FR2747786A1 - INTEGRATED DEVICE FOR OPTICAL MOLECULAR DETECTION AND METHOD FOR SAME - Google Patents

Abstract

A substrate (12) defines a binding site (14) for receiving a molecular receptor (16). A light source (18) and an optical detector (20) are incorporated in the substrate (12). The light source (18) illuminates the binding site (14) through the substrate (12) to produce an optical indication of a molecule (19) hybridized to the molecular receptor (16). The optical detector (20) detects the optical indication through the substrate (12). Preferably, the substrate (12) defines a first side on which the binding site is defined, and a second side opposite the first side on which the light source and the optical detector are integrated. A transparent electrode can be included at the binding site for field-assisted hybridization and de-hybridization.

Description

Headline
INTEGRATED DEVICE FOR OPTICAL MOLECULAR DETECTION
AND METHOD FOR THIS DETECTION
Field of the invention
The present invention relates to integrated devices for optical molecular detection and methods of such detection.

Technological background
Recently, an increased effort has been made to develop microarrays for molecular detection. In general, a molecular detection chip includes a substrate on which is arranged an arrangement of binding sites. Each binding site (or hybridization site) has a respective molecular receptor which binds to, or hybridizes with, a molecule of predetermined structure. A sample solution is deposited on the detection chip, and the molecules contained in the sample bind to or hybridize with one or more of the binding sites. The particular binding sites at which hybridization occurs are detected, and the presence of one or more molecular structures in the sample is subsequently deduced.

Molecular detection chips are of great interest for gene sequencing. These chips, often called DNA chips, use a grouping of binding sites each presenting respective single-stranded DNA test tubes. A sample of single-stranded DNA fragments called
Target DNA, is deposited on the DNA chip. The DNA fragments attach to one or more of the DNA test pieces in a hybridization process. It is possible to determine a nucleotide base sequence in the DNA fragment by detecting the DNA test tubes with which a DNA fragment has hybridized.

 To accelerate the hybridization process, a local concentration of target DNA can be increased at predetermined sites by means of an electric field.

In this case, each site is associated with an electrode intended to selectively generate an electric field close to it. The electric field is generated by applying an electric potential between an electrode located at the site and a counterelectrode located in a peripheral part of the chip.

To attract the DNA fragments to the site, the polarity of the electric potential is chosen so as to generate an electric field having a polarity opposite to the charge of the DNA fragments. To dehybridize the site, an electric field having the same polarity as the DNA fragments can be generated in order to move the DNA fragments away from the site.

 Different approaches have been used to detect a hybridization phenomenon at a binding site. In one of these approaches, a radioactive label is attached to each molecule of a plurality of molecules in the sample. The binding of a molecule to a molecular receptor can then be detected by detecting the radioactive marker.

 Other approaches to detection use fluorescent labels, such as fluorogens, which selectively produce light when hybridization occurs. These fluorogens are lit by a light absorbing source, external to the substrate.

An external charge coupled device (CCD) camera is used to detect the illuminated fluorescence.

 A DNA chip for optical detection is described in "Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides" [Real-time detection of DNA hybridization and fusion on groups of 'oligonucleotides using light waveguides], Proceedings of the National Academy of Sciences, VOL. 92, pp. 6379-6383.

This DNA chip uses a glass substrate, one surface of which defines the binding sites. The glass substrate has one end adjacent to the surface into which light is injected by means of an external light source. An external CCD camera is used to detect scattered light at the binding sites where hybridization occurs.

Brief description of the drawings
The invention is specifically described in the appended claims. However, other characteristics of the present invention will appear more clearly, and the invention will be better understood by referring to the detailed description below, taken in conjunction with the accompanying drawings, among which:
Figure 1 is a block diagram of an integrated molecular detection device according to the present invention;
Figure 2 is a schematic cross-sectional view of an integrated molecular detection device according to the present invention;
Figure 3 is a flow diagram of an embodiment of a method for detecting hybridization of a molecule with a molecular receptor;
Figure 4 is a schematic perspective view of a preferred embodiment of an integrated molecular detection chip according to the present invention; and
Figure 5 is a schematic cross-sectional view of the preferred embodiment of the integrated molecular detection chip.

Detailed description of a preferred embodiment
The embodiments of the present invention advantageously provide an integrated molecular detection device which incorporates binding sites, a light source, and the electronic optical detection circuits on a single chip. A preferred embodiment uses a microreproduced glass substrate, a light source consisting of a polymer light-emitting diode and an optical image transformer consisting of a thin film transistor for producing a fully integrated chip. By further including transparent electrodes near the bonding sites, the device improves hybridization and dehybridization using an electric field. The embodiments of the present invention can be used for applications in DNA sequencing and detection.

 Figure 1 is a block diagram of an integrated molecular detection device 10 according to the present invention. The integrated molecular detection device 10 comprises a substrate 12 which defines a binding site 14 intended to receive a molecular receptor 16. In general, the molecular receptor 16 is chosen according to the type of molecule which is to be detected. The molecular receptor 16 typically comprises a biological or synthetic molecule which has a specific affinity for the molecule to be detected. For DNA sequencing and / or detection applications, the molecular receptor 16 includes at least one DNA test piece which has a specific sequence of base pairs. It should be noted that the embodiments of the present invention are not limited to the detection of hybridization of DNA molecules. For example, embodiments of the present invention can be used to detect antibody-antigen binding.

 A light source 18 is integrated into the substrate 12. The light source 18 illuminates the binding site 14 through the substrate 12 in order to produce an optical indication of the hybridization or of the binding of a molecule 19 with the molecular receptor 16 .

The optical indication can be provided, for example, by a fluorogen which selectively produces light when hybridization occurs.

 An optical detector 20 is also integrated in the substrate 12. The optical detector 20 detects the optical indication through the substrate 12, and thus detects the hybridization or the binding of the molecule 19.

 In one of the preferred embodiments, the substrate 12 is formed of glass, so that the binding site 14 can be illuminated through the substrate 12, and the optical indication can be detected through the substrate 12. In general, the substrate 12 can be formed of an essentially transparent material to allow lighting and detection through it.

 Figure 2 is a schematic cross-sectional view of another embodiment of an integrated molecular detection device according to the present invention. The integrated molecular detection device comprises a substrate 30 which can be formed from glass. The substrate 30 defines a first face 32 (or upper face) and a second face 34 (or opposite face). As illustrated, the first face 32 is opposite the second face 34 in this embodiment.

 A binding site 36 intended to receive a molecular receptor 37 is defined on the first face 32 of the substrate 30. The binding site 36 can be formed by providing a well by chemical etching or by micromachining in the substrate 30. The molecular receptor 37 is deposited in the binding site 36 using a robotic distribution technique, an automatic assembly technique, or other techniques known to those skilled in the art. For DNA sequencing and / or detection applications, the molecular receptor 37 comprises a chain of at least one nucleotide to detect a molecule having a complementary chain of at least one nucleotide. In this case, the molecular receptor 37 can comprise at least one DNA test tube which hybridizes selectively with predetermined DNA molecules.

 A light source 38 is incorporated on the second face 34 of the substrate 30. In one of the preferred embodiments, the light source 38 comprises a polymer light-emitting diode fabricated on the second face 34 of the substrate 30.

 The bonding site 36 is illuminated by the light source 38 through the substrate 30. In other words, the light generated on the second face 34 of the substrate 30 (by the light source 38) is transmitted through the substrate 30 to reaching the binding site 36 on the first face 32. The lighting of the binding site 36 acts to illuminate an optical indicator of a molecule which has hybridized with the molecular receptor 37. The lighting of the optical indicator acts to produce an optical indication of hybridization.

 The optical indicator can be provided by a fluorogen which becomes selectively active when hybridization occurs. In this case, the light source 38 preferably has a spectral characteristic which corresponds to the absorption bands of the fluorogen. The number of fluorescent molecules can be increased by attaching multiple fluorescent molecules to a bead or similar element which is linked to the molecule. It should be noted, however, that the embodiments of the present invention are not limited to the use of fluorescent indicators, and that other optical indicators can be used for the same purpose.

 An optical detector 40 is incorporated on the second face 34 of the substrate 30 in order to detect the optical indication. The optical detector 40 can comprise a photodiode fabricated on the second face 34 of the substrate 30. The optical detector 40 detects the optical indication, and therefore the hybridization of the molecule, through the substrate 30. In other words, the the optical indication generated on the first face 32 of the substrate 30 is transmitted through the substrate 30 to the optical detector 40 on the second face 34.

 In the embodiment of FIG. 2, the light source 38 and the optical detector 40 are located one next to the other on the second face 34 of the substrate 30. To protect the optical detector 40 from the light emitted by the light source 38, the integrated molecular detection device comprises a filter 42 incorporated on the optical detector 40. In this case, the filter 42 may comprise a colored filter intended to eliminate the fluorescent light coming from the light source 38.

 The integrated molecular detection device optionally comprises a transparent electrode 44 incorporated in the substrate 30 at a location close to the binding site 36. The transparent electrode 44 is used to accelerate the hybridization and the dehybridization of the molecule from the receptor molecular 37 by means of an electric field. Because of its transparency, the transparent electrode 44 allows lighting and detection of the optical indicator on the second face 34 of the substrate 30.

 FIG. 3 is a flow diagram of an embodiment of a method for detecting the hybridization of a molecule with a molecular receptor. Preferably, the method is used in conjunction with a molecular detection device as described in this document. It should be noted, however, that the method can also be used to detect hybridization in other devices.

 As indicated in box 50, the method includes a step of providing a substrate which defines a binding site intended to receive the molecular receptor. The substrate can be provided in a molecular detection device, such as the embodiments of the molecular detection device described in this document. As described above, the substrate is formed of a material, such as glass, which allows the transmission of light.

 As indicated in box 52, the method includes an optional step of producing an electric field by means of a transparent electrode located near the bonding site. The electric field is generated to attract molecules to the binding site in order to improve hybridization with the molecular receptor.

 As indicated in box 54, a step of illuminating the binding site through the substrate is performed to produce an optical indication of hybridization of a molecule with the molecular receptor. If a transparent electrode is included, the binding site and the optical indication are illuminated through the transparent electrode. As described above, the binding site can be illuminated from one face of the substrate which is opposite to the face on which the binding site is defined. The bonding site can be illuminated by a light source, such as a light emitting diode, fabricated on the substrate.

 As indicated in box 56, a step of detecting the optical indication through the substrate is carried out. If a transparent electrode is included, the optical indication is detected through the transparent electrode. The optical indication can be detected from a face of the substrate opposite to the face on which the binding site is defined. The optical indication can be detected using an optical detector, such as a photodiode, fabricated on the substrate.

 As indicated in box 58, the method comprises an optional step consisting in generating an electric field by means of the transparent electrode in order to dehybridize the molecule with the molecular receptor.

 Figure 4 is a schematic perspective view of a preferred embodiment of an integrated molecular detection chip according to the present invention. The integrated molecular detection chip comprises a substrate 60 which defines a plurality of binding sites 62. The plurality of binding sites 62 is formed of wells 64 formed by chemical etching on the upper face 66 of the substrate 60. In this embodiment , the plurality of selective binding sites 62 is arranged in the form of a matrix. In other embodiments, the plurality of binding sites 62 can be arranged in another type of grouping.

 Figure 5 is a schematic cross-sectional view of the preferred embodiment of the integrated molecular detection chip. Specific DNA 70 receptors having specific base pair sequences are deposited in wells 64. Specific DNA 70 receptors can be deposited using techniques which include, but are not limited to, robotic delivery techniques and automatic assembly techniques.

 Integrated into the substrate 60, under each site of the plurality of bonding sites 62, is disposed the respective detector of a plurality of optical detectors 72. Each detector of the plurality of optical detectors 72 comprises an a-Si photodiode manufactured in the lower surface 74 of substrate 60.

The plurality of optical detectors 72 can be addressed in a matrix by using thin-film a-Si or poly-Si transistors to form an imaging group in one-to-one correspondence with the binding sites 62.

 Between each adjacent pair of a row of the plurality of optical detectors 72 is a respective source of a plurality of light sources 76 integrated into the substrate 60. Each source of the plurality of light sources 76 comprises a polymer light-emitting diode fabricated in the lower surface 74 of the substrate 60. The plurality of light sources 76 has a spectral characteristic corresponding to the absorption bands of the fluorogens used to provide the optical indication. A color filter 78 is incorporated on each detector of the plurality of optical detectors 72 in order to obscure the light emitted by the plurality of light sources 76.

 In order to speed up the hybridization and dehybridization processes, each site of the plurality of binding sites 62 has a respective electrode of a plurality of transparent electrodes 80 integrated close to it. The transparent electrodes 80 generate an electric field intended to promote hybridization and dehybridization. The transparent electrodes 80 are individually addressable for automatic addressing and assembly purposes. The integrated molecular detection chip can also include a polymer membrane at each site of the plurality of binding sites 62 to facilitate the binding of molecules with them.

 During operation, the appropriate DNA sequences hybridize to the selective sites of the plurality of binding sites 62 using conventional hybridization, or hybridization assisted by electrical or thermal technique. After hybridization, the undesirable sequences, the binding of which is only partial, are dehybridized using an electric field or thermal desorption.

The plurality of light sources 76 emit light, and the fluorescence of each site of the plurality of binding sites 62 is read by the plurality of optical detectors 72. Phase-sensitive detection techniques can be incorporated by modulating a circuit drive connected to each detector of the plurality of optical detectors 72. These techniques are advantageous for improving the sensitivity of the detection.

 The hybridized sites can then be detected in order to determine the specific nucleotide sequences in the DNA sample. The molecular detection chip can then be completely dehybridized using chemical, thermal or electrical means, so that the hybridization and detection process can be repeated.

 Consequently, a concept is described here, as well as several embodiments comprising preferred embodiments of an integrated optical molecular detection device and a method of this detection.

 Because the various embodiments of the present invention integrate a binding site, a light source, and an optical detector on a single substrate, they offer a significant improvement in that a fully integrated molecular device is produced.

 Furthermore, the various embodiments of the present invention, as described in this document, include a transparent electrode located near each binding site in order to facilitate hybridization and dehybridization by means of a electric field. The transparency of the electrode allows both to illuminate and detect an optical indication through the electrode.

 It will be obvious to those skilled in the art that the invention described herein can be varied in many ways and can take the form of many embodiments which are different from the preferred form specifically presented and described above.

 Accordingly, the appended claims are intended to cover all modifications of the invention which respect the spirit and the scope of the present invention.

Claims (10)

 1. Integrated molecular detection device comprising:  a substrate which defines a binding site for receiving a molecular receptor;  a light source integrated in the substrate, the light source being intended to illuminate the binding site through the substrate in order to produce an optical indication of the hybridization of a molecule with the molecular receptor; and  an optical detector integrated in the substrate, the optical detector being intended to detect the optical indication through the substrate.  2. Integrated molecular detection device according to claim 1, in which the substrate is substantially transparent.  3. Integrated molecular detection device according to claim 1 wherein the substrate is formed of glass.  4. Integrated molecular detection device according to claim 1, in which the substrate defines a first face on which the binding site is defined and a second face opposite to the first face on which the light source is integrated.  5. Integrated molecular detection device according to claim 1, in which the molecular receptor comprises a chain of at least one nucleotide, and in which the molecule comprises a complementary chain of at least one nucleotide.  6. Method for detecting the hybridization of a molecule with a molecular receptor, the method comprising the steps of:  providing a substrate which defines a binding site for receiving the molecular receptor;  illumination of the binding site by a light source integrated in the substrate, the binding site being illuminated through the substrate in order to produce an optical indication of the hybridization of a molecule with the molecular receptor; and  detection of the optical indication through the substrate by means of an optical detector integrated in the substrate.  7. The method of claim 6 wherein the optical indication is detected from the second face of the substrate.  8. The method of claim 7 wherein the optical indication is detected by a photodiode fabricated on the second face of the substrate.  The method of claim 7 further comprising the step of protecting the optical detector from light which illuminates the binding site.  10. The method of claim 6 wherein the optical indication is detected through a transparent electrode located near the binding site.