WO2001018242A1

WO2001018242A1 - Affinity sensor for the detection of biological and/or chemical species and use thereof

Info

Publication number: WO2001018242A1
Application number: PCT/EP2000/008360
Authority: WO
Inventors: Andrea Czaki; Wolfgang Fritzsche; Johann Michael KÖHLER; Jörg REICHERT
Original assignee: Institut für Physikalische Hochtechnologie e.V.
Priority date: 1999-09-08
Filing date: 2000-08-28
Publication date: 2001-03-15
Also published as: EP1216310A1; DE19943704C1

Abstract

The invention relates to an affinity sensor for the detection of biological and/or chemical species which enables rapid, quantitative and simple detection of the presence of biological and/or chemical species in particular on surfaces in the lower to middle micrometer range. The sensor, depending on the embodiment, consists of an optically transparent or a partially reflecting substrate (1) which is provided with several, voneinander beabstandeten microstructured binding surfaces (2), whose surface compared to the diameter of the nanoparticles (3) and above the light optical refractive limit is chosen to be of such a size that said nanoparticles (3), which are provided with coupling partners (4), which have a selective affinity to the binding surfaces (2) or (DNA) sequences specifically bound thereto such that they can bind durably to the binding surfaces (2) or specifically bound to the said sequences (DNA), daß auf one or more binding surfaces (2) eine so large number of nanoparticles (3) unter Ausbildung von nanoparticle occupation (31) anbindbar can be connected, daß die von der nanoparticle covearge (31) covered surface relative to the surface Fläche einer Bindefläche (2) can comprise at least 0.1 %, whereby all binding surfaces together (2) zusammen in such active surfaces are arranged, such that they together with the object of a standard optical light microscope with a numerical mit aperature between 0.1 .and 0.9, for determining the nanoparticle occupation (31) generated optical absorption, reflection or scattering or by a device for the determination of plasmonen resonances.

Description

Affinity sensor for the detection of biological and or chemical species and its use

description

The invention relates to an affinity sensor for the detection of biological and / or chemical species, which is particularly suitable for tasks in combinatorial chemistry, for diagnostic tasks, such as in medicine or for the development of new active substances, e.g. for medication or selective pesticides.

For the identification of biological species, individuals and diseases, evidence through molecule-molecule interaction has proven to be particularly useful. Binding events on biochips are usually measured using

Fluorescent markers. However, this method has several essential ones

Disadvantage:

very sensitive detector arrangements are required for reading, which must also strictly separate excitation and fluorescent light from one another,

- The quantification is problematic because the quantum yields of fluorescence are very dependent on the environment (Tanke, HJ .; Herman B. Fluorescence Microscopy, Springer Verlag (1998)). For this reason, quantitative analytical statements are often not at all or can only be achieved with a very high level of measurement effort (Rost FWD Quantitative Fluorescence Microscopy, Cambridge University Press. (1991), so that, for example, evaluation is only possible when measurements on several chips are included (Ffacia, JG ; Brody, LC; Chee, MS, Fodor, SC; Detection of heterozygous mutations in BRCA1 using high density olionucleotide arrays and two color fluorescence analysis, Nature Genetics 14, 441-447 (1996). However, even qualitative detection often fails.

The detection of binding events, especially on highly integrated biochips with small binding areas

Fluorescence measurement is time consuming and only with expensive optical ones Establish reading facilities. The longer exposure and exposure times required for quantitative measurements

Accumulation times also cause a photochemical degradation of the dyes, which worsens the signal in the course of the measurement and makes quantification difficult. The intensity of the fluorescent light per excitation light quantity and binding event also depends on the specific chemical environment of the chromophores used for labeling. Therefore, from batch to batch and from test to test and even within a sample, there are considerable deviations in the measurement signals, which considerably impair the actually required quantification of the measurement signals. A slow photo- and thermochemical degradation of the chromophores also leads to the fact that measurement samples cannot be stored for a longer period, i.e. they cannot be archived and used for comparative measurements at a later date.

With readout of binding events on biochips in addition there is the problem that for assays at a sufficiently large number of discrete milαOSüTjkturierten bonding surfaces (typically about 100-1000 bonding areas) or for SBH chip (typically 10,000 to 10 ⁶ bonding surfaces), the total area of all the individual Binding areas on a chip must not be too large, so that when incubating with a test solution it is not necessary to take too long times for the diffusive spreading of concentration fronts or the spontaneous adjustment of attachment and deodorization equilibria with the coupled molecular transport steps.

Small total areas would also be advantageous for a parallel optical readout, so as not to have to work with lens diameters that are too large for the readout optics that make the method more expensive. By reducing the total area, the absolute number of fluorophores is reduced when the fluorescent dye is labeled with the same density of binding groups, which further complicates the already complicated fluorescence measurement with small amounts of substance on solid surfaces. For this reason, the resolution limit of the optical image (approx. 0.4 ... 1 μm) cannot be fully exhausted in the established fluorescence methods. Instead, it is mostly used Individual areas of 100 μm and more worked (Chee, M. et al .; Accessing Genetic Information with High-Density DNA Arrays, Science 274 (1996), 610-614). With this area size (for equally large spaces), an area requirement of 4 mm ^{2 is achieved} with 100 binding areas, which is already considerably too high for fast assays due to the required diffusion times. With 10 ⁶ binding surfaces, the total area required would be approx. 200 mm ^• 200 mm, which is far above reasonable dimensions if biochips are to be used and evaluated as standard.

US Pat. No. 5,556,756 describes a gold sol that can be used in analytical test kits. Since the analysis is based on a color change of the surface, particles between 1 nm and 5 nm are preferably used. The method described there has the disadvantage that, on the one hand, relatively large test areas have to be used and, on the other hand, permeable membranes have to be used in order to be able to carry out rinsing processes. This solution is not suitable for marking small binding areas in the middle and lower micrometer range.

Furthermore, it is generally known that functionalized nanoparticles can be specifically attached to biomolecules (Nicholl, D., Genetic Methods, Spectrum Academic Publishing House Heidelberg (1995), pp. 24-27). Such binding events are used, among other things, to obtain sequence-specific information on a scale of a few to 1 nm from biogenic macromolecules immobilized on solid surfaces in basic research with the aid of ultramicroscopic techniques such as tunnel, force and transmission electron microscopy. The fact is used that individual particles are attached to discrete regions of a single large molecule. Ultramicroscopic methods are indispensable for the detection of these individual particles. However, these are too complex, that is, both in terms of investment and handling, too complicated and too expensive to be used for series or routine analyzes. In addition, it is known that, in principle, submicromete particles can also be detected by optical methods. WO 98/57148 AI describes a method and an arrangement in which the changes in the angle of the surface plasmon resonance associated with the addition of such small particles to a thin metal film can be determined. This process requires a complex SPR arrangement and is also dependent on the existence of a metal layer on an otherwise transparent substrate that prevents the passage of light.

There is an urgent need for affinity sensors for the detection of biological and / or chemical species which avoid the disadvantages mentioned above and which can be used for series or routine analyzes.

The invention is based on the object of specifying an affinity sensor which enables rapid, quantitative and low-cost detection of the presence of biological and / or chemical species, in particular on areas in the lower to medium micrometer range.

The object is achieved by the features of the first claim. Advantageous configurations are covered by the subordinate claims.

Reliable detection of binding events on surfaces in the lower to medium μm range is achieved in the context of the invention by the production of nanoparticle layers which are formed by markers, the density of which is particularly by absorption but also by reflection or scattering light measurements or by plasmon resonance measurements. measurements is quantifiable. In the case of very small, electrically conductive nanoparticles, the size-specific absorption of light can advantageously be used, as a result of which multiple markings are also possible using several monodisperse groups of nanoparticles with different diameters. The selective labeling of immobilized biomolecules has been surprising proven unproblematic. Gold particles have proven to be particularly favorable for marking. When using gold particles, such an affinity sensor can also be integrated in a device for determining the plasmon resonance. A selective binding can also be demonstrated in the case of relatively large particle diameters which already provide quite comfortable scattered light signals.

The readout of binding events on biochips can be carried out particularly advantageously by the areal marking provided in the present invention with in particular metallic nanoparticles.

The invention will be explained in more detail below with the aid of schematic exemplary embodiments. Show it:

1 shows a schematic top view of an affinity sensor,

2 shows a partial area of the AfBnitätssensor according to Fig. 1 in a side view, Fig. 3 is a scanning force microscope image of a nanoparticle layer, which is formed by a coating with 30 nm gold particles, and Fig. 4 shows a high-microscopic image of an individual

3, which by the nanoparticle

Layer caused absorption compared to the substrate supporting it illustrates.

FIG. 1 shows an example of an affinity sensor for the detection of biological and / or chemical species consisting of an optically transparent substrate 1, which is provided with a plurality of microstructured binding surfaces 2 spaced apart from one another. The type and design of the binding surfaces depends on the specification of the binding events to be determined and can be implemented as such by customary professional action, which is why no further explanations are required here. In the example, the bonding surfaces 2 are spaced apart by free-structured areas 11 of the substrate 1. The dimensioning of the side lengths of the individual bonding surfaces 2, which are square here takes place in such a way that it is set above the light-optical diffraction limit; in the example 2 μm, in any case> 0.5 μm. In the example, the side length of the optically transparent substrate 1 should be 3 mm, with the active surface area 22, namely the surface of the substrate 1, which is provided with the binding surfaces 2, only being of the order of 1 mm ² for certain applications ( e.g. rapid diagnostics) can also easily take up areas below 0.01 mm ² , the only important thing is that the individual binding surfaces 2 are given a surface that lies above the light-optical diffraction limit, with the active surface area 22 occupied by all binding surfaces 2 from common lenses a numerical aperture between 0.1 ... 0.9, preferably 0.2, optical microscopes should be detectable. If one omits this advantage created by the invention, it is also possible to use expensive lenses with a larger aperture, in which case larger active surface areas 22 can also be used.

FIG. 2 shows a partial area of the affinity sensor according to FIG. 1 in a side view, the same parts being designated with the same reference symbols as in FIG. 1. The nanoparticles 3 used in the context of the invention are provided with coupling partners 4 which have a high selective affinity for the binding surfaces 2 or, as shown in the example according to FIG. 2, sequences (DNA) specifically bound thereon. The following example is intended to explain:

Oligonucleotides with, for example, 20 base pairs are immobilized on the mils-patterned areas of the substrate 1 and thus form the binding areas 2 described above. The affinity sensor thus prepared is mixed with a solution to be tested which contains DNA of unknown composition. This only binds to the binding surfaces 2, which carry oligonucleotides complementary to sections of the DNA. After rinsing, in order to remove unspecifically bound DNA, the substrate surface of a colloidal solution which contains nanoparticles, in the example consisting of gold with a particle diameter of 30 nm, is exposed. The surface of the nanoparticles is provided with a coupling partner 4, in the example an oligonucleotide, the sequence of which is such that it is not complementary to one of the binding surfaces 2 on the substrate 1 but is complementary to a conservative nucleotide sequence of the target molecules to be examined, which occurs in the same way in all target molecules in question (bound DNA). The state of the affinity sensor obtained after hybridization is illustrated in the partial section in FIG. 2.

After the hybridization, a density of approx. 40 particles / μm ^{2 was} obtained in the example, the diameter of 30 nm being given to the gold nanoparticles 3 used in the example. With a cross-sectional area of the individual nanoparticles of approx. 700 nm ² , nanoparticle layers with a nanoparticle coating 31 of approx. 3% are obtained, which are immediately and clearly visible in the light microscope. These structures correspond to the pre-stabilized binding areas 2. The diameter ranges of the nanoparticles 3 can be defined within wide limits depending on the test assay to be designed in each case. The diameters of the nanoparticles can be set in the order of 2 ... 600 nm. Depending on the material selected for the nanoparticles, a diameter range of 15 ... 60 nm is preferably selected. Metallic nanoparticles are preferably used in the context of the invention since they absorb light in a very effective manner, as a result of which nanoparticle occupations of the order of magnitude of 0.1% can be detected in transmitted light in the light microscope. This makes it possible to work just above the diffraction limit (1 μm .. 10 μm) even when the individual binding surfaces 2 are extended. In this way, 100 binding surfaces 2 can be placed on active surfaces 22 of significantly less than 0.1 mm in side length, 10,000 binding surfaces 2 on less than 1 mm ² . Even for highly integrated affinity sensors with 100,000 binding areas 2, a total area of less than 1 mm ^{2 appears to} be feasible. In this way, very fast-working assays suitable for the smallest sample amounts can be set up, so that only sample amounts of less than 1 nl and less highly integrated assays of up to about 1 μl are required for low-integrated assays. It is within the scope of the invention for the nanoparticles 3 also to contain metal-containing composite particles in the size range mentioned also use nanoparticles made of a semiconductor material, such as CdSe, InP, as well as coated nanoparticles, for example with ZnSe. It is also within the scope of the invention to use nanoparticles made of plastic, which preferably contain inclusions made of an absorbent dye, metal or semiconductors.

What is important in the selection of said nanoparticles 3 with regard to their material composition and size is that the nanoparticle coatings 31 formed by them with lenses of conventional light-optical microscopes, for the purpose of determining the optical absorption, reflection or scattering caused by the nanoparticle coating 31, also in the case of the above-described ones low densities of the nanoparticles per binding surface 2 can be detected when the entire active surface 22 is recorded. Likewise, the arrangement of the binding surfaces 2, which accommodate the aforementioned nanoparticle coatings 31, can be provided on the surface of a prism in the manner described above, as a result of which such an affinity sensor is accessible to a device for detecting the plasmon resonance.

For the selection of the nanoparticles 3 serving as markers, the size of the nanoparticles should be selected so that, on the one hand, they make a very effective contribution to signal generation even with a few binding molecules, whereby they must not be too small, but on the other hand, in order to specifically couple , must not be too large, a cooperative measurement effect being achieved by coupling several nanoparticles 3 to a single binding surface 2. With decreasing particle size, the measurement signal becomes worse, with increasing particle size the specificity of the binding decreases. The former leads to a reduction in the absolute signal, the latter to an increase in the background signal. The specific selection of the particles in question lies within a reasonable range for the person skilled in the art. For the use described above. In the example described, Au particles with a diameter range between 15 nm and 60 nm were found to be particularly preferred. To illustrate the connections achieved, FIG. 3 shows a scanning force microscope image of a nanoparticle coating 31 on a 5 × 5 μm surface, which is covered by a coating of 30 nm Gold particles is formed, wherein in FIG. 4 a highly microscopic transmitted light image of an individual binding surface, which illustrates the absorption caused by the nanoparticle coating 31 with respect to the substrate 1 carrying it.

In summary, the proposed affinity sensor offers the following advantages over the fluorescent dye marking and reading customary in the prior art:

1. Easy handling of the affinity sensor is guaranteed, since no toxic, carcinogenic and environmentally harmful dyes are used for marking.

2. The signal is independent of the chemical environment, which is why the affinity sensor can be used for quantitative measurements.

3. There are no material changes (fading or the like) during the measurement. Therefore, even when the active area 22 is scanned, the entire assay can remain illuminated without changes in the measurement signals. 4. Short incubation times are guaranteed, since the active area can be defined very small even for a large number of individual binding areas.

5. The proposed structure of the affinity sensor provides a favorable signal-to-noise ratio, as a result of which short measuring times can be achieved.

6. The possibility of forming a large number of individual binding areas on a very small active area 22 enables reading by means of simple microscopic apparatus. In contrast to the prior art, no fluorescent optics and no large objectives are required.

7. The affinity sensors, which are provided with the nanoparticle assignments, have a long-term storage stability, since the particle assignments do not undergo any changes of their own accord, which enables their archiving, which is important in medicine, in particular forensic medicine, environmental technology, and the

Process control technology and research opens up new opportunities. 8. The quantifiability and storage stability of the affinity sensors make it possible to set up calibration or even calibration standards.

Due to the size ratio of the active area 22 to the individual binding areas 2 that can be achieved with the present invention, a high degree of parallelism is ensured, so that the proposed affinity sensor is also suitable for fast assays and for small sample quantities and up to approximately 10 ⁶ binding areas 22 in an area of 1 mm ² can have, which is particularly advantageous for tests with molecular library chips, such as biochips, DNA chips, etc. The proposed affinity sensor can thus be used in particular for tasks in combinatorial chemistry, for diagnostic tasks in medicine, for the development of new active substances, for the development of new medicinal substances or for the development of selectively acting pesticides.

All of the features shown in the description, the subsequent claims and the drawing can be essential to the invention both individually and in a combination with one another.

Claims

claims

1.Affinity sensor for the detection of biological and / or chemical species consisting of a substrate (1) which is optically transparent or partially reflective, depending on the type of reading, and which is provided with a plurality of spaced apart microstructured binding surfaces (2), the surface of which in relation to the diameter of nanoparticles ( 3) and above the highly optical diffraction limit is so large that the nanoparticles (3), which are provided with coupling partners (4), which have such a selective affinity for the binding surfaces (2) or sequences (DNA) specifically bound thereon that they enter into a permanent bond with the binding surfaces (2) or sequences specifically bound thereon (DNA) in such a way that on one or more binding surfaces (2) such a large number of

Nanoparticles (3) with the formation of nanoparticle coatings (31) can be attached such that the area occupied by the nanoparticle coating (31) can be at least 0.1%, based on the area of a binding surface (2), all binding surfaces (2) together in one such active surface area (22) are arranged so that they together by

- A lens of a conventional light-optical microscope with a numerical aperture between 0.1 ... 0.9, for the purpose of determining the optical absorption, reflection or scattering generated by the nanoparticle coatings (31) or

- Can be detected by a device for determining the plasmon resonance.

2. Affinity sensor according to claim 1, characterized in that the diameter of the nanoparticles used (3) in the

Order of magnitude of 2 ... 600 nm.

3. Affinity sensor according to claim 2, characterized in that the diameter of the nanoparticles used (3) is preferably set in the order of 15 ... 60 nm.

4. Affinity sensor according to claim 2, characterized in that the diameter of the nanoparticles used (3) is preferably set in the order of 2 ... 15 nm.

5. Affinity sensor according to claim 2, 3 or 4, characterized in that metals, in particular gold, are used for the nanoparticles (3).

6. Affinity sensor according to claim 2, 3 or 4, characterized in that metal-containing composite particles are used for the nanoparticles (3).

7. Affinity sensor according to claim 2, 3 or 4, characterized in that those made of a semiconductor material, in particular CdSe, InP, are used for the nanoparticles (3).

8. Affinity sensor according to claim 2, 3 or 4, characterized in that for the nanoparticles (3) those made of a plastic, which preferably contain inclusions made of an absorbing dye, metal or semiconductors, are used.

9. Affinity sensor according to claim 4, characterized in that nanoparticles (3) of several different size classes or different compositions are used, which can be read in different spectral ranges.

10. Affinity sensor according to claim 1, characterized in that the individual binding surfaces (2) are set in the order of 0.4 microns ² to 1 mm ² .

11. Affinity sensor according to claim 10, characterized in that the individual binding surfaces (2) are of the order of 1 μm ² .

12. Affinity sensor according to claim 1 and 11, characterized in that up to 10 ⁶ individual bonding areas in the order of 1 micron ^{2 are provided} on a surface area (22) of the transparent substrate in the order of 1 mm ² .

13. Use of an affinity sensor according to the preceding claims for tasks in combinatorial chemistry, for diagnostic tasks in medicine, for the development of new active substances, for the development of new medicinal substances or for the development of selectively acting pesticides.