IL128230A - Method of screening active compounds using a scanning near field optical microscopy instrument - Google Patents

Abstract

Verfahren zum Wirkstoff-Screening, wobei man den Wirkstoff über die mittels einer rasteroptischen Nahfeldmikroskopiervorrichtung (SNOM-Vorrichtung) beobachtbare Änderung eines Signals (S) nachweist, sowie dafür geeignete Einrichtung, umfassend die SNOM-Vorrichtung, eine Einzel- oder Mehrfachprobenaufnahmevorrichtung und eine Steuereinrichtung.

Description

128230X2 a ? ii*ny pn^ii nwptnpm watt* ¾no*¾> ni >j«. sna disi ίΐυ ¾> Method of screening active compounds using a scanning near field optical microscopy instrument BASF Aktiengesellschaft C.U5307 Method of screening active compounds The present invention relates to a method of screening active compounds, in particular a method of mass screening in the active compound sector. The present invention furthermore relates to the use of a near field microscopy instrument, and to an apparatus comprising a near field microscopy instrument and a control device. The present invention furthermore relates to a nanotitration instrument.

The discovery of new basic structures, ie. molecular structures, such as ligands, agonists or active compounds, which have been improved with respect to their action on enzymes or receptors, DNA nucleases or polymerases, proteases, for example thrombin, membrane receptors, nuclear receptors or binding proteins, is even today still a laborious and time-consuming search process.

Its success depends to a great extent on chance, since a very large number of substances must be scanned through until active substances against the target molecule - for example a receptor - are found. Investigation of the bioactivity of a large number of substances - for example from 100,000 to 1,000,000 - takes a long time if it is remembered that activity tests with, for example, cells or enzymes require long incubation times. Such a large number of substances can therefore only be tested and investigated rapidly if test is likewise highly sensitive and very fast.

In many cases, fluorescence methods are used in such tests. Although these are very sensitive, they are also susceptible to faults. In addition, such test methods are also very since it must be taken into account that a multiplicity of further treatment steps is also necessary from the actual incubation to the measurement process.

Here, these include, in particular, washing steps for reducing the background fluorescence and thus improving the signal-to-noise ratio. In general, it may be stated that improvements in the sensitivity of the detection method slow the speed of a test. A solution to the two problems at the same time by a single test method or by a corresponding apparatus has hitherto appeared impossible.

Recently, the discovery of scanning tunneling microscopy (STM) by Binnig and Rohrer [G. Binnig, H Rohrer, C. Gerber, E. Weibel, Phys, Rev. Lett. 49 (1982), 57] resulted in a rapid development in the area of ultra-high-sensitivity scanning probe methods.

In scanning tunneling microscopy, a metallic tip is moved line by line (scanned) over a conducting or semiconducting sample. The tunneling current produced at very small distances between the tip and sample allows, as a regulating variable, a height profile and/or electronic structure profile of the surface under investigation to be recorded. This allows an investigation of structures at an atomic resolution of about 0.1 nm under standard laboratory conditions. In 1986, this was applied to nonconducting samples through scanning atomic force microscopy, (SAFM) [G. Binnig, C.F. Quate, C. Gerber, Phys. Rev. Lett. 56 (1986) 930]. This allowed the investigation of polymeric materials [G. Krausch, Μ, Ηίρρ, M Boltau, O. Marti, J. Mynek, Macromolecules 28 (1995) 260] and biological structures, even under physiological conditions [C. Bustamante, D. Keller, Physics Today, December 1995, 32]. In SAFM, the scanning is carried out using a silicon or -silicon nitride tip, usually pyramidal, on a flexible cantilever. The deflection of the cantilever is generally measured optically and used to reconstruct the height profile.

If, instead of the tip described, a pointed optical fiber into which laser light, for example, is fed is moved as probe over the sample, and the light reflected or transmitted depending on the sample properties is detected with the aid of photodetectors, scanning near field optical microscopy (SNOM or NSOM) is achieved [H. Heinzelmann, D.W. Pohl, Appl. Phys. A 59 (1994) 89; E. Betzig, J. Trautman, Science 257 (1992) 189]. By circumventing the diffraction limitation which occurs in conventional optics, these methods, using all common optical contrast mechanisms, such as absorption, reflection, polarization, fluorescence, etc., allow very high local resolution down into the nanometer region. In fluorescence microscopy, a sensitivity is achieved which allows detection of individual fluorescent molecules [E. Betzig, RJ. Chichester, Science 262 (1993) 1422]. This method is shown diagrarnmatically in Fig. 1.

Besides the SNOM set-up described by way of example above, further set-ups are described in the prior art. For example, tetrahedral tips ( oglin J. et al., Photons and Local Probes. Proc. of the NATO Advanced Research Workshop. Ed. Mart O. et al. Kluwer Academic Publ. 1995) or, for example, photoconducting tips (Akamine S. et al., Proceedings. IEEE Micro Electro Mechanical Systems 1995, page 155) can be used.

These techniques have only been used for sporadic investigations in the biological area, for example S. Smith et al., Proc. SPffi-Int. Soc. Opt. Eng. 1855, 81 (1994) (Scanning Probe Microscopies Π) or D.M.N. Jondle et al., Chromosome Research 3, 239 (1995). The latter reference described investigation of the ultrastructure of polytene chromosomes from the salivary gland of the fruit fly by S AFM.

It is an object of the present invention to provide a method of screening active compounds in which a multiplicity of samples can be investigated in a very short time. This method should be not only time-saving, but also require as little work as possible compared with the methods customary hitherto.

We have found that this object is achieved by a method in which the active compound is detected from the change in a signal [S] which can be observed by means of a scanning near field optical microscopy instrument (SNOM instrument). The invention thus also relates to the use of an SNOM instrument in screening methods and to an apparatus comprising a near field microscopy instrument, a single or multiple sample holder and a control device for selecting the individual compartments of the single or multiple sample holder by means of the SNOM instrument.

Preferred embodiments of the invention are the subject-matter of the subclaims.

In the drawings: Fig. 1 shows the basic structure of an SNOM instrument using an optical fiber, - - Figs. 2a) and b) show the process steps which occur in the novel screening method using the SNOM before [a)j or after [b)] addition of a solution containing the active compound W2 and further active compounds Wx to the complex of receptor Ri and fluorescence-labeled active compound Wi; Figs. 3a) and b) show the same process steps as in Figs. 2a) and b), but with the locations of receptor Ri and active compound Wi interchanged; Fig. 4 shows the process steps that occur in an embodiment of the novel process in the form of a sandwich technique; Fig. 5 shows a further embodiment of the novel process, in which the unknown active compound Wx is detected indirectly from a change in conformation of the receptor.

A SNOM instrument, as schematically shown in Fig. 1, shows in principle a pointed optical fiber (1), into which for example Ar-laser light from a light source (3) is fed and conducted over the probe (2) by means of a control unit (4) and further a photodetector (5) with detection electronics (6) for the light reflected or transmitted by the probe (2).

As already mentioned, the invention also relates to a method of screening active compounds, in particular in the area of mass screening, in which SNOM technology is used. With the aid of this technology, it is possible to detect the fluorescence of mdividual molecules if the distance between the probe and sample is in the region of a few nm. However, the use of SNOM technology in the novel method is in principle not restricted to the area of fluorescence methods; rather, any detection method in which the new active compound to be discovered causes a change in an optical signal can be used. Consequently, the optical signal can, apart from fluorescence, also be absorption, polarization or a change in the wavelength dependence of these properties.

The SNOM technology employed in the novel process is likewise not restricted to - as shown in Figs. 1, 2 and 3 - the optical near field being provided by means of an optical jBber. All scanning optical instruments can be used so long as they provide a near field by means of which similar resolutions can be achieved as are possible using an optical fiber. These include, for example, the abovementioned tetrahedral or photoconducting tips.

For the screening of active compounds, SNOM can advantageously be employed, for example, in the following manner: Firstly, a receptor Rl (Fig. 2) or a reference active Wl compound (Fig. 3) is immobilized on a suitable surface of a substrate 2. The term receptor here is very broadly drawn, including, for example, fibrinogen, endothelin, DNA or RNA, proteins and lipids in the broadest sense, but also entire cells or pieces of tissue. This substance to be immobilized is also referred to below as molecule A. The substrate can be a silicon wafer with a vapor-deposited gold coating, a modified, for example silanized, silicon surface or another planar substrate, for example mica, graphite, etc. It is also possible to use biological substrates, but it may then be necessary to immobilize them in advance on the abovementioned inorganic substrates. The immobilized receptor (Ri) (Fig. 2) or reference active compound (Wi) (Fig. 3) is occupied by a fluorescence-labeled active compound (Wi-F) or fluorescence-labeled receptor (Ri-F) and as it were blocked. If an active compound W2 of higher affinity which forms stronger bonds is added [Fig.2b)] or [Fig. 3b)], the reference active compound (Wi) is expelled from the receptor binding site, and the active compound W2 is bound. The fluorescence-labeled active compound Wi is thus expelled from the surface (Fig. 2b). The same process occurs in the opposite case, if the reference active compound Wi is fixed to the substrate, and the two active compounds Wi and W2 compete for binding to a non-fixed, but fluorescence-labeled receptor (Fig. 3). In both cases, the fluorescence recorded by the optical fiber, which is seemingly directly above the receptor, is reduced or even drops back to zero. However, it can also be merely modulated, for example modified in its wavelength dependence. In this case, it is not the intensity, but the color of the fluorescence that changes.

A particular advantage of the novel method using SNOM technology is that fluorescent molecules moving freely in the solution owing to difEusion play virtually no part, because they are not detected by the SNOM probe. The volume resulting from the relevant information of use of SNOM is extremely small owing to the high distance dependence of the signal. It extends from a few nm3 to a maximum of a few 10,000 nm3. Freely diffusing fluorescent molecules thus play no part in this method, having a very good signal-to-noise ratio. This in turn has the crucial advantage over the highly sensitive measurement methods known to date that time-consuming aftertreatment of the sample, for example by repeated washing, is unnecessary.

In view of this, it is also clear that it is not crucial in the novel method that the near field is provided by an optical fiber. Any method that provides an appropriate near field results in the abovementioned advantages.

A further embodiment of the novel method is shown in Fig. 4. This differs from the method shown in Figs. 1 to 3 described above through the fact that the new active compound Wx is detected directly. Firstly, the unknown active compound W* binds to the receptor R_. This new active compound Wx has a domain (*) through which it differs from the known active compound Wi. In order to detect this domain, a known molecular probe, for example an antibody labeled with fluorescein isothiocyanate is available. This fluorescence-labeled antibody is allowed to bind to the complex of active compound Wx and Ri. In this way, the complex can be detected directly via the detected fluorescence.

A further embodiment of the novel method is shown in Fig. 5. This embodiment differs from that shown in Fig. 4 through the fact that the fluorescence-labeled antibody does not bind directly to the unknown active compound Wx, but rather causes a change in conformation in the receptor Ri during binding thereto (allosteric effect). This change in conformation exposes new structural domains on the receptor. A biological molecular probe, for example a fluorescence-labeled antibody, is allowed to bind to these domains. In this way, the new active compound Wx can be detected indirectly through the fluorescence it causes.

In the cases described above, the active compounds can be added from suitable solvents, for example aqueous or ethanolic salt solutions or suitable solvents. The measurement itself can be earned out in the solvent or on the dried sample. The analysis of the active compounds need not be carried out individually; instead, mixtures of active compounds, including mixtures with other compounds, can be analyzed in the same way (cf. Fig. 2). Immobilization using ELISA plates [96-well microtitration plates) and corresponding sample and solution handling systems, for example automatic pipettes for nano-structured systems, is possible. It is of course also possible for receptors fixed to cellular structures to be analyzed in the same way.

The immobilization of the molecule - be it receptor, ligand or another molecule, for example a neuroreceptor or a receptor on immune system cells or other cellular binding proteins on the substrate - can take place covalently or noncovalently. Noncovalent immobilization takes place, for example, by direct adsorption. However, the immobilization can also take place via covalent bonds if the substrate has an appropriate activated surface. For example, the substrate surface in liquid phase adsorption (self-assembly [S. Akari et al., Adv. Mater. 7 (1995) 549]) can be activated by application of a thiolcarboxylic acid. The thiol binds to the substrate, for example a gold surface, by adsorption, with the acid group facing outward.

Neither must the binding of molecule A take place directly to the substrate surface or the thiolcarboxylic acid. It can also be indirect. For example, a spacer can be arranged between the abovementioned thiolcarboxylic acid and molecule A, for example a protein, -receptor, ligand, etc. A spacer of this type is aminohexanoic acid or spermidin. These molecules can bind via their amino groups to the abovementioned thiolcarboxylic acid.

It is followed by binding of the molecule, i.e. the receptor active compound, ligand or the like, to the substrate surface. In the abovementioned case, this binding takes place covalently via the spacer.

If the insertion of a spacer between the substrate surface and the molecule A to be analyzed is still inadequate for certain reasons, for example owing to unfavorable steric conformations, the step of insertion of a spacer can also be repeated a number of times.

- - For example two or more molecules of the abovementioned spacer aminchexanoic acid can be linked to one another. The only limit to the number of linked spacers is that immobilization in the true sense of the word no longer occurs if the distance between the surface of the substrate and the molecule A to be bound thereto via the spacer is too great, since a very long spacer is naturally corres-pondingly flexible and thus makes the use of SNOM impossible.

Owing to the measurement principle, it is very particularly advantageous that parallelization of the method is possible. Instead of a single optical fiber, it is possible to use a plurality of fibers simultaneously. Detection of the fluorescence can then take place via fiber couplers and/or detector arrays. It is thus possible to read out directly a number of wells, for example of an ELISA plate, or even the entire plate with in general 96 detection sites.

However, this is advantageously carried out using the novel apparatus comprising a near field microscopy instrument, a single or multiple sample holder and a control device for addressing the individual compartments of the single or multiple sample holder by means of the SNOM instrument. It is of course also possible for the control process to be reversed, ie. to address the near field microscopy instrument by means of the mobile multiple sample holder, in a similar way as the specimen stage is moved in conventional microscopes.

The multiple sample holder in this instrument is advantageously a multiwell microtitration plate, as also used, for example, in ELISA tests. A multiple sample holder of this type preferably has miniaturized dimensions in order to increase the speed of the novel active compound screening method. In this case, the lower limit for the size (diameter) of the individual compartments is determined merely by the size of the optical fiber. The lower limit is thus about 10 nm.

There is in principle no upper limit. However, with regard to optimizing the speed of the screening method, it is not very effective for the size of the compartment in the miniaturized instrument to exceed a few hundred πι in particular 500 um. A preferred - - upper limit is 150 μτη- In a holder miniaturized in this way, it is furthermore no longer necessary, in order to optimize the speed, for the individual compartment containing the sample to have the form of a well, as known from conventional microtitration plates. Suitable structures are recesses, cups or metallic bumps, produced, for example, by lithography and/or etching or stamping processes, for example in or on silicon or other materials, for example plastics or glass. It is also conceivable to use vapor-deposited structures or those produced by self-assembly methods. Microstructures produced by the LIGA process can also be used.

For even faster scanning of a compartment, k is sufficient for it to have the shape of a cup, trough or flat recess. The best design in this respect for the multiple sample holder employed in the novel method or in the novel apparatus consists simply of a very flat planar disc onto which the samples to be investigated are placed in the form of small droplets. If the substrate used is an Si wafer onto which the samples are placed in the form of droplets, it is preferable for the areas in question to differ in surface structure from the environment. For example, in the case where the wafer has areas with a vapor-deposited gold coating, it is preferable for the individual compartments to have a gold base, while the hydrophobic silicon separates the individual compartments from one another.

The entire apparatus comprising the near field microscopy instrument, the single or multiple sample holder with compartments for samples and the control device, can have been modified in such a way that the control device controls not the SNOM instrument, but the multiple sample holder, in a similar way as the microscope stage is moved in the area of light microscopy using conventional microscopes - as already mentioned above.

The control mechanism itself can be similar to that known to the person skilled in the art from other computer-controlled instruments, for example stepping-motor control systems.

- - In general, the individual compartments of a multiple sample holder of this type can be arranged in any desired manner, but in particular in a rectangular or circular structure.

Examples An example of the novel process is given below: Fluorescence was detected using a commercial SNOM instrument (for example Aurora, TopoMetrix Inc.). The substrate used was an ELISA plate with a vapor-deposited gold coating or an Si wafer. A thiolcarboxylic acid was applied thereto by liquid-phase adsorption (self-assembly). The thiol binds to the gold surface, and the acid groups face outward.. The relevant receptor, for example avidin or thrombin, was immobilized thereon via a peptide bond.

This was carried out by derivatizing the carboxyl-modified surface or tip by means of a spacer as mentioned above (for example aminohexanoic acid or spermidin) or another ligand. To this end, the carboxyl groups of the substrate or gold tip were incubated for 5 minutes with 100 mM N-hydroxysuccinimide (C4H5NO3) and N-(3-dimethyl-aminopropyl)-N-emylcaitodiimide (hydrochloride) CsHisCINs and with aminohexanoic acid or spermidin (in each case 100 mM).

The aminohexanoic acid-derivatized surface was then re-activated - as described above -by means of N-hy(iroxysuccinimide and N-(3 -dimethylaminopropyO-N-ethylcarbo-diimide and then reacted again with the aminc X>ntaining ligand (for example thrombin or another protein) in a concentration of, for example, 100 Hg/ml. However, it is also possible to bind low-molecular-weight ligands, for example the following thrombin inhibitor, to the surface.

- II - If the spacer selected was spermidin, the free amino group thereof can be reacted with an activated carboxyl group of the ligand. This activited carboxyl group can likewise be prepared by the process described above. However, other processes can also be used, as known, for example, from peptide synthesis for activating carboxyl groups: a) activation of carboxylic acids to give the mixed anhydride using chloroformic acid, EEDQ, UDQ b) activation of the carboxylic acid to give the symmetrical anhydride using carbodiimides c) activation as an active ester, for example using caAodiimide, uranium salts of benzotriazole or uranium salts in general and possibly pentafluorophenol, hy Fluorescence-labeled avidin or thrombin was then added and bound to the reference active compound, with formation of a fluorescent complex directly on the surface of the substrate. This individual-molecule fluorescence was detectable by SNOM. If a mixture with a more effective active compound was then added, the labeled avidin or thrombin was freed from its bond and diffused away from the surface, with a reduction in fluorescence. However, the more effective active compound present in the added mixture need not necessarily bind to the same site as the active compound immobilized on the substrate surface. A reduction in binding of the fluorescein-labeled molecule can also occur through binding to another site on the substrate. The modification of the conformation (allosteric effect) then reduces the affinity of the fluorescence-labeled molecule to the ligand immobilized on the substrate surface, so that only a small proportion of the fluorescent molecules remains bound to the surface, and the fluorescence thus likewise decreases.

As examples of cellular structures which can be used as substrate, mention may be made, in particular, of membrane-immobilized receptors; besides classical signal-transducing receptors (for example receptors coupled to G-protein, ligand- and voltage-dependent ion channels), also receptors on cells of the immune system or on blood platelets (for example GPHb DIa receptors) and pure binding proteins on cells of important cellular targets.

Claims (20)

as smdosed to IPER 13 We claim:

1. A method of screening active compounds, in which the active compound is detected from the change in a signal [S], characterized by an optical contrast mechanism, which change can be observed by means of a scanning near field optical microscopy instrument (SNOM instrument].

2. A method as claimed in claim 1, in which the scanning optical near field is provided by means of an optical fiber (1).

3. A method as claimed in claim 1 or 2, in which the active compound screening comprises the steps of providing a multiple sample holder with a plurality of compartments for samples, and carrying out the following steps on all or some of these compartments, preferably by means of a parallelized SNOM instrument: a) a molecule A is bound directly or mdirectly, covalently or noncovalently, to a substrate (2), b) a molecule B which can be detected from a signal [S] by means of a scanning near field optical microscopy instrument is allowed to bind noncovalently to the molecule A, c) a solution containing an active compound W2 is added, the presence of the active compound W2 is detected from the change in binding of the molecule B and thus of the signal [S].

4. A method as claimed in claim 1 or 2, in which the active compound screening comprises the steps of providing a multiple sample holder having a plurality of compartments for samples, and carrying out the following steps on all or some of these compartments - in parallel or successively: a) a molecule A is allowed to bind to a substrate (2), b) a solution containing an active compound Wx is added, c) the active compound W* is allowed to bind to A with formation of a molecule complex A-X, d) a solution containing a molecular probe FTTC which can be detected from the signal [S] by means of the scanning near field optical microscopy instrument is added, e) the molecular probe FTTC is allowed to bind to the complex A-X, and f) the binding of FITC to A-X is detected by measurement of [S].

5. A method as claimed in any one of the preceding claims, where the signal [S] is a fluorescence signal.

6. A method as claimed in any one of the preceding claims, where the binding of the molecule A to the substrate (2) takes place by adsorption.

7. A method as claimed in any one of the preceding claims, where the substrate (2) is an Si wafer, a microtitration plate with a vapor-deposited Au coating, mica, graphite or a cellular structure, or a combination thereof

8. A method as claimed in claim 7, where the cellular structure is a membrane-immobilized receptor.

9. A method as claimed in any one of the preceding claims, where the molecule A is bound indirectly to the substrate (2) via a spacer, in particular spermidin or aminohexanoic acid.

10. A method as claimed in claim 9, where the spacer is itself bound via a compound adsorbed onto the substrate (2).

11. A method as claimed in any one of the preceding claims, where a parallelized measurement device which includes the SNOM instrument is employed for simultaneous detection of a plurality of sample compartments for active 15 128230/ 2 compound screening.

12. A method as claimed in any one of the preceding claims, where the signal [S] uses an optical contrast, in particular a difference in absorption, fluorescence, polarization or wavelength dependence thereof, for detecting the binding of substance X

13. A method as claimed in any one of claims 10 to 12, where the spacer binding step is repeated a number of times.

14. An apparatus comprising a near field microscopy instrument, a single or multiple sample holder having compartments for samples, and a control device for addressing the individual compartments of the single or multiple sample holder with the near field microscopy instrument.

15. . An apparatus comprising a near field microscopy instrument, a single or multiple sample holder having compartments for samples, and a control device for addressing the near field microscopy instrument by means of the single or multiple sample holder.

16. An apparatus as claimed in any one of the preceding claims, where the single or multiple sample holder is a multiwell microtitration plate.

17. A multiple nanotitration instrument apparatus according to any of Claims 14-16, comprising a planar plate and a plurality of nanocompartments arranged thereon, where the size (diameter) of the compartments is from 10 to 100 nm. 16 128230/2 C

18. A multiple nanotitration instrument as claimed in claim 17 , where the nanocompartments are in the form of wells.

19. A multiple nanotitration instrument as claimed in claim !7 , where the nanocompartments are delimited from the remaining areas of the planar plate in that their size (diameter) is determined by a chemical structure or composition which is different from that of these remaining areas.

20. A multiple nanotitration instrument as claimed in any one of claims 17 to 19 , where the planar plate is hydrophobic silicon, and the nanocompartments are defined by a change in the surface structure.