conductivity.
• a selection is made for a duration of at least one flow step 354 in the first flow direction.
• the duration selected is a decreasing function of the amperage of a peak of this measured amperage or of a peak of a derivative of this measured amperage.
• step 354-358 the desalting and nucleic acid concentration process is complete and the sample is extracted from the device.
• an optional step 351 for releasing the nucleic acids, with or without purification may precede the iterations of steps 354 to 358.
• a change of analysis buffer is carried out, the new buffer analysis buffer having a pH lower than that of the analysis buffer previously used.
• the new buffer analysis buffer having a pH lower than that of the analysis buffer previously used.
• the change of buffer /pH of step 359 is preferable before carrying out steps 360 to 363. If there are no proteins in the sample, steps 352 to 358 are carried out with an analysis buffer at a pH close to neutrality, or slightly acidic, and, after the last iteration of steps 354 to 358, one goes directly to step 360.
• a laminar flow of the sample is carried out in a capillary provided with a local restriction of its section, in the first direction of flow.
• the sample is subjected to an electrical potential difference whose action on the nucleic acid molecules is opposite to the first direction of flow and causes the retention of nucleic acid molecules in the capillary.
• a separation by laminar flow of the sample is carried out in the capillary in the first direction of flow.
• the sample is subjected to an electrical potential difference less than or equal to the first potential difference, the action of which on the nucleic acid molecules is opposite to the first direction of flow and causes a retention partial nucleic acid molecules in the capillary.
• the difference electrical potential is decreasing during step 361, either in stages, or continuously decreasing, that is to say with a negative and non-zero time derivative.
• a measurement of a temporal fluorescence profile of the nucleic acid molecules is carried out and a step of converting the temporal fluorescence profile into a concentration profile into nucleic acid molecules of different lengths.
• the fluorescence profile of a standard sample is used, the concentration of which is known for each length of nucleic acid molecule to compensate for the experimental variations that exist from one day to another. , in terms of DNA passage time in front of the detector and fluorescence intensity.

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