WO2011110338A1

WO2011110338A1 - Fibre-optic device for emitting and receiving light, system, method and computer programme product

Info

Publication number: WO2011110338A1
Application number: PCT/EP2011/001158
Authority: WO
Inventors: Reinhard Bittner
Original assignee: Beckman Coulter, Inc.
Priority date: 2010-03-09
Filing date: 2011-03-09
Publication date: 2011-09-15
Also published as: DE102010010741A1

Abstract

The invention relates to a fibre-optic device for emitting and receiving light, in particular for exciting and observing fluorescence. Said device comprises a fixing device for a fibre-optic bundle and at least one fibre-optic bundle, the latter being fixed and heated by means of the fixing device for said bundle. The invention also relates to a system for exciting and measuring fluorescence, to a method for detecting target molecules and to a computer programme product.

Description

Optical fiber device for emitting and receiving

Light, system, procedures and computer program product

description

The present invention relates to a temperature-controllable optical waveguide device for emitting and receiving light, a system for exciting and measuring fluorescence on at least one liquid receiving area, a method for the qualitative and / or quantitative detection of target molecules in a sample to be analyzed, and a computer program product.

State of the art

Optical systems are used in the field of laboratory diagnostics and medical research for the quantitative and qualitative determination of target molecules in patient samples. A variety of such optical systems, such as systems for photometric absorption measurement and fluorescence analysis are already known.

Fluorescent label molecules are added to a sample to be analyzed in the commonly performed fluorescence-based methods for detecting nucleic acids. A distinction is made between non-specific labeling methods, e.g. by means of fluorescent DNA intercalators and sequence-specific detection methods in which DNA or RNA probes complementary to the desired nucleic acid are used.

In general, excitation of the label molecules by means of light of predetermined wavelength and detection of the fluorescence emitted by the fluorescent label molecules enables the qualitative determination of a possible chemical reaction and, by evaluating the measured light intensities, also a quantitative analysis of this reaction. The Polymerase Chain Reaction (PCR) is a procedure for DNA amplification with a cyclic character. By means of this reaction, which is highly relevant for practice, even the smallest amounts of a nucleic acid molecule optionally present in the sample can be amplified. In the meantime, the use of fluorescently labeled primers and probes for the simple fluorescence-based qualitative and quantitative detection of DNA has become widespread in PCR and other DNA amplification reactions.

In view of current developments in the field of DNA analysis, it is of increasing scientific as well as economic interest to reduce as much as possible the amounts of starting material used and the reaction volume for the amplification. This development has already progressed to the use of individual cells as starting material. The sample volumes used in the single cell analysis amount to only a few microliters. Processing such small sample volumes places a special requirement on a system for determining fluorescence in chemical reactions.

In many of these systems for the qualitative and quantitative measurement of fluorescence volumes of about 50 microliters to a few milliliters are usually used for the sample material to be analyzed. In addition, the starting material is usually provided in small sample tubes or in wells of multi-well plates, which, however, is unsuitable for the field of single-cell amplification. Likewise, manipulation of the sample during amplification and fluorescence measurement is not possible since these containers must remain closed during the amplification phase.

When using liquid samples, it has proven to be problematic that products evaporating from the sample can form deposits on the surfaces of optical measuring devices, which, in addition to reduced sensitivity, can also cause measurement errors in the determination of the fluorescence. This fouling effect is all the more serious if the liquid samples are additionally heated above ambient temperature during the analysis. Object of the invention

It is the object of the invention to provide an improved apparatus as well as a system and method for exciting and / or observing fluorescence in liquid or wet samples contained in unclosed sample containers with the samples and apparatus in one closed volume and contamination of the device during the implementation of the method should be avoided or at least reduced.

This object is achieved according to the independent claims. Advantageous embodiments are the subject of the dependent claims.

Optical fiber device according to one aspect

One aspect of the present invention relates to an optical waveguide device for emitting and receiving light, in particular for excitation and observation of fluorescence, with an optical fiber bundle fixing device and with at least one optical waveguide bundle,

wherein the at least one optical fiber bundle is fixed by means of the Lichtleiterbündelfixierungsvorrichtung and is heatable by means of Lichtleiterbündelfixierungsvorrichtung.

According to one aspect of the invention, the contamination of the optical fiber bundle during the excitation and observation of fluorescence by tempering the optical fiber bundle by means of the Lichtleiterbündelfixierungsvorrichtung is avoided or reduced. Pollution of the optical fiber bundle is understood as meaning, for example, the condensation of molecules or of molecular aggregates on the light emitting bundle. These molecules or molecular aggregates can be formed, for example, by the evaporation of wet or liquid samples, for example during the excitation and observation of fluorescence. The evaporated molecules or molecular aggregates are distributed in the closed volume in which Sample and device are located. A tempering of the optical fiber bundles avoids or reduces the condensation of the molecules or molecular aggregates on these.

In particular, in the excitation and observation of fluorescence in heated liquid or wet samples, a corresponding heating of the optical fiber bundles of the optical fiber device effectively prevent condensation of liquid on the optical fiber bundles and thus ensure the undisturbed excitation and observation of fluorescence.

The term fluorescence is understood to mean the spontaneous emission of light during the transition of an electronically excited system into a state of lower energy. Certain substances may e.g. be excited by the emission of light to emit fluorescence. As a rule, the emitted light is lower in energy than the light previously absorbed by these substances.

An optical waveguide device according to the invention comprises at least one optical fiber bundle. Each optical fiber bundle is composed of at least two optical fibers which are suitable for the forwarding of optical signals. The at least one optical fiber bundle can be heated by means of a heatable

Lichtleiterbündelfixierungsvorrichtung be spatially fixed relative to a sample to be observed. The light guides according to the invention are preferably glass fibers and / or polymer optical fibers with a diameter between 10 micrometers and 500 micrometers, preferably between 20 and 250 micrometers, and more preferably with a diameter of about 50 micrometers.

The at least one optical fiber bundle of the optical fiber device is fixed in the Lichtleiterbündelfixierungsvorrichtung.

In one embodiment, the optical fiber fixing device is designed as a continuous, preferably metallic plate. It has in an advantageous grid on several continuous openings, which are just so large that they each receive and fix a fiber optic bundle. In a further embodiment, the optical fiber fixing device can also consist of more than one part or element. Thus, the individual optical fibers of the optical fiber bundle can be fixed, for example, first by a thermally conductive holder. This version consists for example of a metallic material. It can be embodied, for example, as a pinch socket known to the person skilled in the art. The optical fiber bundle fixing device according to this embodiment comprises a plurality of such sockets and a thermally conductive plate, which in an advantageous grid has a plurality of continuous openings for receiving in each case a socket.

For example, the thermally conductive sockets may protrude beyond the sample side surface of the thermally conductive plate toward the sample to be observed, preferably 2 to 20 mm, more preferably 3 to 15 mm, and most preferably about 7 mm. This is advantageous in order to influence the temperature of the sample to be observed as little as possible by the temperature control of the Lichtleiterbündelfixierungsvorrichtung.

Embodiments of the optical fiber device

According to one embodiment, the optical fiber fixing device comprises at least one heating device.

Such a heating device, which consists at least partially of a heat-conducting, preferably metallic material, may e.g. be designed as electrical resistance heating, Peltier element or as a tempered liquid flow-through heating element.

In one embodiment of the light guide device, the light guide bundle fixing device is arranged on an end region of at least one optical fiber bundle. On the one hand, this enables a precise placement and, on the other hand, a targeted temperature control of the end region of the at least one optical fiber bundle of the optical waveguide device relative to a sample to be observed.

The sample-side end region of an optical fiber bundle, in other words the sample-side optical fiber bundle end region, is understood to be the region of the optical fiber bundle which is directly surrounded by the heatable optical fiber bundle fixing device. The corresponding final surface of the optical fiber bundle is referred to hereinafter as the sample-side end surface of an optical fiber bundle or, in other words, as the specimen-side optical fiber bundle end surface.

At the sample-side end surface, optical signals can be recorded in or emitted from the at least one optical fiber bundle. The ends of the individual optical fibers of an optical fiber bundle are arranged substantially flush with one another in its end region. In other words, all optical fibers of an optical fiber bundle terminate substantially at the same height in the end region of an optical fiber bundle, so that the end surfaces of the optical fiber bundles are substantially planar.

According to one embodiment, the Lichtleiterbündelfixierungsvorrichtung is designed as a heater.

Consequently, the Lichtleiterbündelfixierungsvorrichtung and in particular the end portion of the at least one optical fiber bundle can be tempered without the aid of an additional heating device. The

The optical fiber fixing apparatus may be used in this embodiment as e.g. even act as electrical resistance heating or be an electrical resistance heater.

According to one embodiment, the optical waveguide device has at least one optical fiber bundle, which has at least one excitation optical waveguide bundle comprising at least one excitation light guide and at least one emission light guide bundle with at least one emission light guide.

By way of example, the excitation light guides of the at least one excitation light guide bundle of the light guide device run in such a way that light from a light source can be guided onto a sample to be observed by means of the excitation light guide bundle. By contrast, the emission light guides of the at least one emission light guide bundle of the light guide device run in such a way that light from the sample to be observed can be guided, for example, to a detector. An optical fiber bundle comprises at least one excitation optical fiber bundle and at least one emission optical fiber bundle. The excitation optical fiber bundles and the emission fiber bundles are brought together at the sample-side end region of the optical fiber bundle facing the sample to be observed. The excitation optical fiber bundles and emission optical fiber bundles are fixed there by means of the optical fiber bundle fixing device in such a way that a sample to be observed can both be illuminated by the optical fiber bundle and light emitted by this specimen can be received in the optical fiber bundle.

Advantageously, the excitation light guide and the emission light guide are homogeneously mixed at the end region of the light guide bundle so that the number of excitation light guides and emission light guides are approximately the same in each region of the cross section of the light guide bundle end region. This ensures a homogeneous delivery of light by means of the optical fiber bundle to a sample to be observed and a homogeneous absorption of light by means of the optical fiber bundle starting from a sample to be observed.

According to one embodiment, excitation optical fiber bundles or emission optical fiber bundles comprise 1 to 5000, preferably 1 to 1000, particularly preferably 1 to 500 and most preferably 10 to 300 excitation light guides, or 1 to 5000, preferably 1 to 1000, particularly preferably 1 to 500 and most preferably 10 up to 300 emission light guides. The excitation fiber bundles and For example, emission optical fiber bundles of an optical fiber bundle may contain about the same number of excitation optical fibers and emission optical fibers. A corresponding optical fiber bundle can thus comprise, for example, in each case 50 excitation light guides and emission light guides, which are uniformly mixed in the region of the cross section of the optical fiber bundle end region.

In one embodiment variant, the at least one optical fiber bundle comprises more emission light guides as excitation light guides.

Preferred ratios between excitation light guides and emission light guides are between 1: 1, 1 and 1:10, preferably between 1: 1, 5 and 1: 5 and particularly preferably about 1: 2.

By providing more emission light guides relative to excitation light guides, the sensitivity of the fluorescence measurement device can be increased without significant losses in fluorescence excitation.

According to one embodiment, the light guide device comprises at least one light source and at least one detector, wherein the at least one excitation light guide bundle is associated with the at least one light source and at least one detector is associated with the at least one emission light guide bundle.

In other words, the at least one light source may be spatially associated with the at least one excitation optical fiber bundle. The at least one detector may be spatially associated with the at least one emission light guide bundle.

The at least one light source comprises, for example, a laser, a laser diode, an LED, a gas discharge lamp, and / or an incandescent lamp.

For example, the optical fiber device may comprise the at least one light source and the at least one detector in addition to the optical fiber bundle, wherein the Optical fiber bundle comprises at least one excitation optical fiber bundle and at least one emission optical fiber bundle.

This assignment advantageously has the effect that light from at least one light source can be conducted via the at least one excitation conductor bundle to at least one sample to be observed and light emitted by this sample via the at least one emission light bundle to at least one detector by means of the light guide device. Likewise, light from a plurality of light sources can be directed via several excitation light guide bundles simultaneously to one or more samples to be observed, and from there can be conducted simultaneously to a plurality of detectors via a plurality of emission light guide bundles.

According to one embodiment, the at least one optical fiber bundle comprises N excitation optical fiber bundles, to which N light sources are assigned, and M emission optical fiber bundles, which are assigned to a common detector. The variables N and M are each a natural number with values between 2 and 1000, preferably between 5 and 500, more preferably between 10 and 250 and most preferably with the value 48. The variables N and M can have different values. The variables N and M can have identical values.

For example, a detector may be implemented as a photomultiplier tube, avalanche photodiode, photodiode, CCD or CMOS chip, or generally in the form of any light-sensitive substance, or may comprise a photodiode, a CCD or CMOS chip, or generally any light-sensitive substance. The detector can be read out by means of a read-out device during operation of the light guide device.

According to one embodiment, the detector is temperature-controlled.

The detector can, using a suitable temperature control element in the operation of the light guide device on a range of 5 ° C to 60 ° C, preferably 15 ° C heated to about 35 ° C and / or cooled. During operation of the optical waveguide device, it is particularly preferred to keep the temperature of the detector substantially constant. In other words, the detector is tempered such that its setpoint temperature does not differ from its actual temperature within a certain period of time by more than 5 ° C., preferably not more than 2 ° C., and more preferably not more than 1 ° C. As Temperierungselemente come, for example, Peltier elements, based on temperature-controlled liquids and / or air mass heating or cooling technologies or facilities in question. Such a detector allows, for example, the setting of a substantially constant temperature at the detector to compensate for changes in the ambient temperature, which could result in a falsification of the measurement results.

System according to one aspect

One aspect of the invention relates to a system for exciting and measuring fluorescence on at least one liquid receiving region with an inventive optical waveguide device, a substrate whose surface comprises at least one liquid receiving region, and with a substrate tempering device,

wherein the substrate is tempered by the substrate temperature control device and the optical fiber device is arranged relative to the substrate such that the at least one optical fiber bundle of the optical fiber device and the substrate are non-contact.

A substrate in the sense of the present description may be an article which is thermally conductive and on whose surface samples for fluorescence analysis can be positioned. Glass and / or plastic slides may be exemplary substrates. The glass and / or plastic slide may, for example, be essentially cuboid, wherein one surface of the cuboid substrate may be the surface on or on which one or more samples for fluorescence analysis can be positioned. According to the invention, the term liquid-receiving region means an area of the surface of a substrate on or on which a liquid to be observed can be specifically positioned. Such liquid receiving areas according to the invention can be, for example, superficial cavities and / or liquid receiving areas obtained by hydrophobic and / or hydrophilic modification of the surface structure. For example, a plurality of liquid receiving areas are provided on the substrate, which are separated from each other, for example, by suitable elevations of the substrate surface and / or by hydrophobic coatings of the substrate surface. Systems according to the invention having a plurality of liquid receiving areas preferably have from 2 to 1000, more preferably from 10 to 250 and most preferably 48 liquid receiving areas. It is also possible to arrange 2, 3, 4, etc. substrates which, in each case or together, have 2 to 1000, particularly preferably 10 to 250 and most preferably 48 liquid receiving areas.

An exemplary Substratemperierungsvorrichtung allows the variable temperature of the substrate or substrates and the samples positioned thereon to be observed. An exemplary substrate temperature control device may comprise any desired heating device and any desired cooling device. For efficient heating or cooling of the substrate by means of the substrate temperature control device, there is sufficient thermal coupling between the substrate and the substrate temperature control device, which can be ensured, for example, by the largest possible contact between the substrate temperature control device and the substrate. When tempering planar or planar substrates, a substrate tempering device accordingly comprises, in particular, a planar surface or area. This surface or surface may be suitable or designed for positioning and temperature control of the substrate. In a particular embodiment of the substrate temperature control device, a planar receptacle for positioning and temperature control of the substrate is provided on one of the outer sides of the substrate, preferably on the upper side of the substrate. The top can also be used as upper Surface or surface are called. In the operating state of the system, the substrate is arranged such that the upper side of the substrate and the Lichtleiterbündelendfläche the associated at least one optical fiber bundle are opposite. In other words, end faces of the excitation light guides through which light is emitted to the substrate and end faces of the emission light guides through which light from the sample is received in the emission fibers may be arranged substantially parallel to the upper surface of the substrate. Under parallel in this sense is also to be understood that depressions, bumps, etc. may be present on the substrate and, for example, remain between the depressions, bumps, etc. planar surface areas of the top. The planar surface areas of the upper surface of the substrate and the end surfaces of the excitation light guides and end surfaces of the emission light guides may be substantially parallel.

Possible . Embodiments of such a substrate tempering device include, for example, devices for carrying out the polymerase chain reaction (PCR), e.g. the device TC4000 / FTC4 / Flat from the company Techne or the device T1 thermocycler with in-situ block of the company Biometra. As particularly suitable for the tempering of planar substrates, e.g. the use of the device "AmpliSpeed" Beckman Coulter Biomedical GmbH (Munich, Germany) proved. Thus, one aspect of the invention may also include the use of a light guide device according to the invention in or with a substrate tempering device, in particular in conjunction with a cuboid substrate.

The system according to the invention for exciting and measuring fluorescence on at least one liquid receiving region is designed such that the at least one optical fiber bundle of the optical fiber device and the substrate are positioned without contact relative to one another so that a gap filled with gas is formed between the at least one optical fiber bundle and the substrate.

In one embodiment, all the optical fiber bundles of the optical fiber device and the Substrate positioned corresponding to each other without contact. The intermediate space advantageously prevents contamination of the at least one optical fiber bundle with liquid samples applied to the substrate. Soiling of the system during the successive examination of several samples on different substrates, ie when the substrates change, can thus be effectively avoided.

Embodiments of the system

In one embodiment of the system, the at least one optical fiber bundle fixed by the optical fiber fixing device ends above the substrate, and the shortest distance between the end surface of the at least one optical fiber bundle and the substrate is between 0.1 mm and 20 mm, preferably between 0.5 mm and 10 mm , more preferably between 1 mm and 5 mm, and most preferably 3 mm.

The term 'above' in this context means that the distance between the substrate surface and the end face of the at least one optical fiber bundle is adjusted along the perpendicular to the sample-side surface of the substrate. This also applies if the substrate has depressions, unevenness, etc. and, for example, flat surface areas remain between the depressions, unevennesses, etc. The distance between the sample-side substrate surface and the optical fiber bundle end surface may be set along the normal to the planar surface areas of the surface of the substrate and the optical fiber bundle end surface. In other words, the distance is the distance along the normal between the optical fiber bundle end surface and the planar surface regions of the surface of the substrate.

The setting of an optimum distance between the substrate surface and the end of the optical fiber bundle in the sense of the present description is intended to ensure that contamination of the optical fiber bundle ends, ie the fiber optic bundle end surfaces, with sample liquids or sample material is avoided becomes. At the same time, a sufficient amount of light can be emitted to excite fluorescence from the at least one optical fiber bundle end face, the sample being positioned in a region of the substrate surface to be illuminated. Furthermore, a sufficient amount of light for measuring the specific fluorescence of fluorescent samples on the substrate surface can be picked up by the optical fiber bundle end face. The recording of nonspecific fluorescence, which in contrast to the above-mentioned specific fluorescence does not originate from the sample illuminated with the optical fiber bundle, but for example from an adjacent sample, into the optical fiber bundle can be avoided according to the described embodiment by the choice of a suitable distance of the optical fiber bundle end to the substrate ,

In this case, the distance of the optical fiber bundle end to the substrate to be used depends on how large the surface of the substrate to be illuminated by an optical fiber bundle, e.g. exactly one liquid intake area is. It is likewise dependent on in which maximum exit angle or acceptance angle α the light emerges from the optical fiber bundle relative to the optical axis of the optical fiber bundle and at what distance optionally a plurality of regions to be observed independently are provided on the surface of the substrate.

For example, the cone of light emerging from an optical fiber bundle neither illuminates substantially more nor substantially less than the area of the substrate surface to be observed, for example precisely one liquid absorption region or a defined subregion of a liquid absorption region. In this case, the distance between the optical fiber bundle end and a region to be observed on the substrate surface is given by the following formula: a = (Dd) / (2 ^* tan a) [Formula I] where:

a is the distance between the fiber optic bundle end and one to be observed Area on the substrate surface is. The distance a can also be called the ideal distance.

α is the maximum exit angle of the light from the optical fiber bundle relative to the optical axis of the optical fiber bundle.

d is the mean diameter of the optical fiber bundle end face.

D is the mean diameter of the area to be observed on the substrate surface.

The maximum exit angle α may be dependent on the material of the optical fibers used and is e.g. in the case of step fibers usually in a range between 1 1, 5 ° and 25.5 °.

The distance between the optical fiber bundle end surface and the substrate surface may also differ by up to 50% from the ideal distance a to be determined according to formula 1. However, it is by no means less than the maximum height of a liquid or wet sample applied to the substrate surface in order to prevent soiling of the optical fiber bundle end surfaces. When observing liquid or moist samples by means of tempered fiber bundle ends, a sufficient distance between the liquid sample to be observed and the tempered fiber bundle end must also be maintained in order to reduce unwanted temperature influences on the sample.

Since with increasing distance of the optical fiber bundle end surface to the substrate increased stray light and / or nonspecific fluorescence, eg from adjacent liquid receiving regions, may enter the optical fiber bundle end surface, the preferred distance range between the fiber optic bundle end surface and the substrate is also limited upwards. If a plurality of liquid receiving regions are to be observed on the substrate independently of one another by means of a plurality of optical fiber bundle ends, the preferred distance of the corresponding optical fiber bundle end surfaces from the substrate is also determined by the distance of these liquid receiving regions from one another on the substrate. The closer the observed liquid receiving areas on the substrate to each other, the more less is the preferred distance of the respective optical fiber bundle end faces from the substrate.

In one embodiment, the surface of the at least one liquid receiving area has modified wetting properties and comprises a central, preferably circular hydrophilic reaction area, a preferably annular hydrophobic central area surrounding the reaction area and a preferably annular hydrophilic outer area surrounding the central area. The outer area is in turn surrounded by a hydrophobic area.

The substrate surface in the region of such a liquid receiving region is preferably substantially planar or planar. Particularly preferred are completely planar or planar substrates with several liquid receiving areas, for example 7.5 cm long and 2.5 cm wide glass or plastic slides with 48 liquid receiving areas. When using substrates having a plurality of hydrophobically structured liquid receiving areas, the areas between the individual liquid receiving areas advantageously have a hydrophobic surface structure.

The reaction area is preferably arranged in the center of the respective liquid receiving area. It serves to apply a sample and / or reaction liquid to be observed, which is prevented from flowing on the surface by the hydrophobic, preferably annular hydrophobic region surrounding the reaction region. For protection against evaporation, liquid samples and / or reaction liquids positioned on the reaction zone are advantageously covered with an oil-like liquid. The hydrophobic substrate surface surrounding the annular hydrophilic outer region of the liquid-receiving region prevents bleeding of the oily liquid from the region of the liquid-receiving region. The above statements apply mutatis mutandis to a large number of liquid intake areas for each liquid intake area. When using substrates with liquid receiving regions which have wetting properties modified in this way, the optimum distance a between the at least one fiber optic bundle end surface and the substrate surface is preferably set so that the light emerging from the fiber optic bundle end surface strikes substantially exclusively the central, preferably circular, hydrophilic reaction region. so that essentially only the fluorescence occurring in the reaction region is measured. The area D to be observed on the substrate surface in this case corresponds to the mean diameter of the hydrophilic reaction area of the liquid wetting areas with modified wetting structure. In compliance with the above-mentioned restrictions, the optimum distance a between the end of the optical fiber bundle and the substrate surface (according to formula 1) can also be exceeded or fallen below by up to 50%.

In one embodiment of the system for exciting and measuring fluorescence on at least one liquid receiving area, at least one liquid receiving area is assigned an optical fiber bundle end fixed by the optical fiber fixing device, the liquid receiving area and the optical fiber bundle end area of the associated optical fiber bundle having substantially the same dimensions and the liquid receiving area and the associated optical fiber bundle being so aligned with each other, that from the Lichtleiterbündelendfläche emerging light strikes the associated liquid receiving area.

When using liquid receiving regions which have a reaction region as part of the liquid receiving region, for example, the reaction region of the liquid receiving region and the optical fiber bundle end surface of the associated optical fiber bundle can have substantially the same dimensions. In other words, the reaction area of the liquid receiving area and the associated optical fiber bundle end face have an in Substantially identical diameter, or - for non-circular surfaces - a substantially identical average diameter.

For the purposes of this description, the criterion of essentially the same dimensions should also be met if the average diameter of the liquid absorption region or the average diameter of the reaction region of the liquid absorption region is 0.25 to 4 times the average diameter of the optical fiber bundle end of the associated optical fiber bundle end , Since light emerging from the optical fiber bundle end is scattered relative to the optical axis of the optical fiber bundle, it is advantageous if the average diameter of the liquid receiving region is slightly larger than the average diameter of the optical fiber bundle end of the associated optical fiber bundle end.

For example, in one embodiment, the circular reaction area of a liquid receiving area has a diameter of 1.6 mm, while the diameter of the optical fiber bundle end of the associated optical fiber bundle end is 1 mm. By setting a distance of about 3 to 3.5 mm between the optical fiber bundle end and the substrate can be ensured when using the above-mentioned dimensions that light emerging from the Lichtleiterbündelendfiäche essentially only on the reaction region of the liquid receiving area and from this outgoing fluorescence effectively with the associated fiber optic bundle end can be detected.

For example, a liquid receiving area and a fiber optic bundle end are each associated with each other such that the center of the liquid receiving area and the center of the Lichtleiterbündelendfiäche the associated Lichtleiterbündelendes come to lie on a common vertical to the substrate surface. The liquid absorption region and the associated optical fiber bundle end can also be displaced relative to one another parallel to the substrate surface, but only to the extent that a normal passing through the center of the optical fiber bundle end surface of the optical fiber bundle end Substrate surface, the substrate in the region of the liquid receiving area, preferably in the range of the reaction region of the liquid receiving area would cut. The optical fiber bundle ends are preferably aligned with the substrate in such a way that the light exiting from the center of the optical fiber bundle end surface strikes the surface of the substrate facing the optical waveguide device substantially perpendicularly, but maximally at an angle of 60 °.

Method according to one aspect

A further aspect of the invention relates to a method for qualitatively and / or quantitatively detecting target molecules in a sample to be analyzed, in particular using a system according to the invention, comprising:

a sample providing step, wherein a sample to be analyzed is provided on at least one liquid receiving area on a substrate,

a substrate tempering step, wherein the substrate is tempered by means of a substrate tempering device,

a Lichtleiterbündeltemperierungsschritt, wherein at least one optical fiber is tempered by means of Lichtleiterbündelfixierungsvorrichtung,

an exposure step, wherein the at least one sample to be analyzed is exposed by means of an optical waveguide device,

a detection step, wherein light from the sample to be analyzed is received by means of the light guide device, and

an evaluation step, wherein the light received in the detection step is used for the qualitative and / or quantitative detection of the target molecules optionally present in the sample to be analyzed.

Qualitative detection serves to detect the presence or absence of target molecules, while quantitative determination can provide information on the number or frequency of target molecules in the observed sample. In the context of this description, target molecules may be any inorganic or organic molecules which either have fluorescent properties themselves or can be labeled by means of fluorescent dyes. The detection of fluorescently labeled molecules is done by the Detection of the fluorescent dye used for labeling. Examples of possible target molecules are deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), proteins and / or any other metabolites and products of cellular metabolism.

In the sample providing step, at least one liquid or wet sample containing target molecules to be assayed is positioned on a liquid receiving area, preferably on the reaction area of a liquid receiving area. Depending on the analysis to be carried out, it is possible to provide either fluorescence samples or non-fluorescent samples which are labeled with a fluorescent dye only later in the process but before the detection step. It is also possible to provide both fluorescent and non-fluorescent samples on a substrate.

For tempering the substrate, this is brought into thermal contact with the substrate temperature control device in the substrate tempering step and brought to at least one desired temperature at least once. The temperature control can be a cooling, a heating or a constant temperature control of the substrate. Constant temperature control means in this context that the substrate is heated so that its target temperature of the actual temperature within a certain period of time by not more than 5 ° C, preferably not more than 2 ° C and more preferably not more than 1 ° C is different.

Before, after or at the same time as the substrate tempering step, the optical fiber bundle tempering step is carried out, wherein the optical fiber bundle ends are tempered by means of the optical fiber bundle fixing device to a constant or sequentially to several different desired temperatures. The optical fiber bundle ends can be heated, cooled or kept at a constant temperature in this step. The fiber optic bundle ends can in this step several times heated and / or cooled several times and / or kept at a constant temperature between heating and / or cooling steps. If the substrate is heated in the substrate tempering step, heating of the fiber bundle ends can also be carried out, for example, in the optical fiber bundle tempering step. According to the invention, a condensation of evaporating sample liquid on the optical fiber bundle ends and thus an impairment of their optical properties is reduced, in particular avoided. If the heated optical fiber bundle ends do not significantly influence the substrate temperature by the emission of heat, the optical fiber bundle ends can also be kept at a temperature above the substrate temperature in a substrate tempering step to simplify process control during any cooling phases that may be present.

During the exposure step, light is emitted from an optical fiber bundle end face to at least one sample to be analyzed by means of the light guide device. This can be done once or several times at any time interval.

During and / or after the exposure step, the fluorescence emanating from the sample to be analyzed is recorded in the detection step by means of at least one optical fiber bundle and passed via the optical fiber device to at least one detector which detects the type and / or intensity of the fluorescence as fluorescence information. In the context of the method according to the invention, any number of detection steps can be carried out, preferably in each case during an exposure step. Before and / or after an exposure step, if appropriate, a measurement of the background signal can also be carried out. This is advantageous in order to be able to exactly quantify the fluorescence induced by the exposure of a sample.

The fluorescence information received at the detector in the detection step is used in the evaluation step for the qualitative and / or quantitative detection of the investigated target molecules. They can be read out visually directly at the detector. Alternatively or additionally, one connected to the detector Data processing system used for their forwarding, storage and / or evaluation. In the course of the procedure any number of evaluation steps can be carried out. In particular, an evaluation step can be carried out as the last step of the method.

Ausführunqsvarianten the method

According to an embodiment of the method, the sample providing step comprises applying the sample to be analyzed to the reaction area of the liquid receiving area and applying a protective liquid to the liquid receiving area.

Preferably, first the sample is applied to the reaction area and then the protective liquid is applied to the liquid receiving area surrounding the reaction area. The protective liquid is preferably a liquid which inhibits the evaporation of the sample to be analyzed, for example an oil-containing liquid. Advantageously, the protective liquid prevents or reduces vaporization of an underlying aqueous sample and / or reaction liquid.

According to one embodiment, the method comprises a detector temperature control step in which at least one detector of the optical waveguide device is temperature-controlled and the temperature of the detector is kept essentially constant.

The detector is preferably heated or cooled by means of a suitable tempering element at the beginning of the process by means of a Detektoremperemperierungseinrichtung to a predetermined temperature and maintained at about this temperature during the remaining process. A constant temperature of the detector has the advantage that detector-temperature-related measurement errors and / or measurement errors due to temperature drift effects in the detector can be avoided or reduced. Suitable detector temperatures are generally in a range of 15 ° C to 35 ° C. Particularly preferred is a setting of Detector temperature in the range of room temperature.

The optical fiber bundle fixing device is tempered according to an embodiment of the method to a temperature between 50 ° C to 1 10 ° C, preferably between 60 ° C to 80 ° C, more preferably at 70 ° C.

Preferably, the Lichtleiterbündelfixierungsvorrichtung and the substrate temperature control are synchronously adjusted in time to approximately the same temperatures, so that the temperatures of the substrate and the sample-side Lichtleiterbündelenden permanently by less than about 20 ° C, preferably less than about 10 ° C, more preferably about 5 ° C different from each other. To simplify the control of the device, the optical fiber bundle ends may be adjusted to a constant temperature, which is preferably higher than the average temperature of the substrate. This temperature is preferably adjusted before the substrate is tempered by means of the substrate temperature control device.

In one embodiment of the method, the substrate tempering step comprises at least one tempering cycle with at least one heating and / or at least one cooling process.

Such Temperierungszyklen are particularly advantageous in certain procedures such as when performing a PCR. If a sample positioned on the substrate is to be subjected to a PCR, for example, the substrate may first be heated to 95 ° C., for example, and then tempered in several tempering cycles. Each tempering cycle includes, for example, a suitable primer annealing temperature, a suitable chain extension temperature, and a suitable denaturation temperature which, depending on the experiment, for example in the range of about 30 ° C to 70 ° C (primer annealing) or about 50 ° C to 75 ° C (chain extension) or may be about 85 ° C to 100 ° C (denaturation). In each case, precisely one liquid receiving region or one reaction region contained in a liquid receiving region is exposed by exactly one light source by the optical waveguide device.

Furthermore, preferably, a plurality of liquid receiving regions can be exposed simultaneously by a light source and / or more than one light source can expose a liquid receiving region or its reaction region. In this way, for example, in the grid arranged on a substrate samples can be exposed line by line or column by column or in any other groups at the same time. Likewise, it is possible by means of such an optical waveguide device that light emitted by a plurality of light sources of different wavelengths is simultaneously directed to a single sample.

The fluorescence emanating from the sample to be analyzed in several liquid receiving areas or reaction areas can be detected, each with a separate detector. In one embodiment, the fluorescence of two or more liquid receiving areas or reaction areas is received or detected by the same detector. Embodiments are also possible in which light is received by exactly one liquid pickup area or reaction area of two or more detectors.

Computer program product according to one aspect

One aspect relates to a computer program product comprising computer-interpretable instructions stored on a computer-readable medium and / or as a computer readable data stream which, when loaded into the memory of a computer and executed by a computer, cause the computer to perform the method of the invention.

In order to control the method according to the invention, the computer-interpretable instructions or commands are stored in the memory of the optical waveguide device or a connected data processing system, for example, a computer, loaded and executed therein. A computer program product may be stored on a data carrier, eg an optical data carrier, such as a CD, DVD, a magnetic data storage, such as a hard disk, a semiconductor memory, a magneto-optical storage, etc., or provided as a data stream interpretable for a data processing system.

The present invention is not limited to the above aspects and embodiments. Rather, individual features of one or more of the abovementioned aspects and / or embodiments or variants can be combined with one another to form further aspects and / or embodiments or variants of execution independently of the respective aspect and / or the respective embodiment or variant embodiment.

Description of the figures

An exemplary embodiment of the invention is described below with reference to exemplary figures, wherein

FIG. 1 shows a side view of an optical waveguide device for emitting and receiving light with exemplary cross sections of the optical waveguide bundles,

Figure 2 is a plan view of one located on a surface

Liquid intake area shows

Figure 3 shows a side view of the exemplary system for exciting and measuring fluorescence on at least one reaction area, and Figure 4 shows a detail of the system of Figure 3.

Figure 1 shows a schematic side view of the light guide device for emitting and receiving light. The optical waveguide device has at least one forked optical fiber bundle 10, which is divided into an excitation optical fiber bundle 12 and an emission optical fiber bundle 14. These forked optical fiber bundles 10, may also be referred to as Y-fibers or Y-fibers, and may for example consist of individual glass fibers, or polymer-optical fibers, or a mixture of these. The one excitation light guide bundle end 16 of the excitation light guide bundle 12 is fixed in a light source side mount 18 such that the excitation light guide bundle end 16 along the optical axis each an excitation light frequency filter 20, a Lichtfokussierungselement 22, and a light source 24 is assigned. The excitation light frequency filter 20, may for example comprise a frequency filter and / or a bandpass filter. The light focusing element 22 may, for example, be an optical lens made of glass or transparent polymer or an internally mirrored hollow channel. The light source 24 may be, for example, a laser or a laser diode, an LED, an incandescent lamp, a gas discharge lamp. In one embodiment, not shown, for example, a light source with monochrome light can be used to avoid the use of excitation light frequency filters. The illustrated cross-section 26 of the excitation fiber bundle end 16 together with the frame 18 has a diameter of a few hundred microns to a few millimeters, for example from 100 microns to 5 millimeters, preferably from 500 microns to 3 millimeters, more preferably 2 millimeters

According to the illustrated embodiment, the other excitation fiber bundle end 28 of the excitation optical fiber bundle 12 is fixed together with the one emission fiber bundle end of the emission optical fiber bundle 14 in a sample-side socket 30, that this socket 30 can be arranged in a Lichtleiterbündelfixierungsvorrichtung 32. In a further embodiment, the optical waveguide device may comprise two or more optical fiber bundles 10 (not shown here), wherein these are respectively fixed in a corresponding socket 30 according to the above statements. Preferably, the Lichtleiterbündelfixierungsvorrichtung 32 is made of a thermally conductive material, for example metal, preferably made of aluminum or another light metal. In an embodiment, in the optical fiber fixing device 32, a plurality of holes are provided in a predetermined pattern, which are just large enough to each one of the two or more Sockets 30 record. However, the socket 30 and the Lichtleiterbündelfixierungsvorrichtung 32 may also be made for example in one piece. Likewise, for example, the optical fibers can be fixed directly in the Lichtleiterbündelfixierungsvorrichtung 32 without socket 30. The illustrated cross-section 34 of a sample-side optical fiber bundle end 28 in the socket 30 shows a homogeneous distribution of the individual optical fibers from one excitation and one emission optical fiber bundles 12,14 and has a diameter of a few hundred microns to a few millimeters, for example from 200 microns to 5 millimeters , preferably from 1 millimeter to 3 millimeters, more preferably 2.5 millimeters.

In the embodiment illustrated here, optical fiber bundle fixing device 32, as well as sample-side socket 30 and the specimen-side optical fiber bundle end 28 fixed therein are in thermal contact. The optical fiber fixing device 32 comprises at least one sample-side heating device 36, preferably an electrically operated heating element, e.g. a Peltier element or a resistance heating element. However, other heating elements are also conceivable, for example based on pre-tempered fluids. By means of the sample-side heating elements 36, the

Lichtleiterbündelfixierungsvorrichtung 32, optionally present sample-side sockets 30 and the sample-side optical fiber bundle ends 28 are tempered.

In the detector-side emission optical fiber bundle end 38, in the embodiment illustrated in FIG. 1, all of the emission optical fiber bundles 14 of the optical fiber bundles 10 of the device are all fixed in a detector-side holder 40. This embodiment therefore has only one detector-side emission optical fiber bundle end 38 which is assigned to a detector 44. In the illustrated embodiment, an emission light frequency filter 42 is also disposed between the emission fiber bundle end 38 and the detector 44. As a detector 44, for example, a sensitive photodiode or a CCD, or CMOS sensor comes into question. The detector 44 can also be tempered in the embodiment shown here in order to to avoid thermal interference in the measurement signal. For this purpose, a detector-side temperature control element 46 is arranged in spatial proximity to the detector in order to be able to hold the detector at a constant temperature. The detector-side temperature control element 46 may be, for example, a Peltier element. In principle, the tempering element 46 can also consist of a combination of any heating element, eg a resistance heater, and any cooling element, eg a fan. The illustrated cross-section 48 of the emission fiber bundle ends 38 in the detector-side frame 40 has a diameter of a few millimeters to a few centimeters, for example from 1 millimeter to 2 centimeters, preferably from 5 millimeters to 1, 5 centimeters, more preferably about 10 millimeters.

FIG. 2 shows a plan view of a liquid receiving area 52 located on a surface of a substrate 50. The substrate may be, for example, a glass or plastic slide. According to a preferred embodiment, a plurality of such liquid receiving regions 52 may be arranged in an advantageous grid on the substrate. Only one example of such a configuration of the substrate with 48 liquid receiving areas is represented by the AmpliGrid marketed by Beckman Coulter Biomedical GmbH (Munich, Germany).

The liquid receiving region 52 comprises a central, substantially circular hydrophilic reaction region 54, whose diameter in an advantageous embodiment is approximately 1.6 mm. This reaction region 54 is surrounded by a substantially annular, hydrophobic middle region 56 and an annular hydrophilic outer region 58 surrounding this central region. The surface of the substrate 50 surrounding this outer region 58 is essentially hydrophobic.

FIG. 3 shows a schematic side view of the system 60 according to the invention for exciting and measuring fluorescence. The system 60 shown in one embodiment shows an optical fiber device 62 shown in FIG Emitting and receiving light in conjunction with a substrate 50. On the surface thereof is at least one liquid receiving area 52. By means of at least one light conductor bundle 10, which is fixed, for example, with a sample-side frame 30 of Lichtleiterbündelfixierungsvorrichtung (not shown here completely), light on the Liquid receiving area 52 is discharged and received light from this. In the embodiment shown here, the substrate 50 is positioned parallel on a substrate tempering device 64 and is in direct thermal contact with it. The substrate temperature control device 64 comprises at least one heating device 66 and at least one cooling device (not shown here). As an example of a substrate temperature control device, the AmpliSpeed marketed by Beckman Coulter Biomedical GmbH (Munich, Germany) may be cited. With such a temperature-controllable system 60, a method according to the invention for the quantitative and / or qualitative detection of target molecules in a sample to be analyzed can be carried out.

FIG. 4 shows a detail of the side view shown in FIG. 3 of the system 60 for exciting and measuring fluorescence. According to one embodiment, FIG. 4 shows the geometric arrangement of a specimen-side optical fiber bundle end 28 fixed in a sample-side holder 30 relative to the reaction region 54 of a liquid-receiving region 52 on a substrate 50. As shown here by way of example, the holder 30 of the optical fiber-fixing device (not shown here) is positioned in this way in that the optical fiber bundle 10 with a liquid receiving region 52 is advantageously located on the same optical axis 70. The optical fiber bundle end surface 68 is also disposed above a liquid receiving area so as to form a right angle with the optical axis 70. This ensures that excitation light emanating from the optical fiber bundle end face 68 strikes the liquid receiving region 52. The light emitted by a fluorescent sample positioned in the liquid receiving region is received in the optical fiber bundle 10 at the optical fiber bundle end surface 68. The physical nature of the individual optical fibers in the optical fiber bundle 10 define the emission or acceptance angle α of the optical fiber bundle and thus according to generally known mathematical principles the illuminated surface. The distance a between the optical fiber bundle end surfaces 68 and the surface of the substrate 50 is thus advantageously selected so that the illuminated surface completely covers the reaction region 54. In the example shown here, the reaction area 54 has a diameter D, the optical fiber bundle end area 68 has a diameter d. This results in a distance for the optical fiber bundle ends to the surface of the substrate of a = (Dd) / (2 ^* tan (a)).

In essence, however, the distance a is greater than the total height of the liquid present in the liquid receiving region 52, ie the sample which has been overcoated with a hydrophobic liquid, in order to avoid direct contact and associated fouling of the fiber bundle ends.

List of labels:

10 optical fiber bundles

12 excitation fiber bundles

14 emission optical fiber bundles

16 light source side excitation fiber bundle end

18 light source-side socket

20 excitation light frequency filter

22 light source side optical lens

24 light source

6 Cross section of an excitation fiber bundle end with socket

8 Sample-side fiber optic bundle end

0 Sample-side version

2 optical fiber bundle fixing device

4 cross section of a sample-side optical fiber bundle end with socket

6 heating device of the Lichtleiterbündelfixierungsvorrichtung

8 detector side emission fiber bundle end

0 detector-side socket Emission light frequency filter

detector

Detector temperature control element

Cross section of the emission optical fiber bundle ends with the substrate frame

Liquid receiving area

reaction region

hydrophobic middle area

hydrophilic outer region

System for exciting and measuring fluorescence

Optical fiber device

Substrattemperierungsvorrichtung

Heating device of the substrate tempering device Lichtleiterbündelendfläche

optical axis

Claims

Applicant: Beckman Coulter Inc. "Light-guide device for emitting and receiving light, system, method and computer program product" Our mark: B 3139WO - hy claims

1 . Optical fiber device for emitting and receiving light, in particular for excitation and observation of fluorescence, with

- A Lichtleiterbündelfixierungsvorrichtung (32) and with

at least one optical fiber bundle (10),

wherein the at least one optical fiber bundle by means of

Lichtleiterbündelfixierungsvorrichtung is fixed and by means of

Lichtleiterbündelfixierungsvorrichtung is heated.

2. Optical fiber device according to claim 1 wherein the

Optical fiber bundle fixing device (32) comprises at least one heating device (36).

3. optical fiber device according to claim 1 or 2, wherein the

Optical fiber bundle fixing device (32) is arranged at an end region of the at least one optical fiber bundle (10).

4. Optical fiber device according to at least one of the preceding claims, wherein the Lichtleiterbündelfixierungsvorrichtung (32) is designed as a heating device (36).

5. optical fiber device according to at least one of the preceding claims, wherein the at least one optical fiber bundle (10) at least one excitation optical fiber bundle (12) with at least one excitation optical fiber and at least one

Emission optical fiber bundle (14) with at least one emission light guide comprises.

6. optical fiber device according to claim 5, wherein the at least one optical fiber bundle (10) comprises more emission light guide as excitation light guide.

7. optical fiber device according to claim 5 or 6 with at least one light source (24) and at least one detector (44), wherein the at least one

At least one light source is associated with the excitation light guide bundle (12) and at least one detector is associated with the at least one emission light guide bundle (14).

8. optical fiber device according to claim 7, wherein the detector (44) is temperature-controlled.

9. System for exciting and measuring fluorescence on at least one

Liquid intake area (52) with

- An optical fiber device (62) according to one of the preceding claims,

- A substrate (50) whose surface comprises at least one liquid receiving area, and with

a substrate tempering device (64),

wherein the substrate is tempered by means of the substrate temperature control device and the optical waveguide device is arranged relative to the substrate such that the at least one optical fiber bundle (10) of the optical waveguide device and the substrate are non-contacting.

10. The system of claim 9, wherein the at least one of the

Lichtleiterbündelfixierungsvorrichtung (32) fixed optical fiber bundles (10) above the substrate (50) ends and the shortest distance between the end face of the at least one optical fiber bundle and the substrate between 0.1 mm and 20 mm, preferably between 0.5 mm and 10 mm, especially preferably between 1 mm and 5 mm, and most preferably 3 mm.

1 1. The system of claim 9 or 10, wherein the at least one

Liquid receiving region (52) comprises a central, preferably circular hydrophilic reaction region (54), a preferably annular hydrophobic central region (56) surrounding the reaction region and a preferably annular hydrophilic outer region (58) surrounding the central region and wherein the outer region is surrounded by a hydrophobic region.

12. The system according to claim 9, wherein at least one liquid receiving region is assigned an optical fiber bundle end fixed by the optical fiber bundle fixing device, the liquid receiving region and the optical fiber bundle end surface of the associated optical fiber bundle Have substantially the same dimensions and the liquid receiving area and the associated optical fiber bundles are aligned with each other so that from the Lichtleiterbündelendfläche (68) emerging light on the associated

Liquid intake area meets.

13. A method for qualitatively and / or quantitatively detecting target molecules in a sample to be analyzed comprising:

a sample providing step, wherein a sample to be analyzed is provided on at least one liquid receiving area (52) on a substrate,

a substrate tempering step, wherein the substrate (50) by means of a

Temperature control device (64) is tempered,

- A Lichtleiterbündeltemperierungsschritt, wherein at least one optical fiber (10) is tempered by means of Lichtleiterbündelfixierungsvorrichtung (32)

an exposure step, wherein the at least one sample to be analyzed is exposed by means of an optical waveguide device (62),

a detection step, wherein light coming from the sample to be analyzed is received by means of the optical waveguide device,

- An evaluation step, wherein the received light in the detection step for the qualitative and / or quantitative detection of optionally in the zu

analysis sample is used existing target molecules.

14. The method of claim 13, wherein the sample providing step is the

Applying a sample to be analyzed on the reaction region (54) of

Liquid receiving area (52) and the application of a protective liquid on the liquid receiving area comprises.

15. The method according to claim 13 or 14, comprising

a detector tempering step, wherein at least one detector (44) of the

Optical fiber device (62) is heated and the temperature of the detector is kept substantially constant.

16. The method according to claim 13 to 15, wherein the

Lichtleiterbündelfixierungsvorrichtung (32) to a temperature between 50 ° C to 10 ° C, preferably between 60 ° C to 80 ° C, more preferably at 70 ° C is tempered.

17. The method according to claim 13 to 16, wherein the substrate tempering step comprises at least one Temperierungszyklus with at least one heating and / or at least one cooling process.

18. The method of claim 13 to 17, wherein two or more reaction areas (54) are exposed simultaneously by a light source (24) and / or two or more light sources illuminate a Reaktionsbe rich.

19. The method of claim 13 to 18, wherein light of two or more

Reaction areas (54) from the same detector (44) is received and / or light from exactly one reaction area of two or more detectors are received.

A computer program product comprising computer-interpretable instructions which, when loaded into the memory of a computer and executed by a computer, cause the computer to perform the method of any one of claims 13 to 19.