WO2007091280A1 - Optical apparatus and method for the inspection of nucleic acid probes by polarized radiation - Google Patents
Optical apparatus and method for the inspection of nucleic acid probes by polarized radiation Download PDFInfo
- Publication number
- WO2007091280A1 WO2007091280A1 PCT/IT2006/000061 IT2006000061W WO2007091280A1 WO 2007091280 A1 WO2007091280 A1 WO 2007091280A1 IT 2006000061 W IT2006000061 W IT 2006000061W WO 2007091280 A1 WO2007091280 A1 WO 2007091280A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- radiation
- excitation
- polarization direction
- nucleic acid
- acid probes
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
-
- 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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6445—Measuring fluorescence polarisation
Definitions
- the present invention relates to an optical apparatus and to a method for the inspection of nucleic acid probes by polarized radiation.
- nucleic acids require, according to different modalities, preliminary steps of preparation of a sample of biological material, of amplification of the nucleic material contained therein, and of hybridization of individual target or reference strands, corresponding to the sequences sought. Hybridization occurs (and the test yields a positive outcome) if the sample contains strands complementary to the target strands.
- the sample must be examined to control whether hybridization has occurred (the so called detection step) .
- various inspection methods and apparatuses are known, for example of an optical or electrical type.
- the methods and apparatuses of an optical type are frequently based upon the phenomenon of fluorescence.
- the reactions of amplification and hybridization are conducted so that the hybridized strands, contained in a detection chamber made in a support, include fluorescent molecules or fluorofors (the hybridized strands may be either grafted to the bottom of the detection chamber or remain in liquid suspension) .
- the support is exposed to a light source having an appropriate spectrum of emission, such as to excite the fluorofors.
- the excited fluorofors emit a secondary radiation at an emission wavelength higher than the peak of the excitation spectrum.
- the light emitted by the fluorofors is collected and captured by an optical sensor.
- the optical sensor is provided with band-pass or interferential filters centred at the wavelength of emission of the fluorofors.
- the difference between the maximum peak of the emission spectrum of the fluorofors and the peak of the excitation spectrum is not very high, and the filters, however selective they may be, can only attenuate the light emitted by the source and subsequently diffused, without, however, eliminating it altogether.
- the materials used for providing the supports often have high reflecting power.
- microfluidic devices for the analysis of nucleic acids integrated in semiconductor chips are increasingly widespread.
- the detection chamber often has the bottom coated with a layer of silicon dioxide and, sometimes, also metal electrodes are present, for example of gold or aluminium. In effect, hence, only a relatively small part of the light emitted by the source is absorbed, whereas a conspicuous fraction is reflected and is potentially capable of disturbing the detection of the light emitted by the fluorofors.
- the aim of the present invention is to provide an optical apparatus and a method for the inspection of nucleic acid probes with polarized radiation which will be free from the limitations described.
- an optical apparatus and a method for the inspection of nucleic acid probes are provided, as defined in Claims 1 and 8, respectively.
- FIG. 1 is a top plan view of a chip for analysis of nucleic acids
- - Figure 2 is a cross-sectional view through the chip of Figure 1;
- FIG. 3 is a simplified block diagram of an optical apparatus in accordance with a first embodiment of the present invention
- FIG. 4 is a schematic illustration of the apparatus of Figure 3 in use, into which the chip of Figures 1 and 2 has been loaded;
- - Figure 5 is a graph that shows quantities regarding the apparatus of Figure 3; and - Figure 6 is a simplified block diagram of an optical apparatus in accordance with a second embodiment of the present invention.
- FIGS. 1 and 2 show a chip 1 in which a chemical microreactor for the analysis of nucleic acids (here DNA) is provided.
- the chip 1 comprises: a substrate 2 made of semiconductor material; inlet reservoirs 4; a plurality of microfluidic channels 5; heaters 6 associated to the microfluidic channels 5; and a detection chamber 7.
- the inlet reservoirs 4 and the detection chamber 7 are defined in a structural layer 9 arranged on the surface of the substrate 2 (for example, the structural layer 9 may either comprise a resist layer deposited on the substrate 2 or a glass chip glued thereto) .
- microfluidic channels 5 are buried within the substrate 2, for example as described in EP-A-I 043 770, EP-A-I 130 631, or in the published patent application US 2005/282221, and extend between the inlet reservoirs 4 and the detection chamber 7. Furthermore, the microfluidic channels 5 are fluidly coupled both to the inlet reservoirs 4, through inlet openings 10, so as to be accessible from outside, and to the detection chamber 7, through outlet openings 11.
- the heaters 6, here including resistive elements made of polysilicon, are formed on the surface of the substrate 2 and extend in a direction transverse to the microfluidic channels 5. Furthermore, the heaters 6 are electrically connectable in a known way to external electric power sources (not shown) and can be driven to release thermal power to the microfluidic channels 5 so as to control the temperature within them cyclically according to predetermined thermal profiles .
- the detection chamber 7 accommodates .a plurality of so called “DNA probes" 12, comprising single stranded reference DNA containing predetermined sequences of nucleotides. More precisely, the DNA probes 12 are arranged in predetermined positions so as to form an array and are grafted to an anchorage layer 14, which forms the bottom of the detection chamber 7. After a step of hybridization, some of the DNA probes, designated by 12', are hybridized, i.e., they are bound to individual complementary DNA sequences, and contain fluorofors 15.
- the microreactor integrated in the chip 1 is prearranged for performing reactions of amplification of nucleic material, for example by PCR (Polymerase Chain Reaction) , and hybridization of the DNA probes 12.
- a biological sample containing nucleic material previously treated is supplied to the inlet reservoirs 4 and fed into the microfluidic channels 5.
- the sample is subjected to thermal cycling in order to amplify the DNA present, in a known way.
- the biological sample is further made to advance as far as the detection chamber 7, where the DNA probes 12 are located. If the biological sample contains a sequence of nucleotides complementary to the DNA probes 12, the latter are hybridized.
- the amplification reactions are conducted so that the hybridized DNA probes 12' will contain fluorofors 15 (shown only schematically) having a characteristic emission wavelength.
- number 20 designates an optical inspection apparatus for the detection of hybridized DNA strands, based upon fluorescence.
- the inspection apparatus 20 comprises a control unit 21, a holder 22 for housing an item of the chip 1, a light excitation device 24 and an optical sensor 25, provided with a collimation and focusing device 26 and a sensing polarizing filter 27.
- Figure 3 moreover shows a detail of the chip 1 loaded into the holder 22 so as to be examined.
- the light source 24 comprises a radiant element 28, for example an incandescent lamp or a LED, which emits incoherent non-polarized radiation W 0 with an emission spectrum such as to excite the fluorofors 15, and an excitation polarizing filter 30 associated to the radiant element 28.
- the excitation polarizing filter 30, of a linear type is positioned so as to intercept the non-polarized radiation W 0 emitted by the radiant element 28 and has a predetermined excitation polarization direction D E (i.e., the radiation emerging from the excitation polarizing filter 30 is polarized according to the excitation polarization direction D E ) . Consequently, the excitation radiation W E that leaves the light source 24 is linearly polarized according to the excitation polarization direction D E .
- the light source 24 is moreover oriented so that the excitation radiation W E reaches the detection chamber 7 of the chip 1 with a predetermined angle of incidence (for example 45°) .
- the optical sensor 25, for example of a CMOS or CCD type, receives the fluorescent radiation W F emitted by the fluorofors 15 in the detection chamber 7 of the microreactor integrated in the chip 1. More precisely, the coll.imation and focusing device 26 is arranged so as to collect the fluorescent radiation W F emitted in a direction substantially perpendicular to the chip 1 and orient it in the direction of the optical sensor 25.
- the sensing polarizing filter 27 has a sensing polarization direction D 3 that is transverse, preferably perpendicular, to the excitation polarization direction D E of the reflected radiation W R .
- the reflection does not modify substantially the direction of the electric field E associated to the excitation radiation
- the direction of propagation K R of the reflected radiation W R is determined by the surface conformation of the chip 1 (as well as, of course, by the direction of propagation K E of the incident excitation radiation) , according to the laws of geometrical optics.
- a part of the reflected radiation W R is thus directed towards the optical sensor 25 along an optical path P.
- the sensing polarizing filter 27 is arranged along the optical path P in a plane substantially perpendicular to the direction of propagation K R of the reflected radiation W R directed towards the optical sensor 25.
- the sensing polarization direction Ds of the sensing polarizing filter 27 is perpendicular to the excitation polarization direction D E (i.e., perpendicular to the direction of the electric field E associated to the reflected radiation W R ) .
- the orientation of the sensing polarizing filter 27 is adjustable so as to achieve the most correct alignment.
- the inspection apparatus 20 operates as described hereinafter. Initially, an item of the chip 1, integrating a microreactor in which a step of hybridization of the DNA probes 12 has been performed, is loaded into the holder 22.
- the control unit 21 activates the light source 24, and the excitation radiation W E emitted reaches the detection chamber 7. A fraction of the excitation radiation W E incident on the chip 1 is absorbed by the fluorofors 15 of the hybridized DNA probes 12', whereas the remaining part is reflected or diffused in various directions, according to the surface conformation of the chip 1.
- the fluorofors 15 are hence excited and emit in an approximately isotropic way an fluorescent radiation W F , which does not preserve the state of polarization of the excitation radiation W E .
- the sensing polarizing filter 27 which is located in front of the optical sensor 25.
- the reflected radiation W R is intercepted and almost completely blocked by the sensing polarizing filter 27, because it is polarized in a direction substantially perpendicular to the sensing polarization direction D 3 .
- the effectiveness of the sensing polarizing filter 27 is higher the closer the sensing polarization direction D s is to being perpendicular to the polarization direction of the reflected radiation W R (i.e., the excitation polarization direction D E ) .
- the fluorescent radiation W F due to the excitation of the fluorofors 15, instead, is in part attenuated, but not eliminated completely (transmitted radiation W ⁇ ) , and can hence reach the optical sensor 25, which detects an image IMG and sends it to the control unit 21.
- the sensing polarizing filter 27 enables practically total elimination of the excitation radiation emitted by the light source 24 and reflected by the chip 1, which represents a disturbance. Consequently, the images detected by the optical sensor 25 are produced substantially only by the fluorescent radiation and enable detection of the hybridized DNA probes 12' in an extremely reliable way.
- an optical inspection apparatus 20 for the detection of hybridized DNA strands, based upon fluorescence comprises the control unit 21, the holder 22, a light source 124, the optical sensor 25, the collimation and focusing device 26, and the sensing polarizing filter 27.
- the light source 124 comprises an emitter element 130, which generates directly a coherent monochromatic excitation radiation W E , polarized according to a excitation polarization direction D E (for example, a laser emitter) .
- the emission is spontaneously polarized, and the use of biasing filters associated to the light source 124 is not required.
- the sensing polarizing filter 27 is once again oriented so that the sensing polarization direction D s is substantially perpendicular to the polarization direction of the reflected radiation W R , which is in practice the excitation polarization direction D E .
- the reflected radiation W R is blocked by the sensing polarizing filter 27, and only the fluorescent radiation W F emitted by the fluorofors 15 is transmitted to the optical sensor 25.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
An optical apparatus for the inspection of nucleic acid probes includes: a holder (22) for housing a chip (1) for analysis of nucleic acids, containing nucleic acid probes (12, 12' ); a light (24), for supplying an excitation radiation (WE) to the holder (22); and an optical sensor (25) for detecting images (IMG) of the nucleic acid probes (12, 12'), when a chip (1) is housed in the holder (22). The light source (24) is configured for polarizing the excitation radiation (WE) according to a excitation polarization direction (DE). Furthermore, the apparatus is provided with a sensing polarizing filter (27), which is arranged so as to intercept a reflected portion (WR) of the excitation radiation (WE), directed towards the optical sensor (25). The sensing polarizing filter (27) has a direction of the sensing polarization (DS) transverse to the excitation polarization direction (DE).
Description
"OPTICAL APPARATUS AND METHOD FOR THE INSPECTION OF NUCLEIC ACID PROBES BY POLARIZED RADIATION"
TECHNICAL FIELD The present invention relates to an optical apparatus and to a method for the inspection of nucleic acid probes by polarized radiation.
BACKGROUND ART As is known, the analysis of nucleic acids requires, according to different modalities, preliminary steps of preparation of a sample of biological material, of amplification of the nucleic material contained therein, and of hybridization of individual target or reference strands, corresponding to the sequences sought. Hybridization occurs (and the test yields a positive outcome) if the sample contains strands complementary to the target strands.
At the end of the preparatory steps, the sample must be examined to control whether hybridization has occurred (the so called detection step) . For this purpose, various inspection methods and apparatuses are known, for example of an optical or electrical type. In particular, the methods and apparatuses of an optical type are frequently based upon the phenomenon of fluorescence. The reactions of amplification and hybridization are conducted so that the hybridized strands, contained in a detection chamber made in a support, include fluorescent molecules or fluorofors (the hybridized strands may be either grafted to the bottom of the detection chamber or remain in liquid suspension) . The support is exposed to a light source having an appropriate spectrum of emission, such as to excite the fluorofors. In turn, the excited fluorofors emit a secondary radiation at an emission wavelength higher than the peak of the excitation spectrum. The light emitted by the fluorofors is collected and captured by an optical sensor. In order to eliminate the background light radiation, which
represents a source of disturbance, the optical sensor is provided with band-pass or interferential filters centred at the wavelength of emission of the fluorofors.
However, the difference between the maximum peak of the emission spectrum of the fluorofors and the peak of the excitation spectrum (also referred to as "Stokes shift") is not very high, and the filters, however selective they may be, can only attenuate the light emitted by the source and subsequently diffused, without, however, eliminating it altogether. It should also be taken into account that the materials used for providing the supports often have high reflecting power. For example, microfluidic devices for the analysis of nucleic acids integrated in semiconductor chips are increasingly widespread. In integrated microfluidic devices, the detection chamber often has the bottom coated with a layer of silicon dioxide and, sometimes, also metal electrodes are present, for example of gold or aluminium. In effect, hence, only a relatively small part of the light emitted by the source is absorbed, whereas a conspicuous fraction is reflected and is potentially capable of disturbing the detection of the light emitted by the fluorofors.
DISCLOSURE OF INVENTION The aim of the present invention is to provide an optical apparatus and a method for the inspection of nucleic acid probes with polarized radiation which will be free from the limitations described.
According to the present invention, an optical apparatus and a method for the inspection of nucleic acid probes are provided, as defined in Claims 1 and 8, respectively.
BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding of the invention, some embodiments thereof are now described, purely by way of non-limiting
example and with reference to the attached plate of drawings, wherein:
- Figure 1 is a top plan view of a chip for analysis of nucleic acids; - Figure 2 is a cross-sectional view through the chip of Figure 1;
- Figure 3 is a simplified block diagram of an optical apparatus in accordance with a first embodiment of the present invention; - Figure 4 is a schematic illustration of the apparatus of Figure 3 in use, into which the chip of Figures 1 and 2 has been loaded;
- Figure 5 is a graph that shows quantities regarding the apparatus of Figure 3; and - Figure 6 is a simplified block diagram of an optical apparatus in accordance with a second embodiment of the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION Figures 1 and 2 show a chip 1 in which a chemical microreactor for the analysis of nucleic acids (here DNA) is provided. The chip 1 comprises: a substrate 2 made of semiconductor material; inlet reservoirs 4; a plurality of microfluidic channels 5; heaters 6 associated to the microfluidic channels 5; and a detection chamber 7.
More precisely, the inlet reservoirs 4 and the detection chamber 7 are defined in a structural layer 9 arranged on the surface of the substrate 2 (for example, the structural layer 9 may either comprise a resist layer deposited on the substrate 2 or a glass chip glued thereto) .
The microfluidic channels 5 are buried within the substrate 2, for example as described in EP-A-I 043 770, EP-A-I 130 631, or in the published patent application US 2005/282221, and extend between the inlet reservoirs 4 and the detection
chamber 7. Furthermore, the microfluidic channels 5 are fluidly coupled both to the inlet reservoirs 4, through inlet openings 10, so as to be accessible from outside, and to the detection chamber 7, through outlet openings 11.
The heaters 6, here including resistive elements made of polysilicon, are formed on the surface of the substrate 2 and extend in a direction transverse to the microfluidic channels 5. Furthermore, the heaters 6 are electrically connectable in a known way to external electric power sources (not shown) and can be driven to release thermal power to the microfluidic channels 5 so as to control the temperature within them cyclically according to predetermined thermal profiles .
The detection chamber 7 accommodates .a plurality of so called "DNA probes" 12, comprising single stranded reference DNA containing predetermined sequences of nucleotides. More precisely, the DNA probes 12 are arranged in predetermined positions so as to form an array and are grafted to an anchorage layer 14, which forms the bottom of the detection chamber 7. After a step of hybridization, some of the DNA probes, designated by 12', are hybridized, i.e., they are bound to individual complementary DNA sequences, and contain fluorofors 15.
The microreactor integrated in the chip 1 is prearranged for performing reactions of amplification of nucleic material, for example by PCR (Polymerase Chain Reaction) , and hybridization of the DNA probes 12. For this purpose, a biological sample containing nucleic material previously treated is supplied to the inlet reservoirs 4 and fed into the microfluidic channels 5. Here, the sample is subjected to thermal cycling in order to amplify the DNA present, in a known way. At the end of the amplification step, the biological sample is further made to advance as far as the detection chamber 7, where the DNA probes 12 are located. If the biological sample contains a
sequence of nucleotides complementary to the DNA probes 12, the latter are hybridized. Furthermore, the amplification reactions are conducted so that the hybridized DNA probes 12' will contain fluorofors 15 (shown only schematically) having a characteristic emission wavelength.
With reference to Figure 3, number 20 designates an optical inspection apparatus for the detection of hybridized DNA strands, based upon fluorescence. The inspection apparatus 20 comprises a control unit 21, a holder 22 for housing an item of the chip 1, a light excitation device 24 and an optical sensor 25, provided with a collimation and focusing device 26 and a sensing polarizing filter 27. Figure 3 moreover shows a detail of the chip 1 loaded into the holder 22 so as to be examined.
The light source 24 comprises a radiant element 28, for example an incandescent lamp or a LED, which emits incoherent non-polarized radiation W0 with an emission spectrum such as to excite the fluorofors 15, and an excitation polarizing filter 30 associated to the radiant element 28. The excitation polarizing filter 30, of a linear type, is positioned so as to intercept the non-polarized radiation W0 emitted by the radiant element 28 and has a predetermined excitation polarization direction DE (i.e., the radiation emerging from the excitation polarizing filter 30 is polarized according to the excitation polarization direction DE) . Consequently, the excitation radiation WE that leaves the light source 24 is linearly polarized according to the excitation polarization direction DE.
The light source 24 is moreover oriented so that the excitation radiation WE reaches the detection chamber 7 of the chip 1 with a predetermined angle of incidence (for example 45°) .
The optical sensor 25, for example of a CMOS or CCD type, receives the fluorescent radiation WF emitted by the fluorofors 15 in the detection chamber 7 of the microreactor integrated in the chip 1. More precisely, the coll.imation and focusing device 26 is arranged so as to collect the fluorescent radiation WF emitted in a direction substantially perpendicular to the chip 1 and orient it in the direction of the optical sensor 25.
The sensing polarizing filter 27, which is also of a linear type, is positioned between the collimation and focusing device 26 and the optical sensor 25 so as to intercept the fraction of excitation radiation WE coming from the light source 24 and reflected or diffused by the chip 1 in the direction of the optical sensor 25 (reflected radiation WR; for reasons of convenience, the term "reflected radiation" will be used hereinafter to indicate both the fraction of radiation incident on the chip 1 that is reflected according to a macroscopically predictable path and the fraction of incident radiation that is diffused in the direction of the optical sensor 25, for example on account of the imperfect homogeneity of the surface of the chip 1) . The sensing polarizing filter 27 has a sensing polarization direction D3 that is transverse, preferably perpendicular, to the excitation polarization direction DE of the reflected radiation WR. In this connection (see Figure 5) , the reflection does not modify substantially the direction of the electric field E associated to the excitation radiation
(parallel to the excitation polarization direction DE) , whereas the direction of propagation KR of the reflected radiation WR is determined by the surface conformation of the chip 1 (as well as, of course, by the direction of propagation KE of the incident excitation radiation) , according to the laws of geometrical optics. A part of the reflected radiation WR is thus directed towards the optical sensor 25 along an optical path P. The sensing polarizing filter 27 is arranged
along the optical path P in a plane substantially perpendicular to the direction of propagation KR of the reflected radiation WR directed towards the optical sensor 25. The sensing polarization direction Ds of the sensing polarizing filter 27 is perpendicular to the excitation polarization direction DE (i.e., perpendicular to the direction of the electric field E associated to the reflected radiation WR) . Preferably, moreover, the orientation of the sensing polarizing filter 27 is adjustable so as to achieve the most correct alignment.
The inspection apparatus 20 operates as described hereinafter. Initially, an item of the chip 1, integrating a microreactor in which a step of hybridization of the DNA probes 12 has been performed, is loaded into the holder 22. The control unit 21 activates the light source 24, and the excitation radiation WE emitted reaches the detection chamber 7. A fraction of the excitation radiation WE incident on the chip 1 is absorbed by the fluorofors 15 of the hybridized DNA probes 12', whereas the remaining part is reflected or diffused in various directions, according to the surface conformation of the chip 1. The fluorofors 15 are hence excited and emit in an approximately isotropic way an fluorescent radiation WF, which does not preserve the state of polarization of the excitation radiation WE.
Consequently, a part of the fluorescent radiation WF and the reflected or diffused radiation WE directed towards the optical sensor 25 reach the sensing polarizing filter 27, which is located in front of the optical sensor 25. The reflected radiation WR is intercepted and almost completely blocked by the sensing polarizing filter 27, because it is polarized in a direction substantially perpendicular to the sensing polarization direction D3. In particular, the effectiveness of the sensing polarizing filter 27 is higher the closer the sensing polarization direction Ds is to being
perpendicular to the polarization direction of the reflected radiation WR (i.e., the excitation polarization direction DE) . The fluorescent radiation WF due to the excitation of the fluorofors 15, instead, is in part attenuated, but not eliminated completely (transmitted radiation Wτ) , and can hence reach the optical sensor 25, which detects an image IMG and sends it to the control unit 21.
Advantageously, the sensing polarizing filter 27 enables practically total elimination of the excitation radiation emitted by the light source 24 and reflected by the chip 1, which represents a disturbance. Consequently, the images detected by the optical sensor 25 are produced substantially only by the fluorescent radiation and enable detection of the hybridized DNA probes 12' in an extremely reliable way.
A different embodiment of the invention is illustrated in Figure 6, where parts that are the same as those already shown are designated by the same reference numbers. In this case, an optical inspection apparatus 20 for the detection of hybridized DNA strands, based upon fluorescence, comprises the control unit 21, the holder 22, a light source 124, the optical sensor 25, the collimation and focusing device 26, and the sensing polarizing filter 27.
The light source 124 comprises an emitter element 130, which generates directly a coherent monochromatic excitation radiation WE, polarized according to a excitation polarization direction DE (for example, a laser emitter) . The emission is spontaneously polarized, and the use of biasing filters associated to the light source 124 is not required.
The sensing polarizing filter 27 is once again oriented so that the sensing polarization direction Ds is substantially perpendicular to the polarization direction of the reflected radiation WR, which is in practice the excitation polarization
direction DE .
Hence, as has already been described, the reflected radiation WR is blocked by the sensing polarizing filter 27, and only the fluorescent radiation WF emitted by the fluorofors 15 is transmitted to the optical sensor 25.
Finally, it is evident that modifications and variations may be made to the apparatus and method described herein, without departing from the scope of the present invention, as defined in the annexed claims .
Claims
1. An optical apparatus for the inspection of nucleic acid probes, comprising: - a holder (22) for receiving a chip (1) for analysis of nucleic acids, containing nucleic acid probes (12, 12' ) ;
- a light excitation device (24; 124), for supplying an excitation radiation (WE) to said holder (22) ; and
- an optical sensor (25) , for detecting images (IMG) of said nucleic acid probes (12, 12'), when one said chip (1) is housed in said holder (22) ; characterized in that said light excitation device (24; 124) is configured for polarizing said excitation radiation (WE) according to a predetermined first polarization direction (DE) and in that it comprises a sensing polarizing filter (27), having a second polarization direction (D3) transverse to said first polarization direction (DE) and arranged so as to intercept a reflected portion (WR) of said excitation radiation (WE) , directed towards said optical sensor (25) .
2. The apparatus according to Claim 1, wherein said second polarization direction (Ds) is substantially perpendicular to said first polarization direction (DE) .
3. The apparatus according to Claim 1 or Claim 2, wherein said light excitation device (24) comprises a radiant element (28) , supplying a non-polarized radiation (W0) , and an excitation polarizing filter (30) , associated to said radiant element
(28) for polarizing said non-polarized radiation (W0) according to said first polarization direction (DE) .
4. The apparatus according to Claim 1 or Claim 2, wherein said source (124) comprises a polarized radiation emitter element (130) .
5. The apparatus according to Claim 4, wherein said polarized radiation emitter element (130) is a laser emitter.
6. The apparatus according to any one of the preceding claims, wherein said light source (24; 124) is oriented so that said excitation radiation (WE) reaches said chip (1) with an angle of incidence (α) of approximately 45° .
7. The apparatus according to any one of the preceding claims, comprising a collimation and focusing device (26) arranged so as to collect a fluorescent radiation (WF) emitted by said nucleic acid probes (12, 12') in a direction substantially perpendicular to said chip (1) and to orient said fluorescent radiation (WF) collected towards said optical sensor (25) .
8. A method for the inspection of nucleic acid probes, comprising the step of sending an excitation radiation (WE) to a chip (1) for analysis of nucleic acids, containing nucleic acid probes (12, 12'); characterized in that it comprises the steps of: - polarizing said excitation radiation (WE) according to a predetermined first polarization direction (DE) ; and - filtering a reflected portion (WR) of said excitation radiation (WE) with a polarizing filter (27), having a second polarization direction (Ds) transverse to said first polarization direction (DE) .
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/278,267 US20090303479A1 (en) | 2006-02-06 | 2006-02-06 | Optical Apparatus and Method for the Inspection of Nucleic Acid Probes by Polarized Radiation |
PCT/IT2006/000061 WO2007091280A1 (en) | 2006-02-06 | 2006-02-06 | Optical apparatus and method for the inspection of nucleic acid probes by polarized radiation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/IT2006/000061 WO2007091280A1 (en) | 2006-02-06 | 2006-02-06 | Optical apparatus and method for the inspection of nucleic acid probes by polarized radiation |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2007091280A1 true WO2007091280A1 (en) | 2007-08-16 |
WO2007091280A8 WO2007091280A8 (en) | 2007-12-13 |
Family
ID=36997684
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IT2006/000061 WO2007091280A1 (en) | 2006-02-06 | 2006-02-06 | Optical apparatus and method for the inspection of nucleic acid probes by polarized radiation |
Country Status (2)
Country | Link |
---|---|
US (1) | US20090303479A1 (en) |
WO (1) | WO2007091280A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009151407A2 (en) | 2008-06-14 | 2009-12-17 | Veredus Laboratories Pte Ltd | Influenza sequences |
WO2010102567A1 (en) * | 2009-03-11 | 2010-09-16 | Industrial Technology Research Institute | Apparatus and method for detection and discrimination molecular object |
US8865078B2 (en) | 2010-06-11 | 2014-10-21 | Industrial Technology Research Institute | Apparatus for single-molecule detection |
US8865077B2 (en) | 2010-06-11 | 2014-10-21 | Industrial Technology Research Institute | Apparatus for single-molecule detection |
US9482615B2 (en) | 2010-03-15 | 2016-11-01 | Industrial Technology Research Institute | Single-molecule detection system and methods |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0455067A2 (en) * | 1990-05-03 | 1991-11-06 | F. Hoffmann-La Roche Ag | Micro-optical sensor |
EP0617273A2 (en) * | 1993-03-26 | 1994-09-28 | F. Hoffmann-La Roche Ag | Optical method and device for analyzing substances on sensor surfaces |
US5712705A (en) * | 1993-11-15 | 1998-01-27 | Carl Zeiss Jena Gmbh | Arrangement for analysis of substances at the surface of an optical sensor |
EP1085315A1 (en) * | 1999-09-15 | 2001-03-21 | CSEM Centre Suisse d'Electronique et de Microtechnique SA | Integrated-optical sensor |
US6211954B1 (en) * | 1996-03-30 | 2001-04-03 | Novartis Ag | Integrated optical luminescence sensor |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE377751T1 (en) * | 1995-05-12 | 2007-11-15 | Novartis Erfind Verwalt Gmbh | METHOD FOR THE PARALLEL DETERMINATION OF SEVERAL ANALYTES USING EVANESCENTLY EXCITED LUMINESCENCE |
EP3415641B1 (en) * | 2004-09-17 | 2023-11-01 | Pacific Biosciences Of California, Inc. | Method for analysis of molecules |
-
2006
- 2006-02-06 WO PCT/IT2006/000061 patent/WO2007091280A1/en active Application Filing
- 2006-02-06 US US12/278,267 patent/US20090303479A1/en not_active Abandoned
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0455067A2 (en) * | 1990-05-03 | 1991-11-06 | F. Hoffmann-La Roche Ag | Micro-optical sensor |
EP0617273A2 (en) * | 1993-03-26 | 1994-09-28 | F. Hoffmann-La Roche Ag | Optical method and device for analyzing substances on sensor surfaces |
US5712705A (en) * | 1993-11-15 | 1998-01-27 | Carl Zeiss Jena Gmbh | Arrangement for analysis of substances at the surface of an optical sensor |
US6211954B1 (en) * | 1996-03-30 | 2001-04-03 | Novartis Ag | Integrated optical luminescence sensor |
EP1085315A1 (en) * | 1999-09-15 | 2001-03-21 | CSEM Centre Suisse d'Electronique et de Microtechnique SA | Integrated-optical sensor |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009151407A2 (en) | 2008-06-14 | 2009-12-17 | Veredus Laboratories Pte Ltd | Influenza sequences |
WO2010102567A1 (en) * | 2009-03-11 | 2010-09-16 | Industrial Technology Research Institute | Apparatus and method for detection and discrimination molecular object |
US9778188B2 (en) | 2009-03-11 | 2017-10-03 | Industrial Technology Research Institute | Apparatus and method for detection and discrimination molecular object |
US10996166B2 (en) | 2009-03-11 | 2021-05-04 | Industrial Technology Research Institute | Apparatus and method for detection and discrimination molecular object |
US9482615B2 (en) | 2010-03-15 | 2016-11-01 | Industrial Technology Research Institute | Single-molecule detection system and methods |
US9777321B2 (en) | 2010-03-15 | 2017-10-03 | Industrial Technology Research Institute | Single molecule detection system and methods |
US8865078B2 (en) | 2010-06-11 | 2014-10-21 | Industrial Technology Research Institute | Apparatus for single-molecule detection |
US8865077B2 (en) | 2010-06-11 | 2014-10-21 | Industrial Technology Research Institute | Apparatus for single-molecule detection |
US9995683B2 (en) | 2010-06-11 | 2018-06-12 | Industrial Technology Research Institute | Apparatus for single-molecule detection |
Also Published As
Publication number | Publication date |
---|---|
US20090303479A1 (en) | 2009-12-10 |
WO2007091280A8 (en) | 2007-12-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8062595B2 (en) | Nucleic acid analysis chip integrating a waveguide and optical apparatus for the inspection of nucleic acid probes | |
JP2022031290A (en) | Systems and methods for assessing biological samples | |
EP2160476B1 (en) | Real-time pcr monitoring apparatus | |
JP2009505076A (en) | Method and system for monitoring multiple optical signals from a single signal source | |
US11366062B2 (en) | Biosensor | |
US8753869B2 (en) | Cartridge for biochemical analyses, system for biochemical analyses, and method of carrying out a biochemical process | |
US20090303479A1 (en) | Optical Apparatus and Method for the Inspection of Nucleic Acid Probes by Polarized Radiation | |
US20090194693A1 (en) | Imaging Apparatus for Combined Temperature and Luminescence Spatial Imaging of an Object | |
US7906321B2 (en) | Integrated semiconductor microreactor for real-time monitoring of biological reactions | |
US20230033349A1 (en) | Method and Device for Optically Exciting a Plurality of Analytes in an Array of Reaction Vessels and for Sensing Fluorescent Light from the Analytes | |
EP3502276B1 (en) | Convective pcr device | |
JP2003344290A (en) | Fluorescence detector with temperature controller | |
WO2017030505A1 (en) | Optical structure and optical light detection system | |
WO2015111443A1 (en) | Nucleic acid analyzing device | |
US20050196778A1 (en) | Nucleic acid analysis apparatus | |
KR100818351B1 (en) | Multiple channel bio chip scanner | |
JP2008180677A (en) | Microchip inspection system, microchip inspection apparatus, and program | |
JP2010533297A (en) | Optical fiber detection system | |
US10451551B2 (en) | Methods for high-throughput fluorescence imaging with sample heating capability | |
JP2012023988A (en) | Method for nucleic acid analysis, apparatus for implementing the method, and reagent set for nucleic acid analysis | |
JPWO2008090760A1 (en) | Fluorescence detection device, microchip, and inspection system | |
JP2023548817A (en) | Calibration of single molecule detection systems | |
JP2023550693A (en) | Single molecule detection system using photobleaching information | |
JP3621927B2 (en) | Biological sample measuring method and apparatus | |
JPWO2008090759A1 (en) | Micro total analysis system |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 06711415 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12278267 Country of ref document: US |