Source: http://www.google.com/patents/US8211693?dq=5251294
Timestamp: 2018-01-21 08:00:00
Document Index: 300473155

Matched Legal Cases: ['application No. 11', '§ 120', 'Application No. 11', '§ 371', 'Application No. 0311204', '§ 119', 'Application No. 10']

Patent US8211693 - Device for separating and/or analyzing several molecular targets dissolved ... - Google Patents
The invention relates to a device for separating and/analyzing several molecular targets dissolved in a complex mixture which is characterized in that it comprises a) a matrix of micro-columns, wherein each micro-column (2) comprises an immobilized molecular probe for retaining a specific molecular target...http://www.google.com/patents/US8211693?utm_source=gb-gplus-sharePatent US8211693 - Device for separating and/or analyzing several molecular targets dissolved in a complex mixture
Publication number US8211693 B2
Application number US 12/700,680
Also published as CA2539995A1, CA2746552A1, CN1882839A, CN1882839B, CN102225325A, CN102225325B, DE602004032156D1, EP1668365A2, EP1668365B1, EP2189792A2, EP2189792A3, US7687257, US20060239864, US20100203540, WO2005036170A2, WO2005036170A3
Publication number 12700680, 700680, US 8211693 B2, US 8211693B2, US-B2-8211693, US8211693 B2, US8211693B2
Inventors Nicolas Ugolin, Sylvie Chevillard, Catherine Ory, Jerome Lebeau
Patent Citations (26), Non-Patent Citations (2), Classifications (18), Legal Events (3)
Device for separating and/or analyzing several molecular targets dissolved in a complex mixture
US 8211693 B2
a) a matrix of micro-columns, wherein each micro-column (2) comprises an immobilized molecular probe for retaining a specific molecular target contained in the complex mixture by specific probe/target linkage,
b) a first capillary network (3) for circulating the complex mixture introduced into the inventive device towards each micro-column of the matrix defined in a),
c) a second capillary network (4) for circulating, after elution, the molecular targets retained on the micro-columns towards a sensor (5) for carrying out the recovery and/or analysis thereof, and
d) if necessary, a sensor (5), preferably in the form of a mass spectrometer, for carrying out the recovery and/or analysis of different molecular targets.
6. The device according to claim 5, wherein the first lower transverse channel makes an angle other than 90° with the network of capillaries located below the plane of the micro-columns.
This application is a Divisional of application No. 11/386,781 filed on Mar. 23, 2006 now U.S. Pat. No. 7,687,257, and for which priority is claimed under 35 U.S.C. § 120. Application No. 11/386,781 is the continuation of PCT International Application No. PCT/PCT/FR2004/002207 filed on Aug. 27, 2004 under 35 U.S.C. § 371. The entire contents of each of the above-identified applications are hereby incorporated by reference. This application also claims priority of Application No. 0311204 filed in France on Sep. 24, 2003, under 35 U.S.C. § 119.
a. a matrix of micro-columns comprising N rows and P columns of micro-columns disposed in the same plane, each micro-column comprising an immobilized molecular type probe which can retain a specific molecular target present in the complex mixture. In accordance with one embodiment of the invention, this is by specific probe/target binding;
b. a first network of capillaries located in a plane parallel to the plane of the micro-column matrix, above the micro-column matrix, said first network allowing movement of a complex mixture introduced into the device towards each micro-column of the matrix defined in a);
c. a second network of capillaries located in a plane parallel to the plane of the micro-column matrix, below the micro-column matrix, said second network allowing movement of molecular targets, after elution, towards one or more detector(s), allowing their recovery and/or analysis;
d. if necessary, a detector allowing recovery and/or analysis of the various molecular targets, preferably a mass spectrometer.
The invention also pertains to a device for separating and/or detecting a plurality of molecular targets in solution in a complex mixture, said device comprising:
a. a network of capillaries allowing movement of a complex mixture introduced into the device;
b. two sets of electrodes disposed either side of the network of capillaries;
a set of functionalized electrodes, the electrodes of which are grafted to probes organized into spots, each probe being capable of retaining a specific molecular target present in the complex mixture, by specific probe/target binding;
a set of non-functionalized electrodes.
The invention also concerns the uses of said device, in particular for separating and/or analyzing DNA or RNA molecules contained in a biological sample.
1) by the difficulty in producing high density arrays with more than 2000 spots. Because of the size of the electrodes and the geometry of the connections used to produce impedance arrays, the hybridization surface becomes very large as soon as the number of spots exceeds 800. However, a large hybridization surface implies a large sample volume which has to cover the hybridization surface, hence the need for a large quantity of biological material to reach the minimum concentration for detection. This is incompatible with experiments in which only a small amount of material is available, such as in diagnostics;
2) by changes in the conformation of the study molecules (probe and/or target), which causes measurement artifacts rendering the measured impedance values impossible to interpret. As an example, deformations in DNA due to the sequence or intramolecular hybridizations causes variations in impedance of the same order of magnitude as for hybridization.
Field effect transistors are used in the prior art as current amplifiers to measure the variation in impedance linked to hybridization of the DNA molecule (ref). The probes are grafted to the transistor gate. When the targets bind, they modify the impedance of the gate and cause a modification to the current between the source (transistor inlet) and the drain (transistor outlet). However, no networked organization has yet been described for that type of detector. The fact of using a field effect transistor as a current amplifier limits the frequency of the alternating currents which can be used to highlight variations in impedance linked to hybridization, further limiting the sensitivity of the detector (ref). Further, in such prior art descriptions, the gate controls the passage of current into the transistor, and the fact of grafting the probes thereto does away with the possibility of using the various terminals of the transistor as electrodes to control the movement of targets and thus to direct hybridization by concentrating the targets at the probes.
a. a matrix of micro-columns comprising N rows and P columns of micro-columns disposed in the same plane, each micro-column comprising an immobilized molecular type probe which can retain a specific molecular target present in a complex mixture by specific probe/target binding;
If appropriate, electrode systems can control/displace the targets in the networks.
As an example, to obtain different migration times for the targets from each micro-column of the same row, the network of capillaries connecting each micro-column to the lower transverse channel may comprise P parallel capillaries, each capillary connecting the P micro-columns of the same row of the matrix to the lower transverse channel and forming an angle with said lower transverse channel which is other than 90°. To further increase the delay, the parts of the capillaries located between the last rows of cells and the transverse channel follow non linear trajectories.
In a preferred embodiment of the device, the first and second network of capillaries are located in planes parallel to the plane of the micro-column matrix, preferably respectively above and below the plane formed by the matrix of micro-columns, and the first network of capillaries, termed the upper network, comprises N parallel capillaries, each capillary connecting the upper transverse channel to P micro-columns of a single row of the matrix, and the second network of capillaries, termed the lower network, comprises P parallel capillaries, each capillary connecting the N micro-columns of one column of the matrix to the lower transverse channel, the angle formed between the two networks of capillaries, lower and upper, being other than 0°, preferably 90°. For this reason, each capillary of the upper network is connected to all capillaries of the lower network via a row of P cells of the micro-column matrix. Reciprocally, each capillary of the lower network is connected to all capillaries of the upper network via a column of N cells of the micro-column matrix.
the targets may be quantified directly on the network of functionalized electrodes. A trihedral prism is fitted to the back of each row electrode of the network of functionalized electrodes to be able to carry out the SPR measurement (see description on SPR detection).
to reduce problems with background noise and detection sensitivity, the SPR measurement may be carried out on the set of non-functionalized electrodes where the face opposite to the capillaries of each electrode is coupled to a trihedral prism (see SPR section). In this detection process, the set of functionalized electrodes may be coupled to a structure where each spot is walled off and delimited by a small cell (FIG. 13). Each cell is a small cup, the base of which is constituted by the functionalized electrode and which opens into the capillary facing the non-functionalized electrode (cell open only to one side of a capillary of the network of capillaries). The walls of the cells prevent the targets of one spot mixing with that of another during migration of one of the targets between the functionalized, electrode and the non-functionalized electrode.
In the context of using cells open only on one side and a single network of capillaries, it is possible to adapt a transverse channel to the system of capillaries and a system for delaying migration (as described for the lower network of capillaries of the double network (see section)). In the context of using a single capillary network, it is possible to do away with the second reservoir and replace it with a channel transverse to the capillary system and a migration delay system (FIG. 14). Hence, the same types of detection described for the system having two superimposed networks of capillaries are applicable to the simplified system.
if the targets are labeled with a fluorescence marker, it is possible to read the hybridized targets directly using a scanner over the set of functionalized electrodes. In similar manner to the use of fluorescence coupled with SPR (excitation of fluorescence molecules by the damped wave of SPR) may be used.
without labeling the targets, the amount of probe/target complexes may be evaluated using fluorescence ligands for the target or the complex. For DNA arrays, acridins may be cited, in particular acridin orange which is an intercalating agent for positively charged DNA. Acridin orange can label single or double strand DNA differently, the free molecules will be eliminated due to the action of the electric fields produced by the systems of electrodes. The set of measurements may be made during hybridization reactions or complexing reactions by dint of the set of electrodes which can manipulate the set of charged molecules. Using this approach, it is possible to evaluate the number of molecules of probes composing the spot and the number of targets which have hybridized; these two measurements allow the real concentrations of the targets in solution to be determined.
To prevent unwanted fluorescence from electrodes, the connections may be produced with ITO type alloys which are transparent in the visible spectrum (indium oxide and tin oxide or any other equivalent alloy, such as . . . ). Since these alloys are highly electrophilic, they must undergo a chemical isolation procedure as described in the chemical isolation section.
hybridization of targets on an electrode;
dehybridization by a chaotropic agent;
maintaining the targets by an electric potential;
reversing the potential to migrate the targets to a non-functionalized electrode;
measuring the SPR on the non-functionalized electrode.
The SPR measurement is made by reflecting light onto the outer face of the distal electrode through prisms disposed on each electrode. It is possible to estimate the dissociation constant by observing the rate at which the measured SPR signal varies with time. By applying an alternating current between a pair of row electrodes, it is possible to measure the association and dissociation probe/target constants. In the particular case of experiments made without denaturing agent, the variations in local concentration induced by the electric field can produce associations and dissociations of probes and targets.
a. introducing a complex mixture containing molecular targets to be separated into a device in accordance with the invention as described above;
b. moving the complex mixture through the micro-column matrix of the device under conditions appropriate for allowing targets to bind specifically to the probes of the micro-columns;
c. eluting targets specifically retained on the micro-column probes;
d. moving targets eluted from the micro-columns towards a detector;
e. recovering and/or analyzing each target using a detector.
In a specific embodiment, the invention pertains to a method for comparative analysis of at least two populations of specific DNA or RNA molecules contained in two biological samples, said method comprising:
a. labeling DNA or RNA molecules derived from a first sample, for example with a heavy isotope, to provide mass differentiation of a labeled DNA or RNA molecule contained in the first sample from that of a DNA or RNA molecule with an identical structure but not labeled contained in a second sample;
b. equimolar mixing of two DNA or RNA populations to be compared;
c. introducing an equimolar mixture into a suitable device of the invention comprising a detector constituted by a mass spectrometer;
d. moving the equimolar mixture through the micro-column matrix of the device under conditions appropriate to allow specific binding of the targets to the micro-column probes;
e. eluting targets specifically retained on the probes of micro-columns;
f. moving targets eluted from micro-columns towards a mass spectrometer;
g. detecting and/or assaying each target, labeled or not labeled, using a mass spectrometer.
In a particular implementation of this method, the device selected to carry it out is constituted in particular by pairs of row electrodes as described above, to control elution and/or migration of targets eluted at each row of the micro-columns. In this case, a preferred implementation of the method comprises the following steps:
a. introducing a complex mixture containing DNA and/or RNA molecules to be separated into the appropriate micro-column device of the invention;
b. moving the complex mixture through the micro-column matrix of the device under conditions appropriate to allow specific binding of targets to the micro-column probes, preferably in a closed circuit;
c. if appropriate, moving a rinsing solution through the micro-column matrix to eliminate non-hybridized or not specifically hybridized molecules, in an open circuit;
d. applying an electric potential difference between the pairs of row electrodes of the micro-columns which is positive at the cell electrodes and negative at the distal electrodes, and applying an electric potential difference between the pairs of transverse channel electrodes, negative at the lower transverse channel, and positive at the upper transverse channel, so that the DNA or RNA molecules are retained in each micro-column after denaturing in step e;
e. moving a denaturing solution through one or more rows of the micro-column matrix under conditions allowing denaturing of the probe/target complexes and thus elution of DNA or RNA molecules;
f. reversing or suppressing the electric differential applied to the row electrode pair of a row of micro-columns of the matrix;
g. moving denatured targets from the micro-columns of a row of the matrix towards a detector;
h. recovering and/or analyzing each target using a detector.
A preferred alternative to the method described above allows the steps for denaturing and migrating the molecular targets to be carried out using only an electric field. Such a method is characterized in that it comprises:
a. introducing a complex mixture containing DNA and/or RNA molecules to be separated into an appropriate device in accordance with the invention;
b. moving the complex mixture through the micro-column matrix of the device under conditions appropriate for allowing specific binding of the targets to the micro-column probes, preferably in a closed circuit, if necessary under the effect of an alternating electric field between the electrodes of the transverse channels and the successive row electrodes, as described above;
c. if necessary, moving a rinsing solution through the micro-column matrix to eliminate non-hybridized or not specifically hybridized molecules, in an open circuit;
d. connecting the lower transverse channel to a first reservoir containing a denaturing solution comprising negative ions capable of migrating in an electric field and connecting the upper transverse channel to a second reservoir comprising a denaturing solution comprising the same negative ions.
e. applying a negative electric potential to the lower and upper transverse channels and the distal row electrodes, and a positive electric potential to the cell row electrodes, so that the negative ions of the solution migrate towards the positively charged micro-columns and cause the molecular targets to denature, the thus denatured molecular targets being immobilized in the micro-columns due to the negative electric potential at the distal row electrodes;
f. applying an electric potential difference between the pairs of transverse channel electrodes, which is negative at the upper transverse channel and positive at the lower second transverse channel;
g. reversing or suppressing the electric differential applied to the row electrode pair of a micro-column row of the matrix to allow the denatured targets to migrate by electrophoresis from the micro-columns of a row of the matrix towards a detector;
The invention also pertains to a method comprising:
a. constituting a device of the invention in which each micro-column contains RNA or DNA targets in proportions representative of a transcriptome;
b. introducing a stoichiometric mixture of probes specifically complementary to the RNA or DNA targets immobilized in each micro-column of the matrix, each specific probe of a target having a molecular weight which differs from the other probes and is thus distinguishable from the others in a mass spectrometer;
c. moving the mixture of probes through the micro-column matrix of the device under conditions appropriate to allow specific binding of the probes to the targets immobilized on the micro-columns;
d. eluting probes specifically retained on the targets immobilized in the micro-columns;
e. migrating probes eluted from the micro-columns towards a detector constituted by a mass spectrometer;
f. analyzing each probe using a mass spectrometer.
As an alternative to the preceding method, it is possible to use the set of probes constituted by specific probes, the size of each specific probe for a target differing from the other probes and/or labeled by a specific fluorescence marker, detection being carried out after capillary electrophoresis of probes using a suitable spectrophotometer.
a. introducing a complex mixture containing molecular targets to be separated into a device of the invention comprising firstly, a network of capillaries allowing the complex mixture to be moved and secondly, two sets of electrodes in accordance with the description above;
The implementation of this method is illustrated in the examples and Figures.
a. a set of magnetic particles on which the set of targets representative of a transcriptome to be analyzed is immobilized; and
b. a set of different types of probes in which each probe type of the set of probes is specific and complementary to one target type to form a stoichiometric mixture with said targets.
The invention concerns a method as herein defined, characterized in that each probe type of the set of probes is specific and complementary to a target type to form a stoichiometric mixture.
1) the dehybridized targets are held in place by the electrode field;
2) the targets migrate by dint of the field to the non-functionalized electrode;
3) SPR detection takes place;
4) The current may alternatively be cut to improve detection.
FIG. 17 illustrates a device with two crossed sets of superimposed electrodes to carry out impedance measurements.
EXAMPLES 1. Example of a Device for Detecting DNA or RNA Molecules Contained in a Biological Sample
The capillaries have a diameter in the range 1 to 100 μm. All of the capillaries of the upper network open into a transverse capillary (31) (upper transverse channel) with a diameter in the range 2 to 1000 μm. The upper transverse channel thus connects all of the capillaries of the upper network; it is perpendicular to the direction of the upper network. The ends of the capillaries of the upper network opposite to the transverse channel stop at the last connection with the Pth and last cells of the cell rows of the micro-column matrix. All of the capillaries of the lower network open into a transverse capillary (41) (lower transverse channel) with a diameter in the range 2 to 1000 μm. The lower transverse channel thus connects all of the capillaries of the lower network. The capillaries of the lower network are produced so that the trajectory between the connection with the first cell of a cell column and the lower transverse channel has a different length from one lower capillary to another. This portion of a capillary of the lower network is termed the delay. The delay is obtained using a lower transverse channel making an angle other than 90° with the lower capillary network. Depending on the angle selected, the delays increase or decrease between successive lower capillaries.
2. Example 2 Pistonless Device
3. Example 3 Preparation of a Micro-Column Containing a Set of Nucleic Acids Representative of a Transcriptome and its Use in Analysis of a Transcriptome
4. Example 4 Use of a Micro-Column Array as Described in Example 1 to Analyze RNA Samples
5. Example 5 Use of a Micro-Column Array as Described in Example 2 to Analyze RNA Samples
6. Example 6 Micro Sequencing Biopolymer Separated and Isolated in Each Micro-Column
A=curved connection electrode (for example a glass sheet with transparent ITO electrodes)
B=functionalized row electrodes (for example a glass sheet with transparent ITO electrodes)
C=reservoir electrode (for example a glass sheet with transparent ITO electrodes)
D=capillary without floor or top (for example hollowed into Kapton): median plane
E=capillary without floor but with top (for example hollowed into Kapton): median plane
F=reservoir without floor or top (for example hollowed into Kapton): median plane
G=non-functionalized row electrodes (for example a glass sheet with transparent ITO electrodes)
H=non-hybridized targets
I=functionalized electrodes
J=non-functionalized electrodes
K=spots of probes: hybridization unit
L=prism
M=Horizontal gate electrode
N=vertical source electrode
O=spot electrode
P=gate
R=drain
T=cell without base or top
S=direction of movement of current
U=network of capillaries with delay
V=connection row electrode for analysis
W=secondary transverse channel.
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International Classification G01N33/543, B01L3/00, C12M1/34, B01J19/00
Cooperative Classification B01L2300/0819, B01L2400/0478, G01N27/44773, B01L3/502761, B01L2400/0406, G01N27/44782, B01L2400/0415, G01N1/405, B01L3/502715
European Classification B01L3/5027H, G01N27/447C5, B01L3/5027B, G01N27/447C4