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EP1807700A2 - Procede de mise enevidence et/ou de determination de la concentration d'au moins un ligand - Google Patents

Procede de mise enevidence et/ou de determination de la concentration d'au moins un ligand

Info

Publication number
EP1807700A2
EP1807700A2 EP05803501A EP05803501A EP1807700A2 EP 1807700 A2 EP1807700 A2 EP 1807700A2 EP 05803501 A EP05803501 A EP 05803501A EP 05803501 A EP05803501 A EP 05803501A EP 1807700 A2 EP1807700 A2 EP 1807700A2
Authority
EP
European Patent Office
Prior art keywords
radiation
ligand
solution
measurement signal
test site
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Withdrawn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
EP05803501A
Other languages
German (de)
English (en)
Inventor
Mirko Dr.Rer.Nat Lehmann
Ingo Dipl.-Ing FREUND (FH)
Holger Dr. Klapproth
Sonja Mohry
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.)
TDK Micronas GmbH
Original Assignee
TDK Micronas GmbH
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 TDK Micronas GmbH filed Critical TDK Micronas GmbH
Publication of EP1807700A2 publication Critical patent/EP1807700A2/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Withdrawn legal-status Critical Current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
    • 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

Definitions

  • the invention relates to a method for detecting and / or determining the concentration of at least one ligand, which is suspected to be contained in a solution to be analyzed, wherein at least one receptor is immobilized on at least one test site on a semiconductor chip the ligand becomes specifically bound when the receptor contacts the ligand, the solution being applied to bind the ligand to the receptor on the test site, the solution being removed from the test site and a .
  • Help liquid is applied to the test site, which contains a chemiluminophore is excited in response to the binding of the ligand to the receptor for emitting a luminescence, and wherein at least one of the luminescence sensitive, integrated in the semiconductor chip radiation receiver a radiation measurement signal is detected ,
  • the invention relates to a method for detecting and / or determining the concentration of at least one ligand which is suspected to be contained in a solution to be analyzed, wherein at least one receptor is immobilized on a semiconductor chip at at least one test site, which binds specifically to the ligand when the receptor contacts the ligand, the ligand-binding solution being applied to the receptor on the test site, wherein at least one luminophore immobilizes to the receptor at the test site, depending on the formation of the ligand in which the test site is irradiated with an excitation radiation which contains at least one wavelength at which the luminophore is excited to emit luminescence radiation, and wherein a radiation measurement is effected with the aid of at least one radiation receiver sensitive to the luminescence radiation and integrated in the semiconductor chip Signal will be.
  • the invention relates to a method for detecting and / or determining the concentration of at least one ligand suspected of being contained in a solution to be analyzed, on a support
  • receptors are immobilized on the surface of a semiconductor chip at test sites, on each of which an optical radiation receiver and a surface occupation sensor are integrated into the semiconductor chip .
  • the solution is applied to the test sites so that it comes in contact with the receptors.
  • the receptors have epitopes that undergo specific binding upon contacting the ligand with them.
  • the semiconductor chip is washed. Then, a help liquid is applied to the test site containing a chemiluminescent which is excited to deliver luminescent radiation depending on the binding of the ligand to the receptor.
  • the surface area of the surface of the semiconductor chip is measured with the receptors using the area occupation sensors at the test sites.
  • concentration of the ligand at the individual test sites is determined according to the law of mass action on the basis of the measurement signals from the radiation receivers and the area occupation sensors as well as a binding consonant.
  • the method has been proven in practice, above all, because it allows a direct measurement of the concentration of ligands over a large concentration range.
  • a disadvantage of the method is that the measurement results are relatively inaccurate in solutions containing the ligands only in low concentration.
  • DE 102 51 757 A1 also discloses a method for determining the concentration of a ligand contained in a solution, in which the binding of the ligand to the receptor is detected by means of a luminophore, which is indirectly bound to a receptor-ligand complex via a N ⁇ ch Stamm ⁇ nti body.
  • a luminophore which is indirectly bound to a receptor-ligand complex via a N ⁇ ch Stamm ⁇ nti body.
  • the semiconductor chip is washed. Thereafter, the luminophores, which are further bound to the receptors via the detection antibodies, are irradiated with excitation radiation.
  • the spectrum of the excitation radiation has an excitation wavelength at which the luminophore is excited to emit the luminescent radiation which detects with the aid of the radiation receiver. Also in this method, the measurement accuracy is still capable of improvement.
  • an auxiliary liquid is understood as meaning a liquid in which the measurement of the luminescence radiation and / or a color change occurring as a function of the binding of the ligand to the receptor takes place.
  • a substitute fluid is understood to be a fluid which, when in contact with the at least one radiation receiver, has approximately the same electrical influence on the measurement signal of the radiation receiver as the auxiliary fluid. No luminescence radiation or color change occurs in the replacement liquid.
  • a specific bond is understood to mean a covalent bond and / or a chemical bond.
  • the liquid is in contact with the radiation receivers during the measurement of the radiation measurement signal Help fluid the dark current or darkening of the Strahlungsempfän ⁇ gers influenced, which can be caused inaccuracies in particular at small radiation measuring signal levels inaccuracies when the dark current or the dark voltage is not taken into account.
  • these measurement inaccuracies are avoided by detecting a dark measurement signal during the contact with the auxiliary liquid and / or a corresponding replacement liquid with the receptor-ligand complex (s) coated with the ligand bound to the receptor the radiation measuring signal is compensated with this.
  • the at least one ligand and the at least one receptor may be biocomponents or biomolecules and, for example, nucleic acids or derivatives thereof (DNA, RNA, PNA, LNA, oligonucleotides, plasmids, chromosomes), peptides, proteins (enzyme, protein, oligopeptides, cellular receptor proteins and their Complexes, peptide hormones, antibodies and fragments thereof), carbohydrates and their derivatives, in particular glycosylated proteins and glycosides, fats, fatty acids and / or lipids.
  • nucleic acids or derivatives thereof DNA, RNA, PNA, LNA, oligonucleotides, plasmids, chromosomes
  • peptides proteins (enzyme, protein, oligopeptides, cellular receptor proteins and their Complexes, peptide hormones, antibodies and fragments thereof), carbohydrates and their derivatives, in particular glycosylated proteins and glycosides, fats, fatty acids and
  • the receptor and / or ligand may be biological cells (microorganisms and / or cells of higher organisms) and / or their sub-components, such as cell organelles.
  • the receptor and / or the ligand can also be chemical substances.
  • the replacement fluid can be found experimentally by a comparison test by detecting the dark measurement signal for different fluids and comparing it with the radiation measurement signal measured in the auxiliary fluid when no ligand is bound to the receptor (s).
  • the liquid in which the dark measurement signal has the largest coincidence with the corresponding auxiliary fluid measurement signal is used as a replacement liquid.
  • the liquids selected for the comparison test must meet the following conditions: a) the specific bonds in the liquid are sufficiently stable, b) the enzymes and / or luminophores are not damaged by the liquid, c) in the liquid, no chemiluminescence radiation visible to the radiation receiver is produced in contact with the receptor-ligand complexes.
  • the at least one ligand is labeled directly and / or via at least one detection antibody bound to the ligand with at least one enzyme (binding assay), wherein the chemiluminophore is selected such that it reacts directly or indirectly in the presence of the enzyme the mediation of a chemical substance contained in the auxiliary liquid is stimulated to emit the luminescence radiation.
  • the enzyme is bound directly to the ligand.
  • the enzyme is linked to the ligand via a bridge of at least two specific bonds. For example, a first specific binding between the ligand and a biotin-labeled
  • the specific linkages may include, for example, streptavidin-biotin linkages, digoxigenin-antidigoxigenin linkages, and / or sense and antisense DNA links.
  • the at least one enzyme is a peroxidase enzyme, preferably horseradish peroxidase, and if the chemiluminophore luminol and the chemical substance are hydrogen peroxide.
  • a peroxidase enzyme preferably horseradish peroxidase
  • the chemiluminophore luminol and the chemical substance are hydrogen peroxide.
  • other enzymes and chemiluminophores may also be used, for example luciferase and luciferin and / or alkaline phosphatase and CDP-Star or 2-chloro-5-B-methoxyspiro [l / 2-dioxetane-3,2 '. - (5'-ChIoO tricyclo [3.3.1.1 3 '7] decane ⁇ -4-yl) -l-phenyl phosphate disodium salt.
  • auxiliary liquid hydrogen peroxide and luminol and the replacement liquid preferably contains a phosphate-buffered saline solution (PBS).
  • PBS phosphate-buffered saline solution
  • ECL ® phosphate-buffered saline solution
  • the corresponding solutions are available at low cost and enable a high accuracy of measurement and verification.
  • the method according to the invention is also suitable for competitive assay systems. At least one competitor labeled with the enzyme is introduced into the solution to be analyzed, after which the solution is applied to the test site.
  • the above-mentioned problem is solved in that the radiation measurement signal is detected while the solution and / or a hilum liquid not containing the luminophore is in contact with the radiation receiver, that during the generation of the luminescence is prevented, a dark measurement signal is measured for the radiation receiver, that upon detection of the dark measurement signal of the radiation receiver is in contact with the solution, the auxiliary liquid and / or a replacement liquid, and that the radiation measurement signal is compensated with the dark measurement signal.
  • the above-mentioned object is also achieved with the features of claim 17.
  • the solution and / or replacement liquid is chosen to be approximately the same has electrical influence on the measurement signal of the radiation receiver (have), as the Schwaflüs ⁇ stechnik.
  • the generation of the luminescence radiation is suppressed by the excitation radiation being switched off and / or the auxiliary liquid being replaced by a substitute liquid which does not contain the luminophore
  • the measurement of the radiation measurement signal in the auxiliary fluid allows a higher quantum efficiency of the luminophore. largely avoided.
  • the method according to the invention can be used in the on-line polymerase chain reaction (PCR). With the help of PCR, it is possible to amplify a targeted portion between two known sequences from a small amount of DNA.
  • the luminophore is contained in the analyte solution or admixed with it. During the measurement of the luminescence radiation, the solution with the free luminophores contained therein is in contact with the radiation receiver.
  • this contact can also take place indirectly via a thin waveguide layer, which is arranged on the radiation receiver and preferably monolithically integrated with this. Even when the dark measurement signal is detected, the radiation receiver can be in indirect contact with the solution, the auxiliary liquid and / or the replacement liquid via the waveguide layer.
  • the at least one ligand is labeled directly with a luminophore (binding assay) directly and / or via at least one detection antibody linked to the ligand, wherein the luminophore is detected during detection of the radiation measurement signal for generating the luminescence radiation with a luminescence is irradiated to the wavelength of the luminescent radiation different excitation radiation for the radiation of catcher is insensitive.
  • the luminophore used is preferably an upwardly converting luminophore in which the wavelength of the luminescence radiation is smaller than the excitation wavelength.
  • Such upward-converting lumi- nophores are known per se, for example from EP 0 723 146 A1.
  • the method is also suitable for competitive assay systems. At least one competitor labeled with the luminophore is introduced into the solution to be analyzed and then the solution is applied to the test site.
  • the abovementioned object is also achieved by a method for detecting and / or determining the concentration of at least one ligand which is suspected of being contained in a solution to be analyzed, a) on at least one test site on at least one semiconductor chip immobilizing a receptor which specifically binds with the ligand when the receptor contacts the ligand, b) applying the ligand-binding solution to the receptor on the test site, c) using a reagent in the test site Contact is made and converted depending on the binding of the ligand to the receptor by a chemical Reak ⁇ tion in an indicator which differs in color from the reagent d) wherein the test site is irradiated with optical radiation, e) wherein at least one sensitive to the chemical reaction color change, in the Halbleiferchip integrated
  • a help liquid is understood to mean a liquid which differs from the solution and does not contain the reagent.
  • a replacement liquid is defined in claim 9 as a liquid which differs from the solution and the auxiliary liquid and which, when in contact with the at least one radiation receiver, has approximately the same electrical influence on the measuring signal of the radiation receiver as the liquid during the detection of the radiation measuring signal is in contact with the radiation receiver.
  • both the radiation measurement signal and the dark measurement signal are measured while the radiation receiver is in contact with a liquid.
  • the specific binding is detected by means of a color change reaction.
  • the indicator may contain, for example, methyl red and / or bromothymol blue, the color of which may change from orange to turquoise, depending on the pH value, from orange to yellow.
  • the ligand may contain a urobilinogen, bilirubin, ketone, ascorbic acid, glucose, protein, blood, pH, nitrite and / or leucocytes.
  • glucose oxidase and peroxidase can be immobilized at the test site. The detection of the ⁇ lucose can take place via a coupled enzyme reaction.
  • the ⁇ lucose is oxidized by the ⁇ -glucose oxidase to form ⁇ -gluconic acid to form hydrogen peroxide.
  • De peroxidase oxidizes a redox indicator with the formed hydrogen peroxide, which turns from green to yellow.
  • the semiconductor chip has a plurality of radiation receivers, a radiation measurement signal and a dark measurement signal preferably being determined for each of these radiation receivers, and the radiation measurement signal associated therewith compensated for each radiation receiver with its associated dark measurement signal.
  • receptors can be immobilized either on all radiation receivers or only on a part of the radiation receiver. With the help of at least one radiation receiver, on which no receptor is immobilized, can Blindmesssign ⁇ l be erf ⁇ sst be. The blank measurement signal can be subtracted from the measurement signal of at least one radiation receiver having at least one receptor in order to compensate for background radiation and / or a background color change which is not produced as a function of the binding of a ligand to a receptor.
  • At least one receptor is immobilized on at least two test sites, a radiation measurement signal and a dark measurement signal being respectively detected for the radiation receivers associated with these test sites, and the individual radiation measurement signals being compensated with the respectively associated dark measurement signal.
  • the measurement results if one carries out for the semiconductor chip only a single dark signal measurement for all Strahlungs ⁇ receiver, have relatively large scattering. These scatterings can be reduced by performing an individual dark signal measurement for each individual radiation receiver and then compensating the radiation measurement signals with the respective dark signals assigned to them, for example by subtracting the dark measurement signal from the radiation measurement signal. The method then allows even greater measurement accuracy.
  • the dark measurement signals can be detected simultaneously for several test stations. Of course, the dark measurement signals for individual Tesfstellen or groups of a plurality of test sites but also nachein ⁇ other be measured.
  • the same receptors are immobilized on at least two test sites on the semiconductor chip, to which the solution to be analyzed is applied, if at least one radiation messsignai detected and compensated with a Dunkeimesssignal for these test sites in each case, and if compensated from the thus determined Measurement signals an average signal is formed.
  • the average signal only with respect to a part of the on the H ⁇ lbleiterchip test skins to form.
  • the radiation receiver is a photodiode, which is connected to charge its junction capacitance in the reverse direction with an electrical voltage source, after which the connection to the voltage source is interrupted, and wherein during the interruption as a radiation measurement signal and / or as a dark measurement signal the electrical voltage is tapped at the photodiode.
  • the biasing of the photodiode in the reverse direction results in a low junction capacitance on the photodiode during the measurement of the radiation or dark measurement signal. This allows a large bandwidth and high sensitivity during the measurement.
  • the dark measurement signal is approximated by a section of a straight line, wherein the slope of the device is determined, and wherein the Sfrahlungsmesssignal is compensated on the basis of the slope.
  • the at least one radiation measurement signal can then be compensated in a simple manner, for example with the aid of an evaluation device integrated in the semiconductor chip.
  • a wash solution is used as replacement liquid, the at least one test site being rinsed off after application of the solution to be analyzed with the wash solution, after which the dark measurement signal is detected, and in which case the wash solution is replaced by the auxiliary liquid and the radiation measurement signal is detected.
  • the dark measurement signal is therefore measured immediately after washing the test site, while the washing solution is still located on the test site.
  • the washing solution thereby fulfills a double function and, apart from washing the semiconductor chip, also serves as a replacement liquid for measuring the dark measuring signal, since the application of an additional replacement liquid is saved, the total time required for the measurement is shortened, which is particularly advantageous for unstable ligands.
  • a reverse procedure is conceivable in which first the dark measurement signal is detected, for example in the replacement solution and then the radiation measurement signal in the auxiliary solution,
  • FIG. 1 shows a semiconductor chip on which a receptor is immobilized
  • FIG. 2 shows a representation similar to FIG. 1, but with a ligand bound to the receptor
  • FIG. 3 shows a representation similar to FIG. 2, but with a detection antibody bound to the ligand
  • FIG. 4 shows a schematic representation of a sandwich ELISA assay (enzyme-linked immunosorbent assay) in which a chemiluminescence radiation is generated
  • FIG. 6 shows a bar graph of the gradients of the dark measurement signals of the individual radiation receivers of a semiconductor chip
  • FIG. 10 shows a semiconductor chip on which a plurality of test sites with receptor are arranged, wherein a solution to be examined is applied to the semiconductor chip, the ligands and labeled with a luminophore
  • receptors 5 are immobilized at test sites 3 which are arranged on the surface of a semiconductor chip 4 (FIG. 1).
  • the semiconductor chip 4 has a plurality of test sites 3; which are arranged in a matrix-like manner preferably in a plurality of rows and columns and are spaced from one another.
  • the receptors 5 are tuned to the ligands 2 in such a way that they form a specific bond with them when they come into contact with them.
  • the immobilization of the receptors 5 can be achieved by the application of an adhesive layer on the semiconductor chip 4, at which the receptors 5 anchor themselves.
  • the adhesive layer can be, for example, a silane layer and / or a polymer layer (cf., EP 1 176423 A1).
  • the receptors 5 can be printed on the test stations and / or applied to the semiconductor chip 4 in dissolved form with a micropipette, which is positioned relative to the semiconductor chip 4 by means of an XY positioning device.
  • the solution to be analyzed is applied to the test skins 3 in order to allow the ligand 2 contained in the solution 1 to bind specifically to the receptors 5 (FIG. 2).
  • the application of the solution 1 can take place with the aid of a pipette not shown in detail in the drawing.
  • the semiconductor chip 4 adjoins a flow measuring chamber, which has an inflow opening, through which the solution 1 is pumped into the flow measuring chamber by means of a micropump.
  • Fig. 3 it can be seen that the solution 1 is then removed, for example by means of a rinsing liquid from the test sites 3, so that then only those Lig ⁇ nden 2 are arranged at the test sites 3, which are specifically bound to a receptor 5.
  • the rinsing liquid can be stored in a reservoir (not shown in detail in the drawing) which can be connected to the test sites 3 by means of a supply device which has a micropump on it.
  • the ligands 2 remaining at the test sites 3 are brought into contact with biotinylated detection antibodies 7 which bind to the ligands 2 but not to free receptors 5.
  • biotinylated detection antibodies 7 which bind to the ligands 2 but not to free receptors 5.
  • receptor-ligand-detection antibody complexes are formed.
  • the detection antibodies 7 are stored together with a carrier liquid in a further reservoir, which can be connected to the test sites 3 via the supply device.
  • any remaining free ligands 2 are removed from the test sites, so that then only free receptors, receptor-ligand complexes and / or receptor-ligand detection antibody complexes are present at the test sites 3.
  • the test sites are rinsed with a rinsing liquid, which is conducted to the test sites 3 from a supply container with the aid of the supply device.
  • test sites 3 are brought into contact with a tracer solution which contains streptavidin, to which the enzyme horseradish is added.
  • Peroxidase 9 is bound.
  • the streptavidin has the property of binding specifically to the biotinylated detection antibodies 7.
  • horseradish peroxidase 9 indirectly binds to the detection antibodies 7 through the mediation of streptavidin.
  • the tracer solution is conveyed from a reservoir to the test sites 3 with the aid of the delivery device.
  • a replacement liquid 10 namely a phosphate buffered saline solution (PBS - phosphate buffered saline
  • the replacement liquid 10 is in a reservoir stored on the supply means with the test sites 3 is connectable.
  • horseradish peroxidase 9 is now only present at the sites where a ligand 2 is bound to a receptor 5 immobilized on the semiconductor chip 4.
  • the receptor-ligand complexes are therefore labeled with horseradish peroxidase 9.
  • an electronic radiation receiver ⁇ for example a photodiode, is integrated in the semiconductor chip 4 at each of the test sites 3. It can clearly be seen that the receptors 5 are each immobilized directly on the radiation receivers ⁇ .
  • the radiation receivers ⁇ are sensitive to a luminescence radiation, which is generated in a later Verfedhrensuze, which will be described in more detail below, depending on the binding of the ligand 2 to the receptor 5.
  • FIG. 5 it can be seen that the radiation receivers ⁇ are charged to a predetermined electrical voltage value at the beginning of the dark measurement, and that the amount of the measuring voltage respectively applied to the radiation receivers 6 continuously decreases during the dark measurement.
  • a slope value for the dark measurement signal 1 1 is determined and buffered.
  • the corresponding slope values in the form of a bar chart for a semiconductor chip with 32 test sites 3, which are arranged in matrix form in four rows and eight columns, are shown graphically in FIG.
  • a second dark measurement signal 12 is additionally shown, which was recorded at dry radiation receiver ⁇ . It can clearly be seen that the second dark measurement signal 12 has a greater gradient than the first one l ⁇
  • the measuring signal or the dark current of the radiation receiver ⁇ is therefore influenced by the presence of the substitute liquid 10.
  • the replacement liquid 10 is removed from the test sites 3 and an auxiliary liquid 13 is applied to the test sites 3 containing hydrogen peroxide and luminol.
  • assistance liquid 13 is available for example under the name // ECL ® solution "in the trade.
  • the ligand is not included, or in a non-relevant for the measurement of concentration.
  • the help liquid 13 is by means of the supply means from a reservoir promoted to the test sites 3.
  • a luminescence radiation 14 is generated depending on the binding of the ligand 2 to the receptor 5, a luminescence radiation 14 is generated. This has a wavelength of about 428 nm.
  • the luminescence radiation 14 is detected in each case with the aid of the radiation receiver ⁇ located at the test site 3, while it is covered with the auxiliary liquid 13.
  • the corresponding radiation measurement signal is denoted by 15 in FIG. It can clearly be seen that the radiation measurement signal 15 drops considerably more than the dark measurement signal 11 due to an electrical current induced in the radiation receiver ⁇ by the luminescence radiation.
  • the radiation measuring signals 15 of the individual radiation receivers ⁇ each have an approximately linear course during or after the detection of the radiation. With the aid of the slope value for the dark measurement signal 11 assigned to the corresponding radiation receiver ⁇ , a value for the slope of the radiation measurement signal 15 relative to the dark measurement signal 11 is determined.
  • FIG. 8 graphically shows the respective relative gradient values in the form of a bar chart for the individual radiation receivers ⁇ . For comparison, the absolute slope values of the radiation measurement signals 15 are shown in FIG.
  • the matrix of the semiconductor chip 4 has a plurality of groups of test sites 3; on each of which the same receptors are immobilized. Thus, columns 1 to 3 form
  • Test sites 3 which are assigned to different groups, each differ in their receptors. From the measured values of test sites 3, which belong to the same group, the arithmetic mean is formed in each case. As a result, metrological tolerances / which may be due to differences in the radiation receivers ⁇ and / or the occupancy of the radiation receivers ⁇ with the receptors 5 are smoothed.
  • FIG. 8 it can be clearly seen that in the first matrix line arranged at the rear in the drawing, a relatively high measuring signal was respectively measured at three measuring points assigned to the same group, and that these measuring signals largely correspond. At the corresponding measuring points of the second line, the measuring signal is considerably lower. An even smaller measuring signal was determined at the first three measuring points of the third line. At the remaining measuring points the measuring signal is approximately zero.
  • a solution 1 to be analyzed is applied to a test site 3 arranged on the semiconductor chip 4, which solution contains the ligands 2 and competitors 1.
  • a test site 3 arranged on the semiconductor chip 4, which solution contains the ligands 2 and competitors 1.
  • radiation receiver ⁇ is integrated into the semiconductor chip 4.
  • Both the ligands 2 and the competitors 1 ⁇ bind specifically, if you come into contact with a receptor 5, specifically to this.
  • Fig. 9 it can be seen that the competitors l ⁇ are labeled with a luminophore 17.
  • the luminophore 17 emits a Luminesze ⁇ zstr ⁇ hlung 14 when it is irradiated with a Anreungsstr ⁇ hlung.
  • the wavelength of the excitation radiation differs from the wavelength of the luminescence radiation 14.
  • the radiation receiver ⁇ are insensitive to the excitation radiation.
  • the test site 3 is irradiated with the excitation radiation in order to excite luminophore 17, which are bound indirectly via the competitors 16 to the receptors 5, for emitting the luminescence radiation 14.
  • a radiation measurement signal 15 is detected at the auxiliary liquid 13 in contact with the radiation receiver mit with the aid of the radiation receiver ⁇ .
  • the excitation radiation is then switched off in order to detect a dark measurement signal 1 1 with the aid of the radiation receiver ⁇ with auxiliary fluid 13 in contact with the radiation receiver ⁇ .
  • the radiation measurement signal 15 is compensated by the dark measurement signal 1 1 is subtracted from the radiation measurement signal 15.
  • the aid of the compensated measuring signal thus obtained, the slope of the radiation measuring signal 15 relative to the dark measuring signal 11 is determined.
  • the compensation can take place with the aid of a corresponding compensation device, which has inputs for the radiation measurement signal 15 and the dark measurement signal 11 and also for the compensated radiation measurement signal.
  • FIG. 10 it can be seen that the receptors 5 immobilized at the individual measuring points 3 undergo a specific binding with different ligands.
  • the ligands are each labeled with a luminophore 17, which is only on the
  • Ligands, but not to free receptors 5 binds.
  • the ligands are applied to the test sites 3 in the solution 1 to be analyzed. After the solution 1 for a period of time sufficient to bind a representative amount of the ligands 2 contained in the solution 1 to the receptors 5, with the test site 3 in Contact 1, the solution 1 is removed from the test site 3 and replaced by a Wegenbergkeit 13 which contains no suitable ligands to the receptors 2. Thereafter, the test site 3 is irradiated with the excitation radiation in order to excite those luminophores 17, which are bound to the receptors 5 via the ligands 2, for the emission of the luminescence radiation 14.
  • the radiation measuring signals 15 are detected at auxiliary liquid 13 in contact with the radiation receivers ⁇ . Thereafter, the excitation radiation is switched off to measure the dark measurement signals 1 1, while the radiation receiver ⁇ lo continue to be in contact with the auxiliary liquid 1 3.
  • a receptor 5 is thus immobilized on a semiconductor chip 4 at a test site 3, said receptor being immobilized with
  • ligand 2 can form a specific binding.
  • the solution is applied to the test site 3.
  • a luminescence radiation 14 or a color change is generated.
  • a radiation measurement signal is generated
  • the 20 15 detects while the auxiliary liquid 13 is in contact with the radiation receiver ⁇ . While the generation of the luminescence radiation 14 is prevented and the radiation receiver ⁇ is in contact with the auxiliary liquid 1 3 and / or a replacement liquid, a dark measurement signal 1 1 for the radiation receiver ⁇ is measured.
  • the radiation measurement signal 15 is provided with the dark measurement signal 11

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Abstract

L'invention concerne un procédé permettant de mettre en évidence et/ou de déterminer la concentration d'un ligand (2) contenu dans une solution (1) à analyser, selon lequel il est prévu d'immobiliser un récepteur (5) sur une puce à semi-conducteur (4) en un point de test, une liaison spécifique pouvant intervenir entre ledit récepteur et le ligand (2). Pour lier le ligand (2) au récepteur (5), la solution (1) est appliquée sur le point de test (3). En fonction de la liaison du ligand (2) au récepteur (5), un rayonnement luminescent (14) ou une modification de couleur est produit(e). Un signal de mesure de rayonnement est détecté à l'aide d'un récepteur de rayonnement (6) intégré dans la puce à semi-conducteur (4), tandis que la solution (1) et/ou un liquide auxiliaire ne contenant pas le luminophore (17) se trouve au contact du récepteur de rayonnement (6). Lorsque la production du rayonnement luminescent est inhibée et que le récepteur de rayonnement (6) est en contact avec le liquide auxiliaire et/ou le liquide de substitution, un signal de mesure d'obscurité est mesuré pour le récepteur de rayonnement (6). Le signal de mesure de rayonnement est compensé avec le signal de mesure d'obscurité.
EP05803501A 2004-10-13 2005-10-12 Procede de mise enevidence et/ou de determination de la concentration d'au moins un ligand Withdrawn EP1807700A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102004050032A DE102004050032A1 (de) 2004-10-13 2004-10-13 Verfahren zum Nachweisen und/oder zum Bestimmen der Konzentration mindestens eines Liganden
PCT/EP2005/010976 WO2006040142A2 (fr) 2004-10-13 2005-10-12 Procede de mise en evidence et/ou de determination de la concentration d'au moins un ligand

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WO2009132667A1 (fr) * 2008-04-30 2009-11-05 Micronas Gmbh Procédé permettant de déceler et/ou de déterminer la concentration d'un ligand

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US20080138829A1 (en) 2008-06-12
DE102004050032A1 (de) 2006-04-27
WO2006040142A3 (fr) 2006-08-10

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