GB2432907A

GB2432907A - Device for detecting the characteristics of organic molecules

Info

Publication number: GB2432907A
Application number: GB0525779A
Authority: GB
Inventors: Martin Hegner; Thomas Braun
Original assignee: Universitaet Basel
Current assignee: Universitaet Basel
Priority date: 2005-12-05
Filing date: 2005-12-05
Publication date: 2007-06-06
Also published as: GB0525779D0

Abstract

The device for detecting characteristics of an organic molecule comprises a cantilever 1, an electrical conductible layer 2 arranged on the cantilever, and a function conserving layer 3 arranged on the electrical conductible layer. The function conserving layer is provided for conserving the function of the organic molecule and may be electrically isolating. An array of cantilevers may be provided and the detection may be differential. Characteristics such as voltage, mass change or structural change of receptors, proteins, hormones or cells can be detected.

Description

Device for detecting characteristics of an Organic molecule

Description

Device for detecting characteristics of an organic molecule

Technical field

The present invention relates to a device for detecting char-acteristics of an organic molecule, for example to detect the characteristics of bio-receptors, receptor ligand interac-tions, peptides, proteins, hormones, agents, virus, phages, or cells.

Background of the invention

Currently a lot of effort is put into developing real-time and label free sensors, which can be used to gain insight into protein expression or protein-protein interactions, drug screening and diagnostics. A field of high interest is the research of proteins, which are embedded within biological cell membranes. Drugs have to be transported or diffuse in the cell, to act on intracellular cell components or they are acting on cell surface proteins, also called membrane pro-teins. Around 30% of all proteins in the human genome are membrane proteins. They fulfill vital functions such as en-ergy conversion, transport and signal transduction. More than 30% of known drugs are acting on G-protein coupled receptors (GPCRs) but only 10% of the known G-protein coupled receptors can be addressed by antagonists. Taking the importance of 2 005/249 2. : ; ; ,. * . : membrane proteins in general and G-prote n coupled receptors in special, into account specifically and efficient instru-ments to study molecular interactions with these proteins are from great importance. Unfortunately, these studies are corn- plicated by two hurdles: First, membrane proteins are diffi-cult to handle, since they have to be stabilized by a lipid membrane or artificial detergents, and secondly, suitable in-strumentation for studying the interaction is missing.

A label-free method, called surface plasmon resonance (SPR) imaging is known from B. P. Nelson, T. E. Grimsrud, M. R. Liles, R. M. Goodman, and R. M. Corn. "Surface plasmon reso- nance imaging measurements of DNA and RNA hybridization ad-sorption onto DNA microarrays", 2001, Anal. chem. 73:1-7.

A further label-free method, called quartz crystal micro bal-ance (QCM) is known from K. Bizet, C. Gabrielli, H. Perrot, and J. Therasse, "Validation of antibody-based recognition by piezoelectric transducers through electroacoustic admittance analysis", 1998, Biosensors & Bioelectronics. 13:259-269.

Both methods rely on changes of physical properties of the protein because of the interaction with the ligand on the sensor surface. With the SPR method the change of the re-fracting index on a gold surface is detected, which can be interpreted as mass increase on the sensor surface, whereas with the QCM method a mass increase can be directly moni- tored. Thus, both methods are limited to measure the mass in-crease on the sensor surface and fail, if the potential ligand has a low molecular weight as it is the case for many ligands of membrane proteins when applied at physiological concentrations.

2 005/249 (it 3:

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, : t S With plasmon waveguide resonance spectroscopy (PWR), which is known from G. Tollin, Z. Salamon, and V. J. Hruby, "Tech-niques: plasmon-waveguide resonance (PWR) spectroscopy as a tool to study ligand-GPCR interactions", 2003, Trends Pharma-col. Sc!. 24:655 -659, protein conformation can be measured indirectly and mass increase can be measured directly, but it cannot be discriminated between protein conformation and mass increase.

Currently, only the binding event to a protein can be moni-tored, but the biological relevance of such an event cannot be acquired, and has to be evaluated in additional complemen-tary experiments. As an example, the binding of an antagonist can be detected by means of a fluorescence label approach or by means of a surface plasmon resonance, but no further in-formation can be gathered whether such a ligand forces trans-membrane structural changes or influences ion channels.

Suimnary of the invention An object of the invention is to provide a device for detect-ing characteristics of an organic molecule, wherein several characteristics of the organic molecule can be detected si-multaneously and in real time.

Advantageously, the device is suitable for different kinds of bio-receptors, especially for membrane proteins.

A further object of the invention is to gather additional in-formation whether a ligand forces transmembrane structural changes or influences ion channels of the membrane.

2 005/249 * I.. * * I *** * * C IS I C S S S a a * a a S S II * * a a a a a S * a a * * I a S * S.55 SC. a a According to one aspect of the invention, the object is achieved by a device for detecting characteristics of an or-ganic molecule.

The device for detecting characteristics of an organic mole-cule according to the invention comprises a cantilever, an electrical conductible layer arranged on the cantilever, and a function conserving layer arranged on the electrical con-ductible layer. The function conserving layer is provided for conserving the function of the organic molecule.

Advantageous further developments of the invention arise from the characteristics indicated in the dependent patent claims.

In an embodiment of the device according to the invention the function conserving layer is electrical isolating.

Preferably, on the device according to the invention the function conserving layer comprises an amphiphilic material.

In an alternative embodiment of the device according to the invention the function conserving layer comprises a semicon-ducting material, for example silicon nitride.

Furthermore, the device according to the invention can com-prise a voltage measuring device connected on one side to the conductible layer, wherein the conductible layer forms a ref-erence potential.

Advantageously, the device according to the invention com-prises an array of cantilevers.

In a development of the device according to the invention, a first cantilever is provided with a reference molecule, and a 2005/249 I Ill * * I * I I IS I * I a S * I I I I S I IS a S S a a a. S a a * a * I I S a S 151 III S second cantilever is provided with the organic molecule, wherein the measuring is carried out differentially using the first and second cantilever.

The method for characterizing an organic molecule with the device according to the invention comprises the following steps. In a first step the function conserving layer is ar-ranged on the cantilever. In a second step a lipid and the organic molecule are added, and in a third step the charac-teristics of the organic molecule are determined.

In another aspect of the method according to the invention the organic molecule is provided on the cantilever by means of an inkjet spotter.

Over and above this, the characteristics of the organic mole-cule can be determined in the method according the invention by measuring the voltage, and/or by measuring the mass change, and/or by measuring the structural change.

Finally, the device according to the invention can be used to detect characteristics of receptors, peptides, proteins, hor-mones, agents, virus, phages, or cells.

Brief description of the drawings

The invention and its embodiments will be more fully appreci-ated by reference to the following detailed description of presently preferred but nonetheless illustrative embodiments in accordance with the present invention when taken in con-junction with the accompanying drawings.

The figures are illustrating: 2 005/249 * fit t I I 1Sf * 8 I 88 I S 8 I I I I 8 I I I II * I I 8 I I I * I I I * I 8 * 8 i.e 8*S S S Figure 1 shows an embodiment of the detector for detecting characteristics of an organic molecule according to the invention.

Figure 2 shows an embodiment of a measuring assembly compris-ing the detector according to the invention.

Figure 3 shows a measuring diagram.

Detailed description of the drawings

In a ligand-containing environment membrane proteins, periph-eral membrane proteins or parts of membrane proteins change two, some-times three physical properties because of the ligand binding. One physical property, which can change, is the mass of the protein ligand complex by the additional mass of the ligand. A second physical property, which can change, is the structure of the membrane protein, by which the signal is conducted from the outside in-wards the cell or to other transmembrane proteins. The third physical property of the membrane protein, which can change, is the membrane-potential due to channel openings of the membrane.

These changes can be tracked by means of a rnicromechanical cantilever 1, as shown in FIG. 1, sensing the nanomechanical changes of the protein in a ligand-containirig environment.

This technique provides a versatile approach for measuring forces on a piconewton scale. The cantilever 1 works as a spring with a width and length in the micrometer range and a thickness typically thinner than one micrometer.

2 005/249 * S 4 II I S 4 S S * S I S I S I 5* * * . I I I I * I I I I 4 I * S I S 45* II. I S As depicted in FIG. 1, on the lower side of the cantilever 1 a piezo-element 1.1 working as actuator is mounted, which can let the tip 1.2 of the cantilever 1 swing. On the upper side of the cantilever 1 a conductive layer 2 is provided. On the upper side of the conductive layer 2 is in turn a function conserving layer 3 with a thickness of at least 0,4nm pro-vided. The function conserving layer 3 serves for conserving the function of the membrane protein, is preferably an isola-tor, and comprises a composition with a bifunctional group, an amphiphilic material, or a semiconducting material such as silicon nitride. On the function conserving layer 3 membrane proteins 8.1 and 8.2 can be positioned and surrounded with a buffer solution 9. Furthermore, a voltage measuring device 6 is provided, which is connected on one side via a connector 4.1 to the conductive layer 2 and dipped on the other side via a conductor and an electrode 4.2 in the buffer solution 9.

In FIG. 2 a measurement assembly is depicted comprising the cantilever 1. The piezoelectric actuator 1.1 lets the tip 1.2 of the cantilever 1 swing periodically with a frequency gen- erated by a frequency generator 12. The response of the can-tilever 1 can be read out using a beam-deflection system. The beam of a vertical cavity surface-emitting laser (the laser is not depicted in FIG. 2) is reflected at the tip 1.2 of the cantilever 1 towards a position-sensitive detector (PSD) 10.

The signal generated by the position-sensitive detector 10, which represents the response of the cantilever tip 1.2, is continuously compared with the excitation signal using a fre- quency analyzer 11. The frequency analyzer 11 records the am- plitude A and phase P in relation to the excitation fre-quency. A light emitting diode 13 can be provided for a bleaching process.

2 005/249 * S.. S * *S* * C I (S I S S 5 4 * I I S S S I SI * S S I I S I * S I I I I S * S II* ISS S S A light emitting diode 13 can be provided for an additional light inducible process, e.g. a bleaching process, which tem-porarily changes physical properties in the organic layer 3.

Alternatively hereto, the deflection can also be read out by means of a piezo-resistor, which is integrated directly in the cantilever 1 and changes its resistance, if the cantile-ver 1 is bend. This can be measured directly by an integrated circuit.

The measuring of at least two of the physical properties or characteristics of the protein in parallel, i.e. simultane-ously, is in the following also called multimode measurement.

In one embodiment of the invention all three above mentioned properties of the membrane proteins, peripheral membrane pro-teins or parts of membrane proteins are measured in parallel.

Advantageously, the multimode measurement takes the natural function and architecture of membrane protein into account.

Measuring several physical properties in parallel has also the advantage that noise can be reduced. Furthermore, it fa- cilitates the interpretation of the measured data and pro-vides more information than in a simple binding experiment.

Since the physical properties measured are directly corre-lated to biological meaningful properties, the results can be quantitatively interpreted.

Detection of mass change: In the following the detection of mass change is described.

The mass change of the membrane protein ligand complex can be measured by the change of the resonance frequency. This meas-2 005/249 * *1S S * S * S S ** S S * I S * S S S * a * i* * a * S I I I * I S * S I * S S S I aSS III S uring method is also called dynamic mode. The principle is outlined in FIG. 1 and 2.

The method allows the detection of natural ligands, antago- fists (drugs) and virus particles, if the corresponding re-ceptor or parts of it are used for the sensitization of the sensor interface. How the sensor interface can be sensitized is described later.

By means of an array of cantilevers it becomes possible to compare several organic compounds simultaneously. The array with the functionalized cantilevers is directly mounted onto the piezoelectric actuator, which is excited periodically by the frequency generator 12. As described above, the response of the cantilevers can be read out using the beam-deflection system. The beam of a vertical cavity surface-emitting laser is reflected at the tips of the cantilevers towards position-sensitive detectors. The response of the cantilevers 1 is continuously compared with the excitation using a frequency analyzer 11 recording amplitude and phase spectra.

The resulting spectra are analyzed by a software for tracking the amplitude peak and the phase-turning point. The amplitude peak, which corresponds to the resonance frequency, and the phase-turning point, which corresponds to eigenfrequency, are extracted and plotted versus the time.

In principle, it is also possible to track the resonance fre-quency by a phase locked loop (PLL).

The dynamic mode method is suitable for example for membrane proteins and was executed with the membrane protein ferric hydroxamate uptake protein component A (FhuA), which was overexposed and reconstituted in densely packed artificial 2 005/249 * tat a * a a,.

* I S II S I I I I S I I I I I It a a I I I I I a * a S I I I I S * . III 11* S S membrane to stabilize the protein. The protein was applied on the cantilever surface 3 by an ink-jet spotting device, which is described later on, and was attached to the cantilever via a bifunctional organic cross-linker dithiobissuccinimidyl un-decanoate (DStJ). The passivation of the cantilever 1, which was not functionalized by ink-jet spotting, was performed by incubating the cantilever array in lmg/ml casein. The devel-opment of the frequency response was followed before, during and after the injection of 3fM active bacterial virus parti- des. A clear shift in frequency upon virus binding was ob-served as can be seen in FIG. 3. The vertical lines in the frequency-time diagram indicate the range within which the virus interaction can be detected.

Many membrane proteins, such as G-protein coupled receptors interact with peripheral membrane proteins such as G-proteins. These G-proteins are leaving the membrane protein after activation by a ligand. The mass decrease can also be monitored by means of the dynamic mode method and can be f a-cilitated by channel structured supporting material on the cantilever or by holes in the cantilever where the proteoli- posomes are stretched over the holes similar to black-lipid membranes.

For virus detection, the titer determination can be combined with a label-free genotype characterization.

The dynamic mode, for which the sensitivity depends on the width of the resonance frequency of the cantilever, has been used in gaseous environments or vacuum, and also in liquids to measure the mass adsorbed on the cantilever in buffer so-lutions.

2005/249 * I., V V * **V * V I II I I I V * S S I I I I SI S I I * I I V * * S I I I S V S * I 55 ISV S S Further information on absolute mass-measurement can be found in T. Braun et al, "Micromechanical mass sensors for bio-molecular detection in a physiological environment", 2005, Physical Review E 72, The American Physical Society, 031907-1:9.

Detection of structural changes: In the following the detection of structural changes of the membrane protein ligand complex by static mode is described.

A structural change of the membrane protein ligand complex can be measured by detecting the surface-stress change in- duced by the conformnational change of membrane protein at-tached natively to the cantilever interface. This method is also called static mode.

For testing bacteriorhodopsin was used. To emulate a receptor ligand interaction, the prosthetic group retinal was removed by hydroxylamine. Upon the removal of the prosthetic group a structural change took place in the membrane-protein, which in turn changed the static bending of the cantilever. To test this reaction, bacteriorhodpsin was immobilized directly on the gold layer of the cantilever via a thiomodification of the membrane protein.

A differential signal of the protein can also be detected be-tween a bacteriorhodopsin mutant, which can not complete the photocycle and the unmodified wild-type protein. Here the protein was immobilized over a bifunctional organic protec-tion layer (DSU) 2005/249 * I,. S S * *t* * S I IS I C C I * I C I S * I II * I I I C S I S * I I I I S C C S * I CII III I The static mode was successfully applied to detect various biological interactions, such as deoxyribonucleic acid (DNA) hybridization and protein-antibody binding.

Detection of membrane potential: In the following the detection of membrane potential is de- scribed. The membrane potential change of the membrane pro-tein ligand complex or the effect of membrane potential change can be measured by controlling the electrical poten- tial between the cantilever and the surrounding buffer solu-tion.

The measurement principle is depicted in FIG. 1. Thereto, the cantilever 1 is coated on the upper side with a conductive layer 2, which can be for example a gold layer. On the upper side of the conductive layer 2 in turn a function conserving layer 3 is provided. To measure the voltage or membrane po- tential the conductive layer 2 is connected via a first con- ductor 4.1 with a voltage measuring device 6. The second con-ductor 4.2 is connected on the one side with the voltage measuring device 6. The other side of the second conductor 4.2 is dipped into the buffer solution 9.

The measurement of the membrane potential can be carried out in two ways. One possibility is to detect the membrane poten- tial indirectly. If the membrane potential has changed be-cause of a ligand, this leads to a structural change of the complex, which can be detected by means of static mode as de-scribed above. The influence of the binding behavior of the receptor can be studied. The second possibility is to detect the membrane potential directly, i.e., if ligands are opening ion channels of the membrane, the change of the membrane p0-2 005/249 * II. S S I 555 * * S VS V S I I

I I I I I I I II

* I I I I I I S * S I I V I I I S * I I.. ItS S S tential can be measured by means of the voltage measuring de-vice 6.

It is also possible to combine the above mentioned methods, i.e. to execute two or three of the methods simultaneously.

Static mode, dynamic mode and the voltage measurement have been combined and are implemented in liquid, i.e. buffer so-lution. Advantageously, no crosstalk between the different modes has been found.

The organic molecule, and the membrane protein in particular, can be provided on the sensor surface by means of an ink-jet spotting dispensing system, which can be for example pur-chased under the name MD-P-705-L from Microdrop, Norderstedt, Germany. Further information hereto can be found in A. Bi-etsch, J. Zhang, M. Hegner, H.-P. Lang, and C. Gerber "Rapid functionalization of cantilever array sensors by inkjet printing", 2004, Nanotechnology 15:873-880. This has the ad- vantage, that the amount of membrane protein for functionali-zation of the sensor can be minimized.

The sensitization of the cantilever surface, which forms the sensor interface, can be done with complete membrane proteins or with parts of them. If only soluble parts are used, the protein can be directly attached to the cantilever surface.

For complete membrane proteins, the protein should be stabi- lized: This is done by reconstitution of the protein in arti-ficial lipids. Immobilization methods for proteoliposomes have been already developed. Alternatively thereto, a new class of detergents can be used as described in J. Popot et al., "Amphipols: polymeric surfactants for membrane biology research", Cellular and molecular life sciences, 60:1559- 1574, 2003. In this case, the protein has to be immobilized similar to soluble membrane proteins. If the first route re-2 005/249 * *I t 1 : : * I; : * *

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* I III I** I flects better the physiological environment of membrane pro-teins, it is the first choice.

Having illustrated and described a preferred embodiment for a novel method and device for detecting characteristics of an organic molecule, it is noted that variations and modifica- tions in the method and the device can be made without de-parting from the spirit of the invention or the scope of the appended claims.

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Claims

: ; . ; a a $ I I * I I a a * I I * I S I I * I I IS. *I* I Claims 1.

Device for detecting characteristics of an organic mole-cule, comprising: -a cantilever (1), -an electrical conductible layer (2) arranged on the cantilever (1), and -a function conserving layer (3) arranged on the elec-trical conductible layer (2), and provided for conserving the function of the organic molecule.

2. Device according to claim 1, wherein the function conserving layer (3) is electrical isolating.

3. Device according to claim 1 or 2, wherein the function conserving layer (3) comprises an amphiphilic material.

4. Device according to claim 1 or 2, wherein the function conserving layer (3) comprises a semiconducting material.

5. Device according one of the previous claims, wherein a voltage measuring device (4.1, 4.2, 6) is pro-vided connected on one side (4.1) to the conductible layer (2), which forms a reference potential.

6. Device according to one of the previous claims, wherein an array of cantilevers (1) is provided.

7. Device according to one of the previous claims, -wherein a first cantilever is provided with a reference molecule, -wherein a second cantilever is provided with the or-ganic molecule, and -wherein the measuring is carried out differentially.

2005/249 16, a,.

: ; . *; ; * a a e S S I * V S S * V I S all III V S 8. Method for characterizing an organic molecule with the device according to one of the previous claims, comprising the following steps: -the function conserving layer (3) is arranged on the cantilever, -a lipid and the organic molecule (8.1, 8.2) are added, -the characteristics of the organic molecule (8.1, 8.2) are determined.

9. Method according to claim 8, wherein the organic molecule (8.1, 8.2) is provided on the cantilever (1) by means of an inkjet spotter.

10. Method according to claim 8 or 9, -wherein the characteristics of the organic molecule (8.1, 8.2) are determined -by measuring the voltage, and/or -by measuring the mass change, and/or -by measuring the structural change.

11. Using the device according to one of the previous claims, to detect characteristics of receptors, peptides, pro-teins, hormones, agents, or cells.

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