EP1428026A1 - Puce a biocapteur fluorescent et ensemble puce a biocapteur fluorescent - Google Patents

Puce a biocapteur fluorescent et ensemble puce a biocapteur fluorescent

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Publication number
EP1428026A1
EP1428026A1 EP02758143A EP02758143A EP1428026A1 EP 1428026 A1 EP1428026 A1 EP 1428026A1 EP 02758143 A EP02758143 A EP 02758143A EP 02758143 A EP02758143 A EP 02758143A EP 1428026 A1 EP1428026 A1 EP 1428026A1
Authority
EP
European Patent Office
Prior art keywords
biosensor chip
electromagnetic radiation
fluorescence biosensor
wavelength range
fluorescence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02758143A
Other languages
German (de)
English (en)
Inventor
Meinrad Schienle
Ralf Brederlow
Franz Hofmann
Martin Jenkner
R. Johannes Luyken
Christian Paulus
Petra Schindler-Bauer
Roland Thewes
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Infineon Technologies AG
Original Assignee
Infineon Technologies AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Infineon Technologies AG filed Critical Infineon Technologies AG
Publication of EP1428026A1 publication Critical patent/EP1428026A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G01N21/6454—Individual samples arranged in a regular 2D-array, e.g. multiwell plates using an integrated detector array
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/064—Stray light conditioning
    • G01N2201/0642—Light traps; baffles

Definitions

  • the invention relates to a fluorescence biosensor chip and a fluorescence biosensor chip arrangement.
  • Bio and genetic engineering is to be able to detect biological molecules such as DNA (deoxyribonucleic acid) or RNA, proteins, polypeptides etc.
  • biological molecules such as DNA (deoxyribonucleic acid) or RNA, proteins, polypeptides etc.
  • DNA molecules deoxyribonucleic acid
  • detection methods are becoming increasingly important in the industrial identification and evaluation of new drugs of organic and genetic engineering origin. These detection methods open up diverse applications, for example in medical diagnostics, in the pharmaceutical industry, in the chemical industry, in food analysis as well as in environmental and food technology.
  • a DNA is a double helix, which is made up of two cross-linked helical single chains, so-called half-strands. Each of these half-strands has a base sequence, the genetic information being determined by means of the order of the bases (adenine, guanine, thymine, cytosine).
  • Half strands of DNA have the characteristic property of being very specific in binding only to very specific other molecules. It is therefore a prerequisite for docking a strand of nucleic acid to another strand of nucleic acid that the two molecules are complementary to one another.
  • the two molecules must clearly fit together like a key and the matching lock (so-called key-lock principle). This principle, given by nature, can be used for the selective detection of molecules in a liquid to be examined.
  • catcher molecules for example by means of microdispensing
  • catcher molecules are first applied and immobilized on a substrate made of a suitable material, ie are permanently fixed to the surface of the biochip sensor.
  • catcher molecules for example by means of microdispensing
  • it is known to immobilize biomolecules with thiol groups (SH groups) on gold surfaces.
  • Presence of DNA half-strands complementary to the capture molecules can be used.
  • the liquid to be examined for the presence of a specific DNA half-strand must be brought into active contact with the immobilized capture molecules.
  • the DNA half strand hybridizes to the catcher molecule, i.e. he is bound to it. If, as a result of this binding, the value of a physical variable that can be measured is changed in a characteristic manner, the value of this variable can be measured and the presence or absence of a DNA half-strand in a liquid to be examined can be detected in this way.
  • nucleic acids can be used as capture molecules for peptides or proteins that bind specifically to nucleic acids. It is also known to use peptides or proteins as capture molecules for other proteins or peptides that bind the capture peptide or capture protein. It is also important the use of low molecular weight chemical compounds as capture molecules for proteins or peptides that bind to these low molecular weight compounds. Low molecular weight chemical compounds are those chemical compounds that have less than about 1,700 daltons (molecular weight in grams per mole). Conversely, the use of proteins and peptides as capture molecules for low-molecular compounds which may be present in a liquid to be examined is also possible.
  • Electronic detection methods are known for the detection of the binding that has taken place between the capture molecule applied to the substrate and the molecule to be detected that is present in the liquid to be examined.
  • the value of the capacitance can be measured between two electrodes on which capture molecules are immobilized. If molecules to be detected hybridize with the capture molecules, the value of the capacity changes in a characteristic manner and the hybridization event can be detected by means of an electrical signal.
  • a DNA sensor is described for example in [1].
  • the detection sensitivity of such electronic detection methods for DNA molecules is limited. Problems also arise in such a way that sensitive biomolecules (e.g. DNA, proteins) can be decomposed if they come into direct contact with free electrical charges on the surface of electrodes. It is known that many proteins denature outside of a range of pH values characteristic of each protein.
  • a hybridization event can be detected optically when a hybridized molecule has a fluorescent dye capable of emitting fluorescent electromagnetic radiation in a characteristic wavelength range after the fluorescent dye has been excited by absorbing light of a primary wavelength range.
  • biomolecules to be detected containing analytes for example DNA half-strands, are to be coupled to a fluorescence marker via a suitable linker molecule.
  • the biomolecules to be detected fluorescence-labeled in this way have hybridized with the capture molecules immobilized on the sensor surface, and if light of a suitable wavelength is radiated, which can be absorbed by the fluorescence marker, then the irradiated light is absorbed by the fluorescence markers and light quanta of a different wavelength are re-emitted (resonance fluorescence) , The intensity of the fluorescent light re-emitted from the sensor surface is then a measure of the number of docked molecules to be detected.
  • the re-emitted fluorescent light basically has a longer wavelength (and lower energy) than the exciting primary light. This physical effect makes it possible to separate the fluorescent light from the exciting light by using suitable optical filters which absorb, reflect or transmit depending on the wavelength. If these filters are chosen appropriately, for the wavelength of the
  • the intensity of the fluorescent light to be detected is often several orders of magnitude lower than the intensity of the exciting primary light, which complicates the measurement of the fluorescent light and limits the detection sensitivity of the sensor.
  • the intensity of the fluorescent light should be quantitatively detectable over the largest possible range by means of the sensor (high dynamic range).
  • a good spatial resolution is required of a sensor arrangement, since the sensor elements of the arrangement are often equipped with different capture molecules in order to be able to simultaneously detect different molecules to be detected. High demands are therefore placed on the quality of the optics of a reading device.
  • Known readers typically use a laser scanner for excitation and a confocal microscope to detect the emitted light.
  • An optical edge filter is also inserted in the detection beam path, which suppresses the exciting wavelength ("long wave pass").
  • FIG. 1A shows a fluorescence biosensor chip 100 which is known from [2].
  • the fluorescence biosensor chip 100 has a light source 101 which emits light 100a
  • Wavelength range emitted The light 100a emitted by the light source 101 passes through the light source filter 102, as a result of which essentially monochromatic primary light falls on the biochip 103.
  • a biological sample is attached to the biochip 103, the biological molecules having a fluorescent marker.
  • the fluorescence markers of the biomolecules on the biochip 103 are set up in such a way that they absorb the light from the light source 101 transmitted through the light source filter 102. After the light has been absorbed, the fluorescent markers re-emit light of a second wavelength which differs from the wavelength of the incoming light. The re-emitted light has a longer wavelength than the primary light 100a (red shift). That from the fluorescent markers of the biomolecules on the biochip
  • the lens 104 which is set up such that it images the individual light signals in the correct location on the CCD sensor device 106.
  • the sensor filter 105 is set up in such a way that it is transparent to the wavelength of the re-emitted light, whereas it is transparent to the wavelength of the primary light.
  • the CCD sensor arrangement 106 (“Charge coupled device”) registers the fluorescence events on the biochip 103.
  • the adjustment of the fluorescent biosensor chip 100 which is complex due to the optics or the complicated measuring system, is complicated, which means that the user-friendliness of the Fluorescence biosensor chips 100 result. This is disadvantageous.
  • the fluorescence biosensor chip 100 is expensive because it has expensive individual components such as the CCD sensor arrangement 106.
  • a further fluorescence biosensor chip 110 is known from [3], [4] and is shown in FIG. IB.
  • the fluorescence biosensor chip 110 has a light source 111, which emits light purple of a primary wavelength range.
  • the light purple emitted by the light source 111 first passes through an optical element 112 and then through a light source filter 113.
  • the light source filter 113 is set up in such a way that it is only permeable to electromagnetic radiation of a certain wavelength or a certain wavelength range. That through that
  • Light source filter 113 transmitted light is deflected by means of an optical reflector element 114 and thereby passes into cavities 116 of a sample holder 115, in which the biological molecules to be examined are arranged. If a hybridization event has taken place in one of the cavities 116, ie if molecules having a fluorescence marker have hybridized with the capture molecules in one of the cavities 116, suitably chosen fluorescence markers can absorb the light from the light source 111 incident on the cavities 116 and shift them towards longer wavelengths Re-emit wavelength. The primary light and the re-emitted light arrive at the sensor filter 117, which is transparent to light of the wavelengths of the fluorescent radiation, whereas it is essentially impermeable to light of the wavelengths of the primary light.
  • the individual components of the fluorescence biosensor chip 110 can be assembled by the user. It is the spatial separation of the components that leads to a large spatial Expansion leads to a reduction, but the fluorescence biosensor chip 110 is not very easy to use. Furthermore, the fluorescence biosensor chip 110 is too expensive for many applications.
  • the fluorescence biosensor chips known from the prior art have a complicated structure and a complex structure, are large and therefore expensive. Furthermore, the fluorescence biosensor chips known from the prior art are sometimes not very user-friendly.
  • Another sensor chip is known from [5]. This has a photodiode manufactured according to the CMOS process and an integrated Fabry-Perot filter.
  • a Fabry-Perot filter is made up of two semitransparent mirrors which are arranged at a defined distance from one another, the inner surface of the first mirror ideally being totally reflective and the inner surface of the other mirror having a reflectivity only slightly below one. If incident light passes through the first mirror, the light is often reflected on the inner surface of the second mirror and then on the inner surface of the first mirror, then again on the inner surface of the second mirror, etc., with each reflection on the inner surface of the second A small proportion of the mirror is also transmitted through the second mirror. The transmitted individual beams interfere in such a way that the Fabry-Perot interferometer is only permeable to light of certain wavelengths.
  • the biosensor known from [5] is not intended for the detection of biological molecules.
  • a sensor arrangement known from [6].
  • a camera is known on the basis of photodiodes integrated in a substrate, an image point of the image to be recorded by the camera being composed of three photodiodes, which three photodiodes according to the RGB system with a red, a green and a blue Filters are covered.
  • [7] discloses a device and a method with a field light source array for integrated sample acquisition.
  • [8] discloses a method for producing a carrier coated with biologically or chemically functional materials.
  • [9] describes a light emission detection device which has an LCD matrix as a two-dimensional controllable light source and a CCD matrix opposite the LCD matrix for detecting the optical behavior of a respective sample substance located between the LCD matrix and the CCD matrix ,
  • a method and a device for spatially resolved fluorescence-optical detection of substances immobilized on a surface of a planar carrier are known from [10].
  • the amount of probes fixed on spots of a glass plate is determined by using fluorescent material to identify the probes to emit light.
  • the amount of sample hybridized with the probes is determined by causing fluorescent material to identify the sample to emit light.
  • [12] discloses an analysis substrate using the transmission of fluorescent light.
  • the invention is based on the problem of creating a less complex and therefore less expensive fluorescence biosensor chip.
  • the problem is solved by a fluorescence biosensor chip and a fluorescence biosensor chip arrangement with the features according to the independent patent claims.
  • a fluorescence biosensor chip has a substrate, at least one detection device arranged in or on the substrate for detecting electromagnetic radiation, an optical filter layer arranged on the substrate and an immobilization layer arranged on the optical filter layer for immobilizing
  • all components of the fluorescence biosensor chip are therefore integrated in the fluorescence biosensor chip. Because all components of the fluorescence biosensor chip are spatially very closely adjacent, the fluorescence biosensor chip has a very small size. This provides a very compact fluorescence biosensor chip.
  • Immobilization layer which according to the invention serves as a sensor level
  • the detection devices integrated in the substrate which serve for the indirect detection of hybridization events, are typically arranged on the order of magnitude less than 100 ⁇ m from one another, which results in a good spatial resolution of the fluorescence biosensor chip Has.
  • the fluorescence biosensor chip according to the invention is also designed such that it can be produced using standardized CMOS-compatible semiconductor technology processes. The development of expensive machines for producing the fluorescence biosensor chip is therefore unnecessary, as a result of which the fluorescence biosensor chip can be produced inexpensively and with little effort.
  • the individual components of the fluorescence biosensor chip can also be produced from inexpensive materials.
  • the substrate is preferably made of silicon material.
  • the substrate can be a silicon wafer.
  • the at least one detection device of the fluorescence biosensor chip according to the invention has at least one photodiode, which is set up in such a way that electromagnetic radiation of a first wavelength range can be detected.
  • the at least one detection device as a photodiode, which is integrated in the substrate, a sensitive and inexpensive to produce detector for electromagnetic radiation is provided.
  • the optical filter layer is preferably set up in such a way that the optical filter layer emits electromagnetic radiation from a second
  • the optical filter layer is clearly set up in such a way that it absorbs and / or reflects that part of the electromagnetic radiation incident on the surface of the optical filter layer that is to be shielded by the photodiode, since this electromagnetic radiation is not the radiation to be detected.
  • the first wavelength range in which the photodiode is sensitive to the detection of electromagnetic radiation, lies outside the second wavelength range, it is ensured that the electromagnetic radiation to be detected by the photodiode can at least partially penetrate the optical filter layer.
  • the absorption layer suppresses the irradiation of the photodiodes with electromagnetic radiation which is not to be detected by those hybridized on the immobilization layer
  • Molecules originate, for example, scattered light from the environment or primary light for exciting fluorescent markers of molecules to be detected that may be hybridized on the immobilization layer.
  • the Detection sensitivity of the fluorescence biosensor chip can be increased.
  • the optical filter layer preferably has at least one bandpass filter and / or at least one edge filter.
  • a band filter is further understood to mean an optical filter which is essentially opaque to electromagnetic radiation in a wavelength range between a lower limit wavelength and an upper limit wavelength, whereas the band filter is essentially transparent to electromagnetic radiation below the lower limit wavelength and above the upper limit wavelength ,
  • An edge filter is further understood to mean an optical filter that is essentially either opaque to electromagnetic radiation below a cutoff wavelength and is transparent to electromagnetic radiation above the cutoff wavelength, or that is opaque to electromagnetic radiation above a cutoff wavelength and for electromagnetic radiation below the cutoff wavelength is permeable.
  • Filter layer can be a dielectric interference filter with a layer sequence of at least two materials, wherein a first material has a high refractive index and a second material has a low refractive index.
  • the first material with a high refractive index is preferably one of the materials titanium oxide (Ti0 2 ), silicon nitride (Si 3 N, hafnium oxide (Hf0 2 ), zirconium oxide (Zr0 2 ), aluminum oxide (Al 2 0 3 ), polysilicon (polycrystalline silicon) or Indium tin oxide (ITO), but the first material can also be silicon dioxide (Si0 2 ).
  • the first material can be any mixture of the named or other materials, such that the first material has a suitable refractive index.
  • the use of most of the materials mentioned as the first material for the dielectric interference filter has the The advantage that layers of the materials mentioned can be applied using standardized CMOS processes. This has an advantageous effect on the cost of the fluorescence biosensor chip, since it enables the fluorescence biosensor chip to be produced using standardized and sophisticated methods.
  • the second material of the dielectric interference filter with a low refractive index is preferably silicon dioxide (Si0 2 ), which is also compatible with CMOS processes and thus the inexpensive and inexpensive manufacture of the
  • the second material can also be one of the materials titanium oxide (Ti0 2 ), silicon nitride (Si 3 N 4 ), hafnium oxide (Hf0 2 ), zirconium oxide (Zr0 2 ), aluminum oxide (Al 2 0 3 ), polysilicon (polycrystalline silicon) or indium -Tin Oxide (ITO).
  • the second material can be any mixture of the named or other materials, such that the second material has a suitable refractive index.
  • the materials of the dielectric filter of the fluorescence biosensor chip according to the invention are not restricted to the materials mentioned. Any other suitable material with a sufficiently high refractive index can be selected for the first material with a high refractive index, and any other suitable material with a sufficiently low refractive index can be selected for the second material with a low refractive index.
  • the dielectric interference filter for light between a first
  • Cut-off wavelength and a second cut-off wavelength should be as opaque as possible.
  • the interference filter should be set up in such a way that it has a transmission coefficient of ideally zero, realistically as close to zero as possible, for electromagnetic radiation with a wavelength above the lower limit wavelength and below the upper limit wavelength.
  • the dielectric interference filter for electromagnetic radiation with a wavelength below the lower limit wavelength or above that The upper limit wavelength should be as transparent as possible, ie have a transmission coefficient of ideally one for electromagnetic radiation of the wavelength ranges mentioned, realistically as close as possible to one.
  • the dielectric interference filter should have a large edge steepness, that is to say that the transmission coefficient should drop as suddenly as possible from one to zero at the lower limit wavelength and rise as suddenly as possible from zero to one at the upper limit wavelength.
  • the dielectric interference filter is preferably an arrangement of 31 layers with alternating high and low refractive index:
  • the layer thicknesses are given in quarters of optical wavelengths, i.e. in multiples and fractions of «/ 4.
  • the designation 0, 5H denotes a layer made of a high-index ("H" for "high") material, the thickness of which corresponds to half a quarter of the wavelength of the incident light in the medium passing through.
  • 0.5H therefore designates a «/ 8 layer made of the highly refractive material, where • is the quotient of the vacuum light wavelength and the refractive index of the medium.
  • the «/ 8-layer of the high-index material is followed by a * / 4-layer of the low-index material (" L “for” low “). This is followed by 14 • / 4 double layers of alternating high-index material and low-index material.
  • the layer arrangement is in turn closed off by a »/ 8 layer made of the highly refractive material.
  • the layer system described is composed of alternating layers of silicon dioxide material (low refractive index) and silicon nitride material (high refractive index).
  • the wavelength of the reflection maximum can be determined at a fixed angle of incidence of the light.
  • the dielectric interference filter made of 31 layers of silicon dioxide / silicon nitride, light in a wavelength range between approximately 350 nanometers and approximately 390 nanometers is reflected to more than 99%.
  • the optical filter layer of the fluorescence biosensor chip of the invention can also have at least one edge filter.
  • the edge filter is preferably a color filter made from an organic material.
  • Such color filters made of organic materials have a wavelength-dependent absorption coefficient.
  • Such color filters made of organic materials often do not have steep filter flanks, as are required for a large dynamic range, but such filters have the advantageous property of often not having a strong ripple, i.e. to have no oscillatory characteristics in the absorption coefficient-wavelength characteristic.
  • the use of edge filters is therefore particularly advantageous according to the invention if an edge filter is combined with a bandpass filter.
  • the suitable combination of at least one bandpass filter and / or at least one edge filter makes it possible to be able to adjust the absorption properties of the optical filter layer of the fluorescence biosensor chip of the invention flexibly to the needs of the individual case.
  • the optical filter layer can be simply designed.
  • the optical filter layer can be designed to optimize
  • the optical filter layer According to the invention, a desired balance between cost-effectiveness and accuracy of detection can be achieved.
  • the fluorescence biosensor chip preferably also has a circuit layer between the substrate and the optical filter layer, with the circuit layer at least one electrical component is integrated and the circuit layer is electrically coupled to the at least one detection device.
  • the circuit layer By arranging the circuit layer between the substrate and the optical filter layer, it is possible to manufacture the fluorescence biosensor chip with the circuit layer according to a standardized CMOS process. This contributes to the low cost of the fluorescence biosensor chip.
  • the circuit layer essentially serves to electrically read out a hybridization event detected by the detection devices on the immobilization layer. If a hybridization event occurs on the immobilization layer and an electromagnetic fluorescence signal is emitted in the direction of the photodiodes by the hybridized molecules to be detected, then charge separation takes place in the photodiodes, which can be read out electrically by means of the electronic components of the circuit layer.
  • the at least one detection device can be electrically controlled by means of the circuit layer.
  • each individual photodiode can be read out to determine whether it has an electrical signal due to a hybridization event on the immobilization layer.
  • the immobilization layer of the fluorescence biosensor chip has, for example, one or a combination of the materials silicon dioxide, silicon nitride, organic material and / or gold.
  • Catcher molecules are set up in such a way that a molecule which is complementary to the catcher molecule and can be detected can be coupled to the ready-to-bind catcher molecules.
  • the number of molecules to be detected can be greater than the number of molecules on the immobilization layer of a fluorescence biosensor chip immobilized capture molecules. If each of the capture molecules of a fluorescence biosensor chip has hybridized with a molecule to be detected, the fluorescence biosensor chip is in "saturation", ie it has no ready-to-bind
  • Capture molecules can in particular be nucleic acids (DNA or RNA), peptides, polypeptides, proteins or low molecular weight compounds. In chemistry, low-molecular compounds are understood to mean compounds with molecular masses of less than 1,700 daltons (molecular mass in grams per mole).
  • the material or materials from which the immobilization layer is made is or are matched to the capture molecules to be coupled.
  • the capture molecules are immobilized on the surface of the immobilization layer using the microdispensing technique. This automatically (“soap assembly” technique) forms bonds between the material of the immobilization layer and those end groups of the capture molecules which form a chemical bond with the material of the immobilization layer.
  • the material pair gold / sulfur has particularly advantageous properties, so that the connection of sulfur-containing groups (for example thiol end groups) of capture molecules with immobilization layers made of gold material is to be mentioned as a particularly advantageous combination.
  • the catcher molecules are very selective to very specific molecules to be detected which are complementary to the catcher molecules. In other words, only very specific, structurally appropriate molecules to be detected attach to a specific capture molecule. If different capture molecules are attached to the surface of the immobilization layer, a parallel analysis of different substances to be detected is possible.
  • the parallel analysis of different substances to be detected for example different DNA half strands or different proteins, has a time-saving effect and is particularly interesting for "high throughput screening" analyzes.
  • the analysis of a solution of an unknown composition can ideally be carried out in a single analysis step using the fluorescence biosensor chip according to the invention. Such a highly parallel analysis saves time.
  • Detection devices not only incident the light to be detected from the molecules to be detected hybridized with the capture molecules. Rather, stray light from the surroundings or primary light provided for exciting fluorescent markers also falls on the detection devices. This parasitic electromagnetic radiation falsifies the signal of the detection devices. It is therefore desirable to quantitatively measure the strength of this noise signal (or background signal) and to subtract it from the detected signals.
  • This can be achieved according to the invention in that a surface section of the immobilization layer is free of capture molecules, so that a noise signal can be removed from the at least one detection device arranged below this surface section.
  • the noise signal (also called background or background signal) can also be measured simultaneously by several detection devices, which further increases the sensitivity of the detection.
  • the molecules to be detected and / or the capture molecules preferably have a fluorescent marker, the fluorescent marker being set up in such a way that it emits electromagnetic radiation from a third
  • Wavelength range emitted at least part of the third wavelength range lying outside the fourth wavelength range, and at least part of the fourth wavelength range lying within the first wavelength range.
  • the fluorescence biosensor chip of the invention is described clearly below. If no molecules to be detected with fluorescent markers are attached to the capture molecules on the surface of the fluorescence biosensor chip, then externally irradiated light passes through the capture molecules and the immobilization layer essentially without weakening. However, the incident light is reflected by an appropriately selected filter layer and therefore does not reach the photodiodes integrated in the substrate.
  • the molecules to be detected can hybridize with the capture molecules arranged on the immobilization layer of the fluorescence biosensor chip if the capture molecules and the molecules to be detected are based on the key - Match the lock principle.
  • the hybridized molecules to be detected are provided with a suitable fluorescent marker.
  • the capture molecules can also be provided with a fluorescent marker. Fluorescence markers are groups of molecules that emit electromagnetic radiation from a particular
  • Absorb wavelength range (referred to above as the third wavelength range) and after absorption of another electromagnetic radiation Emit wavelength range (called fourth wavelength range above).
  • the fluorescence markers re-emit electromagnetic radiation with increased wavelengths compared to the incident light.
  • Fluorescence markers are usually coupled to molecules to be detected via so-called linker molecules, that is to say the molecule to be detected with molecules coupling to the fluorescence marker (or the capture molecule). If molecules to be detected with fluorescence markers coupled thereto hybridize to capture molecules immobilized on the surface of the immobilization layer, the fluorescence markers are spatially close to the immobilization layer.
  • this electromagnetic radiation can be absorbed by the fluorescent markers, provided that the electromagnetic radiation has at least one wavelength within the third wavelength range within which the fluorescent markers can absorb electromagnetic radiation.
  • the fluorescent markers are placed in an electronic excitation state, which is characterized by a medium lifespan.
  • the fluorescent markers re-emit electromagnetic radiation of a fourth wavelength range, the fourth wavelength range having more long-wave electromagnetic ones
  • Radiation has as the third wavelength range.
  • the light re-emitted by the fluorescent markers has a longer wavelength than the incident light.
  • the intensity of the re-emitted light is typically several orders of magnitude lower than the intensity of the incident light, which is provided, for example, by an external radiation source.
  • the fluorescent light of the fourth wavelength range and the non-absorbed externally incident light pass through the immobilization layer and reach the optical filter layer.
  • the optical filter layer is set up in such a way that the optical filter layer totally reflects electromagnetic radiation of a second wavelength range, at least one Part of the first wavelength range, in which the detection devices can detect electromagnetic radiation, lies outside the second wavelength range.
  • the second wavelength range in which the optical filter layer is totally reflected is set up according to the invention in such a way that the light incident from the outside is essentially reflected and that the light of the fourth wavelength range which is re-emitted by the fluorescent markers is transmitted essentially through the optical filter layer.
  • the electromagnetic radiation of the fourth wavelength range emitted by a fluorescent marker on a specific catcher molecule penetrates the optical filter layer and, ideally after passing through the essentially transparent circuit layer, reaches the photodiode in the substrate which is the smallest distance from the emitting fluorescent marker.
  • the photodiode which is set up in such a way that it can detect electromagnetic radiation of a first wavelength range, is suitable for detecting the electromagnetic fluorescence radiation of the fourth wavelength range, since the inventive one
  • Fluorescence biosensor chip is set up in such a way that at least part of the fourth wavelength range lies within the first wavelength range.
  • the photodiode is suitable for detecting the fluorescence radiation and is therefore suitable for the indirect detection of a
  • hybridization events can be detected by detecting fluorescent radiation, in that after molecules to be detected have been docked onto catcher molecules having fluorescent markers, the sensor level is brought into active contact with a substance that has been set up in such a way that catcher molecules having fluorescent markers without docked to be detected Molecules are detached from the sensor level, whereas catcher molecules with molecules attached to them remain attached to the sensor level even in the presence of the substance. After capture molecules having fluorescent markers are detached without molecules hybridized with them to be detected, only those capture molecules with fluorescent markers to which molecules to be detected are docked remain at the sensor level. These hybridization events are then based on the principle described above by detecting the
  • Fluorescent radiation of the fluorescent markers coupled to the capture molecules is detectable. According to the alternative concept described, it is not necessary to bind fluorescent markers to the molecules to be detected, instead the fluorescent markers can be bound to the capture molecules.
  • fluorescent markers can only be added after the hybridization events. If the fluorescent markers are set up in such a way that they only bind to capture molecules with molecules hybridized to them to be detected (e.g. only bind to double-stranded DNA), the intensity of the electromagnetic radiation emitted by the fluorescence markers is characteristic of the number of hybridization events that have taken place.
  • different fluorescent markers can also be used to detect different molecules with different fluorescent markers.
  • a parallel analysis is thereby possible, by means of which the different components of an analyte can be examined and quantified simultaneously.
  • coumarin (1,2-benzopyrone 2H-1-benzopyran-2-one, C 9 H 6 0 2 ) is used as the fluorescent marker.
  • the fluorescent dye coumarin has the property, when excited with electromagnetic radiation with a wavelength of 370 nanometers, in a wavelength range around 460 Nanometer around to re-emit electromagnetic fluorescent radiation.
  • the fluorescence marker coumarin thus ensures a sufficiently strong red shift of the re-emitted electromagnetic radiation, so that the exciting and emitted electromagnetic radiation can be easily separated from one another.
  • Any other suitable material such as FITC, Cy2, Alexa Fluor 488, BODIPY 493, Rhodamine 123, R6G, TET, JOE, HEX, BODIPY 530, Alexa 532, R-Phycoerythrin, TRITC, Cy3, TAMRA, Texas Red can also be used as fluorescent marker , ROX, BODIPY 630 and Cy5 can be used.
  • the surface of the fluorescence biosensor chip preferably has a matrix-like arrangement of individual sensor fields.
  • each individual sensor field can be read out individually by means of the circuit layer.
  • the sensor fields are arranged as densely as possible. This is advantageous for "high throughput screening" applications.
  • the dense arrangement of sensor fields is associated with the risk that optical crosstalk can occur from one sensor field to an adjacent sensor field.
  • the photodiodes integrated in the substrate map the immobilization layer with the capture molecules immobilized thereon in the correct position. As a result, a photodiode is essentially sensitive to the fluorescent radiation of those capture molecules which are arranged essentially above the photodiode.
  • Optical crosstalk is now understood to mean that electromagnetic fluorescent radiation from a fluorescent marker is not emitted onto the essentially underneath photodiode, but rather is emitted, for example, in the direction of another photodiode arranged to the left or right of this photodiode.
  • a hybridization event on a catcher molecule will be incorrectly detected by a photodiode that is not arranged below the catcher molecule.
  • possibilities are created according to the invention for optical crosstalk between neighboring ones To keep sensor fields low or to prevent them. This has the advantageous effect that a high integration density of sensors on the fluorescence biosensor chip is combined with reduced optical crosstalk.
  • At least one isolation trench for optically isolating adjacent detection devices is preferably introduced into at least one surface area of the fluorescence biosensor chip, which trench extends at least one isolation trench through the immobilization layer into a region of the optical filter layer stretches in such a way that a detection device is arranged below each area between two adjacent isolation trenches.
  • at least part of the surface of the at least one isolation trench is covered with a layer of an absorbent material or at least one of the trenches is filled with an absorbent material, the absorbent material being set up in such a way that at least electromagnetic radiation is generated by means of the absorbent material of the respective wavelength range or the respective wavelength ranges is absorbed or reflected.
  • fluorescence radiation is emitted from a fluorescence marker which is arranged essentially above a first photodiode in relation to the direction of incidence of light, in a direction in which not the underlying photodiode but an adjacent photodiode is arranged, then one can be used between the photodiodes suitably introduced and filled with an electromagnetic radiation absorbing material at least partially filled trench to prevent the electromagnetic fluorescent radiation from a fluorescence marker which is arranged essentially above a first photodiode in relation to the direction of incidence of light, in a direction in which not the underlying photodiode but an adjacent photodiode is arranged, then one can be used between the photodiodes suitably introduced and filled with an electromagnetic radiation absorbing material at least partially filled trench to prevent the electromagnetic fluorescent radiation from a
  • Fluorescence photodiode is detected. Instead of false detection, the fluorescent radiation is absorbed by the absorbent material in the trench. This reduces the risk of optical crosstalk. This is advantageous because it increases the detection sensitivity of the fluorescence biosensor chip and reduces the susceptibility to errors of the fluorescence biosensor chip.
  • Optical crosstalk can be further reduced by providing a barrier layer made of an absorbent material in at least one area of the circuit layer, such that a detection device is arranged below each area between two adjacent barrier layers, the absorbent material being set up in this way that it absorbs or reflects electromagnetic radiation of at least the respective wavelength range or the respective wavelength ranges.
  • the isolation trench is introduced, for example etched, into the immobilization layer and at least partially into the optical filter layer.
  • Fluorescence radiation which is re-emitted by a fluorescence marker at such an angle that the fluorescence radiation does not pass through the isolation trench on its way to a left or right of the photodiode arranged below the fluorescence marker, but through the circuit layer below the isolation trench is running, can be detected by a "wrong" photodiode despite the isolation trench.
  • barrier layers made of absorbent material can be introduced into the circuit layer as described above. These barrier layers have essentially the same function as the absorbent material in the isolation trenches, namely absorbing and / or reflecting fluorescent radiation on the way to a “wrong” photodiode. However, the barrier layer perceives this functionality in the circuit layer, whereas the isolation trenches this functionality in the immobilization layer and perceive in the optical filter layer.
  • the barrier layers in the circuit layer preferably fulfill a double function.
  • the absorbing and / or reflecting barrier layers if they are made of an electrically conductive material, can also perform the function of electronic components in the circuit layer.
  • the barrier layers can be used as electrical leads to the
  • the barrier layers are preferably metallic conductor tracks or through holes introduced into the circuit layer, which are filled with an electrically conductive and electromagnetic radiation absorbing / reflecting material.
  • optical crosstalk between neighboring sensor fields is further reduced, which increases the sensitivity of detection.
  • the double function of the barrier layer according to the invention as a means for reducing optical crosstalk on the one hand and as electrically integrated components on the other hand is economical and space-saving.
  • the invention also provides a fluorescence biosensor chip arrangement with a fluorescence biosensor chip and an electromagnetic radiation source.
  • the fluorescence biosensor chip has a substrate, at least one detection device arranged in or on the substrate for detecting electromagnetic radiation from a first
  • Wavelength range an optical filter layer arranged on the substrate for absorbing and / or reflecting electromagnetic radiation of a second wavelength range, an immobilization layer arranged on the optical filter layer for immobilizing capture molecules, the detection device, the filter layer and the immobilization layer in the fluorescence biosensor chip are integrated.
  • the electromagnetic radiation source is set up in such a way that by means of the electromagnetic radiation source Surface area of the fluorescence biosensor chip can be irradiated with electromagnetic radiation of a third wavelength range.
  • the fluorescence biosensor chip arrangement of the invention essentially has an electromagnetic radiation source.
  • the electromagnetic radiation source is intended to cover the surface area of the fluorescence biosensor chip with electromagnetic radiation from a third party
  • the electromagnetic radiation source is preferably a laser, a light-emitting diode, a gas discharge lamp or an incandescent lamp. If the electromagnetic radiation source is configured as a laser, this enables the surface of the fluorescence biosensor chip to be irradiated with monochromatic, narrow-band light. Monochromatic light can be easily filtered out by means of a filter layer, the optical absorption properties of which depend on the wavelength.
  • the fluorescence biosensor chip arrangement also has a large number of capture molecules which are coupled to the immobilization layer and which are set up in such a way that a molecule which is complementary to the capture molecule and can be detected can be coupled to the capture molecule.
  • the capture molecules are coupled to the immobilization layer as described above with reference to the fluorescence biosensor chip.
  • Each molecule to be detected also has a fluorescent marker, the fluorescent marker being such is set up so that it at least partially absorbs electromagnetic radiation of the third wavelength range and emits electromagnetic radiation of a fourth wavelength range after absorption, at least part of the third wavelength range lying outside the fourth wavelength range and at least part of the fourth wavelength range lying within the first wavelength range. In addition, at least part of the first wavelength range lies outside the second wavelength range.
  • the functionality of the fluorescence biosensor chip arrangement according to the invention is described in more detail below.
  • the surface of the fluorescence biosensor chip arrangement is irradiated with electromagnetic radiation of the third wavelength range by means of the electromagnetic radiation source.
  • the immobilization layer, on which capture molecules are immobilized, is located on the surface of the fluorescence biosensor chip arrangement of the invention.
  • a solution with molecules to be detected is brought into active contact with this active sensor surface. If the molecules to be detected in this solution are sufficiently complementary to capture molecules immobilized on the immobilization layer, the molecules to be detected are hybridized with the capture molecules.
  • the molecules to be detected are coupled to a fluorescence marker, for example, via a linker molecule, the fluorescence marker being set up in such a way that it at least partially absorbs electromagnetic radiation of the third wavelength range. Therefore, after the hybridization of the molecules to be detected to the capture molecules, the light emitted by the electromagnetic radiation source is absorbed by the fluorescent markers to the molecules to be detected.
  • the fluorescence markers are set up in such a way that after absorption of electromagnetic radiation of the third wavelength range, the fluorescence markers emit electromagnetic radiation of a fourth wavelength range, at least part of the third wavelength range outside the fourth Wavelength range. This means that the fluorescent radiation of the fluorescent markers is longer-wave than the previously absorbed radiation of the third wavelength range, which is provided by the electromagnetic radiation source.
  • the optical filter layer is set up such that electromagnetic radiation of the second wavelength range is absorbed and / or reflected by means of the optical filter layer.
  • the optical filter layer completely reflects or absorbs the electromagnetic radiation of the third wavelength range that comes from the external electromagnetic radiation source.
  • the optical filter layer ideally completely transmits the electromagnetic radiation of the fourth wavelength range, which originates from the fluorescence markers.
  • the optical filter layer is set up in such a way that it is completely transparent to the fluorescent light, whereas it is completely transparent to the light of the electromagnetic radiation source.
  • at least part of the fourth wavelength range, in which the fluorescent radiation of the fluorescent markers lies lies within the first wavelength range, within which the detection devices are capable of detecting electromagnetic radiation.
  • Embodiments of the fluorescence biosensor chip arrangement of the invention are described below, by means of which the detection sensitivity of the fluorescence biosensor chip arrangement can be increased.
  • the electromagnetic radiation source can preferably be aligned such that the electromagnetic radiation emitted by the electromagnetic radiation source is at a predeterminable angle to the normal direction of the optical filter layer.
  • the direction in which the electromagnetic radiation from the electromagnetic radiation source impinges on the catcher molecules can clearly be specified, for example by using an electromagnetic radiation source that generates a bundle of parallel light beams and by making this electromagnetic radiation source displaceable, rotatable, pivotable or tiltable ,
  • the part of the exciting light transmitted through the optical filter does not directly hit the photodiode which is arranged essentially below the absorbing and emitting fluorescent marker.
  • the interfering primary light which reduces the detection sensitivity of the fluorescence biosensor chip arrangement is partially “geometrically” shielded.
  • the obliquely incident exciting light can be isolated as described above by means of insulation Trenches and / or barrier layers may be shielded from detection.
  • shadow effects can advantageously be used are used to increase the detection sensitivity of the fluorescence biosensor chip arrangement.
  • the electromagnetic radiation source is set up in such a way that the electromagnetic radiation emitted by the electromagnetic radiation source is emitted in pulses and in which the detection devices are set up in such a way that the electromagnetic radiation emitted by the fluorescence markers is in the time intervals between the Pulses can be detected by means of the detection devices.
  • Absorb the exciting light has a finite, non-zero lifespan. If a short pulse of exciting light is radiated onto the fluorescent markers by means of the electromagnetic radiation source, the fluorescent markers are brought into an excited electron state by absorption of the light. Due to the high speed of light, the incident light not absorbed by the fluorescent markers reaches the detector devices almost instantaneously, the signal of which is not detected at this point in time. In other words, the detection devices are switched off during the pulse. After a time interval which essentially corresponds to the mean life of the excited electron state of the fluorescence marker, a time-delayed electromagnetic fluorescence wave is emitted by the fluorescence markers.
  • the time delay is in the order of magnitude of the natural lifespan of excited electron states (approximately microseconds to nanoseconds). If the measurement signal of the detection devices is only recorded after this time delay, the parasitic detection of exciting light is avoided and only fluorescent radiation is detected.
  • detection devices with sufficiently good time resolution should preferably be selected, for example photodiodes that have a time resolution in the sub-nanosecond range. through Suppression of the detection of the primary light increases the detection sensitivity of the fluorescence biosensor chip arrangement of the invention.
  • FIG. 1A shows a schematic view of a fluorescence biosensor chip according to the prior art
  • Figure IB is an exploded view of another
  • FIG. 2 shows a cross-sectional view of a fluorescence biosensor chip according to a first exemplary embodiment of the invention
  • FIG. 3 shows a cross-sectional view of a fluorescence biosensor chip according to a second exemplary embodiment of the invention
  • Figure 4 is a diagram that schematically shows the dependence of the
  • FIG. 5A shows a top view of a fluorescence biosensor chip according to a third exemplary embodiment of the invention
  • FIG. 5B shows an enlarged partial cross-sectional view along the section line II 'from FIG. 5A according to FIG third preferred embodiment of the fluorescence biosensor chip of the invention
  • Figure 6A is a circuit diagram with a control logic for controlling a sensor field according to a preferred
  • FIG. 6B shows an enlarged view of the control logic for controlling a sensor field in accordance with the preferred one
  • FIG. 7 shows a cross-sectional view of a fluorescence biosensor chip arrangement according to a preferred one
  • a fluorescence biosensor chip 200 according to a first exemplary embodiment of the invention is described below with reference to FIG. 2.
  • the fluorescence biosensor chip 200 has a substrate 201, at least one detection device 202 arranged in or on the substrate 201 for detecting electromagnetic radiation, an optical filter layer 203 arranged on the substrate 201 and an immobilization layer 204 arranged on the optical filter layer 203 to immobilize capture molecules.
  • the detection devices 202, the filter layer 203 and the immobilization layer 204 are integrated in the fluorescence biosensor chip 200, as shown in FIG. 2.
  • Substrate 201 made of silicon material.
  • six detection devices 202 are provided, whereby each of the six detection devices 202 is designed as a photodiode, which are set up in such a way that electromagnetic radiation from a first
  • Wavelength range is detectable.
  • adjacent detection devices 202 are arranged at a distance “d” from one another.
  • the distance “d” is therefore a measure of the one-dimensional spatial resolution of the fluorescence biosensor chip 200 according to the invention.
  • d 2 is a measure of the two-dimensional spatial resolution of the fluorescence biosensor chip 200 according to the invention, ie for the required surface of the fluorescence biosensor chip 200 pro sensor pixels.
  • the optical filter layer 203 is set up in such a way that the optical filter layer 203 absorbs electromagnetic radiation of a second wavelength range, at least part of the first wavelength range lying outside the second wavelength range.
  • the optical filter layer 203 is designed as an edge filter.
  • the edge filter 203 of the fluorescence biosensor chip 200 absorbs electromagnetic radiation below a cutoff wavelength.
  • the optical edge filter 203 is a color filter made of an organic material.
  • the optical filter layer 203 has a thickness “h”, which according to the exemplary embodiment described is of the order of magnitude of 70 micrometers.
  • the thickness “h” of that as an organic edge filter The optical filter layer 203 configured must be large enough to absorb as completely as possible such electromagnetic radiation that should not reach the detection devices 202, and the optical filter layer 203 designed as an organic edge filter should be chosen sufficiently thin to accommodate such electromagnetic radiation which should arrive at the detection devices 202 in order to be detected by the detection devices 202 to a sufficient extent.
  • the immobilization layer 204 shown in FIG. 2 is a thin gold layer.
  • the fluorescence biosensor chip 200 furthermore has a circuit layer 205 between the substrate 201 and the optical filter layer 203, at least one electrical component being integrated in the circuit layer 205, and the circuit layer 205 with the at least one detection Device 202 is electrically coupled.
  • the electrical components that are integrated in the circuit layer 205 are not shown in FIG. 2.
  • the circuit layer 205 is set up in such a way that the detection devices 202 can each be individually electrically controlled by means of the circuit layer 205.
  • the circuit layer 205 has MOS transistors for selecting one of the detection devices 202, which are electrically conductive
  • the circuit layer 205 has a thickness “1”, which according to the exemplary embodiment described is approximately five micrometers.
  • the thickness “1” should be selected to be sufficiently small or the materials should be selected appropriately be that losses due to absorption of electromagnetic radiation to be detected in the circuit layer 205 are low.
  • the fluorescence biosensor chip 200 also contains a multiplicity of capture molecules 206 which are coupled to the immobilization layer 204 and which are set up in such a way that a molecule 207 to be detected which is complementary to the capture molecule 206 can be coupled to each of the capture molecules 206 ready for binding.
  • the capture molecules 206 shown in Fig. 2 are DNA strands.
  • Each molecule 207 to be detected has a fluorescent marker 208.
  • the fluorescence markers 208 are set up in such a way that the fluorescence markers 208 absorb electromagnetic radiation of a third wavelength range and, after absorption has taken place, emit electromagnetic radiation of a fourth wavelength range.
  • the fluorescent label 208 shown in Fig. 2 is coumarin. In that shown in Fig. 4
  • the diagram shows the emission spectrum of coumarin after the fluorescent dye coumarin has been excited with electromagnetic radiation with a wavelength of 370 nanometers.
  • electromagnetic radiation with a wavelength of 370 nanometers.
  • This emission spectrum corresponds to the fourth wavelength range defined above.
  • Fluorescence biosensor chips 200 not only in active contact with molecules 207 to be detected, which are coupled to a fluorescence marker 208. Furthermore, molecules 209 are also in active contact with the catcher molecules 206 on the surface of the immobilization layer 204. These molecules 209 are also coupled to fluorescent markers 210, which however differ from the fluorescent markers 208 coupled to the molecules 207 to be detected in that the fluorescent markers 210 in other Wavelength ranges absorb or fluoresce as the fluorescent markers 208 of the molecules 207 to be detected. In contrast to the molecules 207 to be detected, which are complementary to the catcher molecules 206 and are consequently attached to the catcher molecules, the molecules 209 are not complementary to the catcher molecules 206 and are therefore not able to hybridize with the capture molecules 206.
  • the functionality of the fluorescence biosensor chip 200 is described below.
  • the fluorescence biosensor chip 200 is brought into contact with a solution which, among other things, contains the molecules 207 to be detected with fluorescent markers 208 coupled to them via linker molecules.
  • Molecules 207 complementary to the catcher molecules 206 hybridize with the catcher molecules 206. If appropriate, a suitable rinsing or washing step is carried out.
  • the hybridization event can be detected by irradiation of electromagnetic radiation of the third wavelength range in which the fluorescent markers 208 absorb.
  • the fluorescent markers 208 After absorption has taken place, the fluorescent markers 208 re-emit light of a fourth wavelength range, the re-emitted light being longer-wavelength than the absorbed light. Both the incident light and the fluorescent light pass through the essentially transparent immobilization layer 204 and reach the optical filter layer 203.
  • the optical filter layer 203 designed as an organic edge filter is designed as a blocking filter for the exciting light wavelength (third wavelength range). That is, the light of the incident wavelength is essentially completely absorbed by the optical filter layer 203, whereas the fluorescent light of the fourth wavelength range is transmitted essentially unattenuated by the optical filter layer 203.
  • the fluorescent light After passing through the essentially transparent circuit layer 205, the fluorescent light preferably reaches that of the photodiodes 202, which is arranged essentially below the fluorescent marker 208 from which the fluorescent light was emitted.
  • the photodiodes 202 are set up such that electromagnetic radiation of the first wavelength range can be detected.
  • Fluorescence radiation is within the first wavelength range, the photodiode 202 is able to detect the fluorescence light. This on the one hand detects a hybridization event, on the other hand the intensity of the detected fluorescent light is a measure of the number of molecules attached, i.e. for the degree of complementary action between capture molecules 206 and molecules 207 to be detected.
  • Photodiodes 202 have a very high dynamic range, a high detection sensitivity can be achieved with the fluorescence biosensor chip according to the invention.
  • a high dynamic range is understood to mean that the detector can measure electromagnetic fluorescence radiation of a large intensity range.
  • the spatial resolution of the fluorescence biosensor chip 200 is not achieved, as in the prior art, by means of lens optics, but rather by means of electrical selection of a sensor region on the immobilization layer 204, which is essentially arranged above a specific photodiode 202.
  • a surface section 211 of the immobilization layer 204 is free of capture molecules 206, so that a noise signal can be removed from the at least one reference detection device 202a arranged below this surface section 211. Since no catcher molecules are immobilized on the surface of the immobilization layer 204 above the reference detection device 202a, this can
  • Detection device 202a removes that noise signal or background signal or zero signal which originates from the parasitic electromagnetic radiation and which has to be subtracted from the signals of all other detection devices 202 in order to obtain a signal which is proportional to the intensity of the fluorescent light. This subtraction is carried out by means of an electronic differential circuit.
  • a fluorescence biosensor chip 300 according to a second exemplary embodiment of the invention is described with reference to FIG. 3.
  • the fluorescence biosensor chip 300 has a substrate 301, a detection device 302 arranged in the substrate for detecting electromagnetic radiation, an optical filter layer 303 arranged on the substrate 301 and one arranged on the optical filter layer 303
  • Immobilization layer 304 for immobilizing capture molecules.
  • the detection device 302, the filter layer 303 and the immobilization layer 304 are integrated in the fluorescence biosensor chip 300.
  • the functionality of the fluorescence biosensor chip 300 largely corresponds to that of the fluorescence biosensor chip 200, which is described above with reference to FIG. 2. Therefore, only those features are discussed at this point that are configured differently from the fluorescence biosensor chip arrangement 200 in the fluorescence biosensor chip arrangement 300.
  • the optical filter layer 303 is designed as a band filter.
  • the exact structure of the optical filter layer 303 is described below with reference to FIG. 4.
  • the detection device 302 is designed as a photodiode 302, which is integrated in the substrate 301. As shown in FIG. 3, further integrated circuit elements 304 are introduced into the substrate 301.
  • the silicon dioxide region 304a serves to electrically isolate adjacent photodiodes 302.
  • the n-doped silicon regions 304b, 304c are part of the control electronics with which a specific photodiode 302 can be controlled.
  • the substrate 301 is a p-doped silicon substrate.
  • a circuit layer 306 is arranged between the substrate 301 and the optical filter layer 303, at least one electrical component 306a being integrated in the circuit layer 306, and the circuit layer 306 being electrically coupled to the detection device 302 , As shown in FIG. 3, the integrated circuit elements 306a form a transistor-like arrangement together with the n-doped silicon regions 304b, 304c and the p-doped silicon substrate 301, the detection device 302 being electrically controllable by means of this transistor-like arrangement is.
  • a large number of capture molecules are immobilized on the immobilization layer 305, of which only one capture molecule 307 is shown in FIG. 3 for reasons of simplicity.
  • the capture molecule 307 shown in FIG. 3 is a DNA half-strand, the bases 307a of which are shown schematically in FIG. 3.
  • a molecule 308 which is complementary to the catcher molecule 307 and is to be detected is coupled to the catcher molecule 307.
  • the molecule 308 to be detected has a fluorescent marker 309.
  • the catcher molecule 307 and the molecule 308 to be detected are two mutually complementary DNA half-strands.
  • Electromagnetic radiation of a third wavelength range 310 which is provided, for example, by an external electromagnetic radiation source (not shown in FIG. 3), impinges on the fluorescent marker 309 and is partially absorbed by it.
  • the fluorescence marker 309 re-emits electromagnetic fluorescence radiation of a fourth wavelength range 311, some of the emitted
  • Fluorescence radiation reaches the fluorescence biosensor chip 300.
  • the electromagnetic radiation of the fourth wavelength range 311 strikes the filter layer 303, which is set up in such a way that the electromagnetic Radiation of the fourth wavelength range 311 is at least partially transmitted through the filter layer 303. As shown in FIG. 3, this part reaches the photodiode 302 and is detected there.
  • the electromagnetic radiation of the fourth wavelength range 310 is largely reflected on the optical filter layer 303. In the ideal case, this means that no electromagnetic radiation of the third wavelength range 310 reaches the photodiode 302.
  • Wavelength range 311 penetrates to the detection device 302, whereas the primary light of the third wavelength range 310 does not penetrate to the detection device 302.
  • the optical filter layer 303 is designed as a bandpass filter, which is a dielectric interference filter with a layer sequence of two materials, a first material having a high refractive index and a second material having a low refractive index.
  • the first material with a high refractive index is silicon nitride and the second material with a low refractive index is silicon dioxide.
  • the dielectric interference filter according to the described preferred exemplary embodiment has 31 alternating layers of alternating silicon dioxide and silicon nitride.
  • the present dielectric interference filter is described by the following nomenclature:
  • H is a layer made of the high-index material
  • the layer thicknesses are given in multiples of * / 4 (•: light wavelength in the medium). With * / 4 the fourth part of the light wavelength in the medium is meant, ie the quotient of the light wavelength in vacuum and the refractive index of the respective medium.
  • the filter layer according to the invention has a "/ 8 layer of the high-index material, a" / 4 layer of the low-index material, 14 double layers, each of the double layers consisting of a • / 4 plate of the high-index material and a • / 4- Platelet of the low-index material is built up, as well as a »/ 8-layer of the high-index material.
  • Interference filter with a wavelength dependence of the transmission as shown in Fig. 4, obtained.
  • a dielectric interference filter configured in this way reflects electromagnetic radiation in the wavelength range between 350 nanometers and 390
  • the wavelength of the reflection maximum i.e. of the transmission minimum in FIG. 4
  • the wavelength of the reflection maximum can be set at a fixed angle of incidence of the electromagnetic radiation by adjusting the layer thickness of the individual layers of the dielectric interference filter. Since the calculated transmission has a pronounced transmission minimum in a relatively wide wavelength range between 350 nanometers and 390 nanometers, depending on the wavelength, as shown in FIG. 4, such a filter is also suitable for suppressing the exciting light of broadband excitation sources such as e.g. LEDs are suitable. If spectrally even wider light sources are to be used, for example also at light wavelengths below the left flank at 350
  • an additional filter is required to filter out electromagnetic radiation in the lower wavelength range. This can be implemented, for example, by means of a suitable edge filter.
  • the emission spectrum of coumarin is also drawn as a dashed line, as is obtained after excitation of the dye with electromagnetic radiation of the wavelength 370 nanometers.
  • the emission spectrum of coumarin is relatively broadband, the left flank of the emission spectrum of coumarin is clearly longer-wave than the right limit of the wavelength range in which the optical filter described above reflects almost totally.
  • the long-wave passband of the dielectric interference filter should be made as flat as possible, i.e. it is particularly advantageous to ensure an approximately constant and as high a transmission as possible over the entire fluorescence range of the dye. This can be done by varying the layer thicknesses of the dielectric filter layer and the materials used for this.
  • the dielectric interference filter described is suitable for the fluorescence biosensor chip according to the invention if coumarin is used as the fluorescence marker.
  • the transmission of the dielectric interference filter described is greater than 75% above approximately 415 nanometers and greater than 92% above 450 nanometers.
  • the fluorescent light of the dye coumarin is only slightly weakened when it passes through the optical filter layer.
  • the greatest possible steepness i.e. a sudden increase from a transmission zero to a transmission one
  • FIGS. 5A, 5B The fluorescence biosensor chip 500 shown in FIGS. 5A, 5B is described below.
  • FIG. 5A shows a top view of the fluorescence biosensor chip 500
  • FIG. 5B shows a cross-sectional view of part of the fluorescence biosensor chip 500 shown in FIG. 5A along the section line II '.
  • the fluorescence biosensor chip 500 shown in FIGS. 5A, 5B is a third preferred exemplary embodiment of the fluorescence biosensor chip according to the invention and differs only in some aspects from the previously described fluorescence biosensor chips 200, 300. Furthermore, the complete functionality of the Fluorescence biosensor chips 500 explained, rather only the supplementary features compared to the previously described exemplary embodiments will be discussed.
  • FIG. 5B shows a fluorescence biosensor chip 500 with a substrate 501, at least one detection device 502 arranged in or on the substrate 501 for detecting electromagnetic radiation, an optical filter layer 503 arranged on the substrate 501 and one on the optical filter layer 503 arranged immobilization layer 505 for immobilizing capture molecules.
  • the detection devices 502, the optical filter layer 503 and the immobilization layer 505 are integrated in the fluorescence biosensor chip 500.
  • the substrate 501 is a p-doped silicon substrate.
  • the detection devices 502 are silicon photodiodes integrated in the substrate 501.
  • the optical filter layer 503 is, according to the exemplary embodiment described with reference to FIGS. 5A, 5B, a dielectric interference filter.
  • Immobilization layer 505 is a thin layer of gold.
  • silicon dioxide regions 504 are introduced into the substrate 501.
  • a circuit layer 504 is also arranged between the substrate 501 and the optical filter layer 503, at least one electrical component 506a being integrated in the circuit layer 504 and the circuit Layer 504 is electrically coupled to the at least one detection device 502. This coupling is explicitly shown in Fig. 5B.
  • the integrated circuit elements 506a which are shown in FIG. 5B, are electrically conductive connection means which enable the silicon photodiodes 502 to be coupled to control electronics.
  • the fluorescence biosensor chip 500 also has a multiplicity of capture molecules 507 which are coupled to the immobilization layer 505 and which are set up in such a way that a molecule 508 which is complementary to the capture molecule 507 and can be detected can be coupled to the capture molecule 507.
  • the reference number 507a denotes the individual bases which are those which are designed as a DNA half-strand
  • molecules 508 to be detected which are complementary to the DNA half strands 507, also DNA half strands, are on capture molecules
  • the molecules to be detected are also 508 DNA half strands, the molecules to be detected also have
  • Fluorescence markers 509 are coupled to the molecules 508 to be detected.
  • At least one isolation trench 510 for optically isolating neighboring detection devices 502 is introduced into at least one surface area of the fluorescence biosensor chip 500, which at least one isolation trench 510 extends through the immobilization layer 505 into a region of the optical filter layer 503 extends in such a way that a detection device 502 is arranged below each area between two adjacent isolation trenches 510.
  • the at least one isolation trench 510 is covered with a layer of an absorbent material 511, the absorbent material 511 being set up in such a way that it absorbs electromagnetic radiation.
  • the isolation trench 510 and the absorbent material introduced into the isolation trench 510 511 is explained below with reference to FIG. 5B and in particular the electromagnetic fluorescence radiation 512 schematically shown therein, which is emitted by the fluorescence marker 509 arranged on the left in FIG. 5B.
  • the various detection devices 502 in the substrate 501 correspond to the sensor pixels on the surface of the immobilization layer 505.
  • all those capture molecules 507 immobilized on the surface of the immobilization layer 505 belong to the detection device 502 which Is arranged substantially below this capture molecule 507.
  • the left detection device 502 is provided for the detection of fluorescent radiation which the left immobilized on the surface of the immobilization layer 505
  • Capture molecule 507 runs out.
  • the right-hand detection device 502 shown in FIG. 5B serves for the detection of fluorescence radiation which originates from a fluorescence marker 509 which is bound to a molecule 508 to be detected, which molecule 508 is docked to a capture molecule 507 which is essentially above the right detection device 502.
  • the left fluorescent marker 509 emits electromagnetic fluorescent radiation 512.
  • this fluorescence radiation which is an indirect consequence of a hybridization event on the left capture molecule 507 arranged on the surface of the immobilization layer 505, should be detected by the left detection device 502.
  • the electromagnetic fluorescent radiation 512 is emitted in such a direction that it is not emitted onto the left detection device 502 shown in FIG. 5B, but rather towards the right detection device 502.
  • Fluorescence radiation 512 detected by the right detection device 502 this would falsify the measurement. This phenomenon is referred to as optical crosstalk between two adjacent sensor fields belonging to the left and right detection devices 502, respectively. With the isolation trench 510 partially filled with the absorbent material 511, the undesired phenomenon of optical crosstalk is reduced.
  • the electromagnetic fluorescent radiation 512 is emitted in the direction of the right silicon photodiode 502 shown in FIG. 5B, but this electromagnetic fluorescent radiation 512 must pass the isolation trench 510 and on the way to the right silicon photodiode 502 pass through the partially filled absorbent material 511.
  • the absorbent material 511 is set up in such a way that electromagnetic radiation is thereby absorbed, in particular in the wavelength range of the fluorescent radiation of the fluorescent markers 509 used.
  • the electromagnetic fluorescent radiation 512 is absorbed in the absorbent material 511 in the isolation trench 510 and therefore cannot reach the right detection device 502 shown in FIG. 5B. This reduces optical crosstalk between neighboring sensor fields.
  • the isolation trenches 510 filled with an absorbent material 511.
  • the fluorescence radiation 513 is likewise not emitted in the direction of the detection device 502 which is essentially below it, but rather in the direction of the detection device 502 arranged to the left of the fluorescence marker 509. Due to the geometric conditions shown in FIG. 5B, the electromagnetic fluorescence radiation 513 is not emitted by absorbent material 511 in isolation trench 510.
  • a barrier layer 514 made of an absorbent material is arranged in at least one area of the circuit layer 504, such that a detection device 502 is arranged below each area between two adjacent barrier layers 514, the absorbent material is set up so that it absorbs electromagnetic radiation.
  • the integrated circuit elements 506a can also take on the function of the absorbent barrier layer 514.
  • the integrated circuit elements 506a are to be produced from a material that absorbs and / or reflects electromagnetic radiation.
  • the integrated circuit elements 506a can therefore perform a double function: on the one hand they can serve as electronic circuit elements, on the other hand they can help to reduce the phenomenon of optical crosstalk.
  • FIG. 5A shows a plan view of the fluorescence biosensor chip 500 according to the described exemplary embodiment of the invention.
  • the isolation trench 510 which according to the exemplary embodiment shown is designed as a coherent isolation region, is shown in FIG. 5A.
  • the individual sensor fields 515, 516 which are defined by the regions between the isolation trenches 510 and which are covered with catcher molecules 507, are shown in FIG. 5A.
  • FIG. 5A An essentially matrix-shaped arrangement of sensor fields 601 is shown in FIG. 6A.
  • the representation selected in FIG. 6A essentially corresponds to the representation of the fluorescence biosensor chip 500 in FIG. 5A. Not shown in Fig. 5A and in Fig. 6A in
  • the circuitry is shown in detail, by means of which each of the sensor fields 601 of the fluorescence biosensor chip 600 can be controlled.
  • the controllability of a specific row and the controllability of a specific column of the sensor fields 601 arranged in the form of a matrix is realized by means of the control circuit 602.
  • each individual sensor field 601 can be controlled by means of the row selection lines 603 and the column selection lines 604.
  • the number of row selection lines 603 (six in the example) and column selection lines 604 (six in the example) depends on the number of sensor fields 601. If the number of columns in the sensor field is 2 m , 2 m row selection lines 603 are required. If the number of columns of the sensor fields 601 is 2 n , 2n column selection lines 604 are required for the sequential activation of all columns.
  • the individual row select lines 603 are partially interdependent.
  • Row select lines 603 are labeled ZI, ZI, Z2, Z2, Z3 and
  • Row selection lines 603 Z2 and Z2 at mutually complementary values.
  • the row selection lines 603 Z3 and Z3 are also at mutually complementary values.
  • Sl, Sl, S2, S2, S3 and S3 are designated.
  • the signals at Sl and Sl are always on complementary logic
  • the signals at S2 and S2 are always at mutually complementary values and the signals at S3 and S3 are always at mutually complementary values.
  • Each of the sensor fields 601 is coupled to three of the six row selection lines 603 according to the exemplary embodiment shown in FIG. 6A and is three to the one according to the embodiment shown in
  • the selected sensor array 601a is coupled to first, second and third row select lines 603a, 603b and 603c.
  • the first row selection line 603a is ZI
  • the second row selection line 603b is Z2
  • the third row selection line 603c is Z3.
  • the selected sensor array 601a is coupled to a first, a second and a third column selection line 604a, 604b, 604c. Referring to Figure 6A, these are the first Column selection line 604a S1, the second column selection line 604b S2 and the third column selection line 604c
  • FIG. 6B schematically indicates with two arrows with the reference numeral 606 that the photodiode 605 is set up in such a way that electromagnetic fluorescent radiation can be detected. If electromagnetic radiation 606 arrives at the photodiode 605, the electrical properties of the photodiode 605 change in a characteristic manner and an electrical signal is present at the source of a first transistor 607a coupled to the photodiode 605. This signal can only pass the first transistor 607a if a voltage signal is present at the gate region of the first transistor 607a and therefore a conductive channel is formed between the source region and the drain region, i.e. when on the first column select line 604a
  • Signal with a logical value "1" is present, that is, when a signal with a logical value "1" is present at S1. If this is the case, the electrical signal of the photodiode 605 can pass from the source region to the drain region of the transistor 607a and from there to the source region of the second transistor 607b.
  • Transistor 607b is present can only reach the drain region of the second transistor 607b if a voltage signal is present at the gate region of the second transistor 607b and a conductive channel is therefore formed between the source region and the drain region, ie if the electrical signal applied to the second column selection line 604b has a logic value "1", that is to say if a signal having a logic value "1" is applied to S2. In this case, the electrical signal passes from the source region of the second transistor 607b to the drain region of the second transistor 607b and from there to the source region of the third transistor 607c.
  • the electrical signal present at the source region of the third transistor 607c can only reach the drain region of the third transistor 607c if a voltage signal is present at the gate region of the third transistor 607c and therefore between the source region and the drain -A region is formed a conductive channel, ie when on the third
  • the electrical node 608 shown in FIG. 6B is coupled to the source region of a fourth transistor 609a.
  • the electrical signal present at the source region of the fourth transistor 609a can only then go to the drain region of the fourth transistor
  • the drain region of the fifth transistor 609b arrive when the second row selection line 603b coupled to the gate region of the fifth transistor 609b is occupied with an electrical signal with a logic value "1". This means that at the second designated Z2 Line selection line 603b an electrical signal with a logic value "1" must be present. In this case, the electrical signal present at the source region of the fifth transistor 609b reaches the drain region of the fifth transistor 609b and from there to the source region of the sixth transistor 609c coupled to it.
  • the electrical signal present at the source region of the sixth transistor 609c can only reach the drain region of the sixth transistor 609c if a voltage signal is present at the gate region of the sixth transistor 609c and therefore between the source region and the Drain area a conductive channel is formed, ie when an electrical signal with a logic value "1" is present on the third line selection line 603c, that is, when an electrical signal with a logic value "1" is present at Z3. Only in this
  • the electrical signal present at the source region of the sixth transistor 609c can reach the drain region of the sixth transistor 609c. If this condition is also fulfilled, the second row of sensor fields 601 belonging to the selected sensor field 601a is selected.
  • the selected sensor field 601a is therefore selected if and only if on the first column selection line 604a S1 and on the second column selection line 604b S2 and on the third column selection line 604c S3 and on the first
  • Row selection line 603c Z3 each an electrical Signal with a logic value "1" is present. If there is also an electrical signal with a logic value "0" only on one of the six mentioned selection lines 603a, 603b, 603c, 604a, 604b, 604c, the corresponding sensor field is not selected. If both the row and the column of the selected sensor field 601a are selected, the electrical signal detected by the photodiode 605 arrives at the means for detecting the electrical current 610 or at the means for detecting the electrical voltage 611. This results in a specific selected sensor field 601a can be selected and the strength of the electrical sensor signal applied to the detection device 605 of the selected sensor field 601a can be read out.
  • FIG. 7 shows a preferred exemplary embodiment of a fluorescence biosensor chip arrangement 700, which is explained in more detail below.
  • the fluorescence biosensor chip arrangement 700 has a fluorescence biosensor chip 700a and an electromagnetic radiation source 705.
  • the fluorescence biosensor chip 700a has a substrate 701, six detection devices 702 arranged in the substrate 701 for detecting electromagnetic radiation of a first wavelength range, an optical filter layer 703 arranged on the substrate 701 for absorbing and / or reflecting electromagnetic radiation of a second wavelength range and an immobilization layer 704 arranged on the optical filter layer 703 for immobilizing capture molecules.
  • the detection devices 702, the optical filter layer 703 and the immobilization layer 704 are integrated in the fluorescence biosensor chip 700a.
  • the electromagnetic radiation source 705 is set up in such a way that a surface area of the fluorescence biosensor chip 700a can be irradiated with electromagnetic radiation of a third wavelength range by means of the electromagnetic radiation source 705.
  • the fluorescence biosensor chip 700a has a circuit layer 706 which is between the Substrate 701 and the optical filter layer 703 is arranged.
  • the electromagnetic radiation source 705 is a laser.
  • the fluorescence biosensor chip 700a has a multiplicity of catcher molecules 707, which are coupled to the immobilization layer 704 and which are set up in such a way that the catcher molecules 707 are attached molecule 708 which is complementary to the catcher molecule 707 can be coupled.
  • Each molecule 708 to be detected has a fluorescence marker 709 which is set up in such a way that it at least partially absorbs electromagnetic radiation of the third wavelength range and, after absorption, emits electromagnetic radiation of a fourth wavelength range. At least part of the third wavelength range lies outside the fourth wavelength range and at least part of the fourth wavelength range lies within the first wavelength range. At least part of the first wavelength range lies outside the second wavelength range.
  • molecules 710 with fluorescent markers 711 which are not complementary to the catcher molecules 707 and therefore do not couple to them.
  • circuit layer 206 capture molecule 207 molecule to be detected

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Abstract

L'invention concerne une puce à biocapteur fluorescent et un ensemble puce à biocapteur fluorescent. La puce à biocapteur fluorescent comporte un substrat, au moins un dispositif de détection placé dans ou sur le substrat, qui sert à détecter un rayonnement électromagnétique, une couche de filtrage optique disposée sur le substrat, et une couche d'immobilisation disposée sur la couche de filtrage optique, laquelle sert à immobiliser des molécules pièges. Le dispositif de détection, la couche de filtrage optique et la couche d'immobilisation sont intégrées dans la puce à biocapteur fluorescent. L'ensemble puce à biocapteur fluorescent comporte une puce à biocapteur fluorescent ainsi qu'une source de rayonnement électromagnétique.
EP02758143A 2001-09-17 2002-08-12 Puce a biocapteur fluorescent et ensemble puce a biocapteur fluorescent Withdrawn EP1428026A1 (fr)

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DE10145701 2001-09-17
DE10145701A DE10145701A1 (de) 2001-09-17 2001-09-17 Fluoreszenz-Biosensorchip und Fluoreszenz-Biosensorchip-Anordnung
PCT/DE2002/002954 WO2003027676A1 (fr) 2001-09-17 2002-08-12 Puce a biocapteur fluorescent et ensemble puce a biocapteur fluorescent

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