KAHYA, Neriman, N. (AE Eindhoven, NL-5656, NL)
1. Method of detecting the presence of at least one analyte molecule within a cell comprising the step of reversibly contacting at least one cell with at least one nanowire.
2. Method according to claim 1, wherein one tip of said nanowire is reversibly positioned in vicinity of the outer side of the cell membrane, is positioned into the cell membrane or introduced across said cell's membrane into the cell's interior.
3. Method according to claim 1 or 2, wherein said nanowire is associated with a solid support being capable of positioning the nanowire with respect to said cell.
4. Method according to any of claims 1 to 3, wherein the nanowire has light emitting and/or conductive properties.
5. Method according to any of claims 1 to 4, wherein said method uses the optical and/or conductive properties of said nanowire and wherein energy transfer between said analyte molecule and said nanowire determines the presence of said analyte molecule.
6. Method according to any of claims 1 to 5, wherein the nanowire has a solid core.
7. Method according to any of claims 1 to 6, wherein the nanowire is associated with at least one sensor molecule which is capable of
detecting said at least one analyte molecule within a cell.
8. Method according to claim 7, wherein said sensor molecule is covalently attached to the nanowire.
9. Method according to claim 7or 8, wherein said sensor molecule is selected from the group comprising antibodies, receptor ligands, peptides, sugars, small molecules and nucleic acids.
10. Method according to any of claims 7 to 9, wherein said method uses the optical and/or conductive properties of said nanowire and wherein energy transfer between a complex which is formed from said analyte molecule with said sensor molecule, and said nanowire determines the presence of said analyte molecule.
11. Method according to any of claim 1 to 10, wherein said cell is not in direct contact with the human or animal body.
USE OF NANOWIRES FOR IN VIVO ANALYSIS OF CELLS
SUBJECT OF THE INVENTION
The present invention is concerned with a method of detecting the presence of at least one analyte molecule within a cell by reversibly contacting a cell with at least one nanowire. BACKGROUND OF THE INVENTION
Over the past years, nanotechnology has emerged as a promising technology allowing to reduce the size of detection devices and to increase sensitivity as well as resolution of established detection methods.
In this context, new probes in nanoscale dimensions have been developed. Such nanoprobes can also be used to detect biological interactions such as the interaction between a protein and a ligand (Wang et al (2005), / PNAS, 102(9), 3208- 3212).
However, the use of nanoprobes for detecting biological interactions has mainly been restricted to testing of highly purified biological components in in vitro systems. Only recently, approaches have been undertaken to use nanotechnology in systems which more closely resemble in vivo situations.
For example, it has been shown that single-well carbon nanotubes can be complexed within a DNA helix and introduced into mammalian cells (Heller et al. (2006), Science, 311, 508-511). After entering, such single-well carbon nanotubes can be employed to probe for the presence of toxic metal ions such as e.g. Hg 2+ . These metal ions can induce a conformational change of the DNA which then results in a change of the optical properties of the single-well carbon nanotubes.
However, there is a continuing need for improved or alternative approaches for determining the in vivo status of a biological system such as a cell. OBJECT AND SUMMARY OF THE INVENTION
It is one objective of the present invention to provide a method for determining the status of and/or the presence of analyte molecules within an intact biological system such as a cell.
This objective as well as others which will become apparent from the ensuing description are attained by the subject-matter of the independent claims. Some of the more specific embodiments of the present invention are defined by the dependent claims. The present invention in one embodiment relates to a method of detecting the presence of at least one analyte molecule within a cell comprising the step of reversibly contacting said at least one cell with at least one nanowire. In one of the embodiments of the present invention, one tip of said nanowire can be used to either reversibly probe the surface of a cell's membrane, it can be reversibly positioned within a cell's membrane or it can be reversibly introduced across a cell's membrane into the cell's interior.
To this end, the nanowire can be associated with a solid support which is capable of positioning the nanowire in three dimensions with respect to a cell.
The nano wires which can be used for the purposes of the present invention can have light emitting and/or conductive properties.
In one of the preferred embodiments of the invention, the nanowire will have a solid core. Furthermore, the nanowire will typically have a diameter in the range of about 1 to about 200 nm. The nanowire will moreover typically display a length in the range of about 1 to about 200 μm. The aspect ratio will be typically in the range of 500 to 1000.
In one of the preferred embodiments the invention uses a nanowire which is associated with a sensor molecule that is capable of detecting an analyte molecule within a cell. Such sensor molecules include e.g. proteins such as antibodies, receptor ligands, peptides, small molecule inhibitors, nucleic acids etc.
In all of the aforementioned embodiments of the present invention, the method will preferably use optical and/or conductive properties of a nanowire to record an energy transfer between the analyte molecule and the nanowire in order to determine the presence of the analyte molecule.
Fig. 1 schematically depicts a cell which is deposited on a solid support and reversibly probed with a nanowire.
Fig. 2 schematically depicts a situation where two nanowires are introduced into a neuronal cell. The two nanowires are coupled to a voltage source and thus function as nanoelectrodes.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the finding that one can use a nanowire to reversibly contact a cell in order to determine the presence of at least one analyte molecule within such a cell. The present invention can also be used to monitor a biological process within a cell.
Before some of the embodiments of the present invention are described in more detail, the following definitions are introduced.
As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise. Thus, the term "an analyte molecule" can include more than one analyte, namely two, three, four, five etc. analytes.
The term "about" in the context of the present invention denotes an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of +/- 10%, and preferably +/- 5%.
The term "contacting a cell" in the context of the present invention means that one end of a nanowire can be positioned in close proximity to the outer surface of a cell's membrane, within a cell's membrane or pushed across a cell's-membrane into the interior of the cell. The interior of a cell denotes the cytoplasm, the nucleus as well as other sub-cellular structures such as the Golgi-apparatus, endosomes, the endoplasmatic reticulum etc.
If the outer surface of a cell's membrane is contacted, this refers to a situation where a nanowire is brought into such close proximity to a cell's surface that it is possible to detect e.g. receptor molecule within the cell's membrane as will be described below in more detail. The term "reversibly contacting" a cell means in the context of the present invention that one tip of a nanowire can e.g. be pushed through a cell's membrane in order to probe the interior of a cell and subsequently can be removed from the cell interior. Thus, reversibly contacting means that after the nanowire has been used to determine the presence of at least one analyte molecule on or within a cell it, can be removed without being left or associated with cellular structures.
In order to contact a cell, the nanowire will usually have to be positioned three-dimensionally with respect to the cell. Positioning of the nanowire may be undertaken by optical trapping such as laser nanowire assembly (LNA).
However, in a preferred embodiment of the present invention the nanowire will be associated with a solid support structure which is capable of being positioned three-dimensionally in order to ensure contact of one tip of the nanowire to the cell.
Such positioning of nanowires has been described e.g. by de Jonge et al (Nano Letters (2003), 3(12), 1621-1624). Thus, one may e.g. associate a nanowire with a support tip which can be made from a tungsten wire. Such a support tip can then be transferred e.g. to a scanning electron microscope (SEM) or an atomic force microscope (AFM) which is equipped with a piezo-driven nanomanipulator. Such a nanomanipulator is e.g. available from Omicron Nanotechnology GmbH (Taunusstein, Germany).
The support structure which has been exemplified above to be a tungsten tip can, of course, also be made from other materials such as e.g. Si, SiO 2 , Ti, Ti 2 O 3 , Al, Al 2 O 3 which are eventually coated with gold. Further, the support structure can also take other forms than a tip. The nanowire may e.g. be attached to a microscrew or mounted onto a piezo-driven step motor for an accurate spatial positioning with respect to the cell.
The term "nanowire" for the purpose of the present invention refers to an elongated nanoscale structure that at any point along its length has a diameter of less than 1 μm.
In some embodiments, the nanowires in accordance with the present invention will have a diameter in the range of about 1 to about 200 nm. In some of the preferred embodiments, the nanowires will have a diameter between about 5 to about 150 nm. A diameter of about 10 to about 100 nm or of about 20 nm will be even more preferred.
The length of a nanowire as it is used for the purposes of the present invention will typically be in the range of 1 to 200 μm. A length of about 5 to 150 μm or of about 10 μm will be preferred.
The nanowires in accordance with the present invention will typically have an aspect ratio of about 200 to about 2000. An aspect ratio of about 500 to about 1000 will be preferred. The nanowires which are used in accordance with the present invention comprise nanowires with a solid core and nanowires with a hollow core. The latter type of nanowires is also referred to in the art as nanotubes. Single-well or multi-well carbon nanotubes are typical examples of this latter type of nanowire.
For the purpose of the present invention, the use of nanowires with a solid core is presently preferred. Such nanowires present some advantages compared to e.g. carbon nanotubes in terms of more controllable properties, assembly and manipulation.
The nanowires as used in the present invention are desirably individual nanowires. As used herein, "individual nanowire" means a nanowire free of contact with another nanowire.
A nanowire as it is used for the purposes of the present invention can be of homogeneous or heterogeneous composition. Thus, the nanowire may display over the whole length of the wire a homogeneous distribution of the materials that are used for manufacturing of the nanowires. Alternatively, the nanowire may comprise different portions all of which can differ with respect to the materials from which the portions are formed. In such a
case, the nanowire may display different properties as regards optical properties, electrical properties etc. over the length of the nanowire.
Typically, nanowires of the present invention will be made from semiconductive or conductive materials. Nanowires can thus be made e.g. from Si, SiC, SiGe, GaN, GaP, GaAs, SnC>2, ZnO, InP, InAsP or other well-known materials such as for example carbon. Si nanowires are preferentially used in the context of the present invention.
The person skilled in the art is aware that nanowires can be classified according to their semiconductive/conductive properties into different categories. For example, III -V nanowires are made from materials such as GaAs, InP, InAs, GaP, and
II-VI are made from amterials such as ZnO.
The person skilled in the art is well aware that the optical properties as well as the conductive properties of a nanowire can be changed and adjusted by selecting the appropriate nanowire material. Additionally, a nanowire may be doped with certain e.g. metal compounds in order to influence the conductive properties of a nanowire. The skilled person will also appreciate that the optical as well as the conductive properties of a nanowire will depend not only on its composition but also on its dimensions.
Depending on their dimensions and the materials from which they are made; the nanowire from will display different optical and/or conductive properties. These different properties may be used for detection of an analyte molecule within a cell as will be set forth in further detail below. The person skilled in the art will therefore select a nanowire to be used in the present invention according to the detection principle to be employed as well as dependent on the properties of the analyte molecule to be detected. If a nanowire is used for the purposes of the present invention that is not further modified with a sensor molecule as will be described below, the primary criteria for selection of nanowires will be whether the nanowire itself is capable of interacting with an analyte substance in e.g. the area of the cell where the analyte molecule is mainly localized. A currently preferred type of nanowire that can be used for various embodiments of the present invention (i.e. with or without sensor molecule being
attached to the nanowire) is a typical Si nanowire or nanowires which are made from semiconducting materials of group III-V such as InP or GaAs. Such nanowires will usually have a diameter in the range of about 1 to about 200 nm and will have a length in the range of about 1 to about 200 m. Such nanowires will preferably have a diameter of about 10 nm and a length of about 5 μm. The aspect ratio will be usually in the range of about 500 to about 1000 with abou 500 being currently preferred.
The person skilled in the art is well familiar with producing nanowires. For example, GaP nanowires can be grown in a laser catalytic growth (LCG) process which means that the GaP reactants are generated by laser ablation of a solid GaP target. In this case, the target may comprise a relatively small amount of gold which serves as a catalyst for the nanowire growth.
Another method that can be used is a vapour-liquid-solid (VLS) growth mechanism. Further methods for producing nanowires of distinct composition and dimensions are described e.g. in WO 2005/064639A2. Descriptions for e.g. producing GaAs or InAs nanowires can be found e.g. in the scientific literature (Hiruma et al. (1995), J. Appl. Phys., 77(2), 447-461).
One detailed description of the aforementioned VLS growth method which uses a surface with e.g. gold particles as a catalytic growth centre is described in Duan et al (Advanced Materials (2000), 12, 298). This method can e.g. be used to produce nanowires such as GaAs-, GaP-, GaN-, InP-, GaAs/P-, InAs/P-, ZnS-, ZnSe-, CdS-, CdSe-, ZnO-, or SiGe-nanowires. The diameter of such nanowires can be controlled by the size of the catalytic gold particles. Fine-tuning of the diameters of the nanowires can then be achieved by photochemical etching whereby the diameter of the nanowire is determined by the wavelength of the incident light during etching. Other approaches for producing nanowires which can be used in accordance with the present invention will be obvious to the person skilled in the art.
Nanowires made of Si or III-V, II-VI semiconducting materials such as InP, GaAs or ZnO which are preferably used in some of the embodiments of the present invention can be made according to the method of epitaxial growth, as described in Bakkers et al. (Nature Materials (2004) 3, 769-773). Nanowires in accordance with the invention can also be fabricated as described in Bakkers et al. (Journal of the American
Chemical Society (2003), 125, 3440-3441) or in Morales et al. (Science (1998), 279 (5348), 208-211).
The optical and/or conductive properties of nanowires can be used for detecting a specific analyte molecule within a cell. To this end, the nanowire is positioned as described above with one tip for example close to a membrane where voltage gated K + receptors are located. Subsequently activation of the receptor leads to a change of the electrical status of the cell which can be transmitted by the nanowire which in this case should display affinity for K + ions to a detector device. This device will be linked to the nanowire and be capable of detecting and amplifying the respective signal. Thus, it is possible to use a nanowire which is not further modified with e.g. one of the below described sensor molecules to detect the presence of a certain type of analyte (K + ions) and to monitor a biological process such as gating and activation of a voltage gated receptor in sub-compartments of a neuronal cell's membrane.
Similarly it is possible to position a nanowire in different compartments of a cell and to accurately measure the pH value of this compartment.
For the purposes of the present invention, a biological process within a cell relates to functions and activities that influence the cellular biology of the cell. Such processes thus include rececptor activation, signal transduction, dynamics of cytoskeleton formation etc. In all of the above case, the detection principle relies on the fact that an association of the analyte molecule of interest with or a close approximation of the analyte of interest to a nanowire will result in an energy transfer to the nanowire which can then be recorded as a change in the nanowire's optical and/or conductive properties. The person skilled in the art is well familiar with detection devices that can be attached to a nanowire in order to measure a change in the optical and/or conductive properties of a nanowire upon occurrence of an interaction of the nanowire with another molecule. For optical detection one may use e.g. a charge coupled device camera (CCD). For electrical detection, one may use an amperometric device within the electric circuit to measure the changes in the nanowire conductance. A preferred embodiment of the present invention relates to the use of nanowires in the above described methods which are associated with a sensor molecule.
The type of nanowire that may be used for this purpose may be the same nano wires in terms of dimensions, composition, optical and/or conductive properties and architecture as mentioned above.
Thus, it will be preferred to use a nanowire with a solid core. The nanowires which are attached to a sensor molecule will furthermore typically have a diameter in the above-mentioned ranges with the range of about 5 to about 100 nm. A preferred length will be in the range of about 1 to 100 m , and a preferred aspect ratio will be in the range of 20 to 1000 also in the case where a sensor molecule is attached to the nanowire. Further, nanowires to which sensor molecules are attached will be preferably made of Si or semiconductive III -V or II-VI materials.
The sensor molecules which may be used in the methods of the present invention can be any type of molecule that is able to specifically interact with a target molecule within a cell. Thus, such sensor molecules may e.g. be fluorescent molecules which specifically interact with ions such as e.g. Ca 2+ . The sensor molecules may also be e.g. fluorescently- labelled molecules that can detect a shift in the pH value.
Typical other embodiments of such sensor molecules are nucleic acids which may be either DNA, RNA or modified versions thereof such as thioate-modifϊed DNA or RNA molecules, RNA-aptamers, or peptide-nucleic-acids. Nucleic acid sensor molecules will be typically capable to detect the presence and optionally the concentration of a complementary nucleic acid sequence within a cell.
Other sensor molecules which can be attached to a nanowire for the purposes of the present invention include proteins that are capable of specifically acting with factors within a cell. Proteins may e.g. be an antibody that is known to recognize a certain antigen that is present within molecules that occur within a cell. In case of antibodies the binding portions thereof such as Fv, scFv, Fab, Fab2 and heavy chained variable regions or other parts thereof may be used as sensor molecules.
A sensor molecule may also be a peptide such as a peptide that are known to be a ligand for e.g. membrane-bound or soluble receptors. A sensor molecule can also be a protein that is know to interact specifically with other proteins within a cell. Such sensor proteins include e.g. tubulin or actin binding proteins.
The sensor molecules will also comprise small molecule inhibitors or small molecule compounds which are known to specifically interact with cellular factors.
The sensor molecules may also be mono-, oligo- or polysaccharides or sugars. All of the above-mentioned sensor molecules can be covalently or non- covalently attached to that part of the nanowire which will be brought into contact with the cell.
For covalent attachment of a sensor molecule such as an antibody it may be necessary to modify the surface of the respective portion of the nanowire. In the case of a protein such as an antibody covalent attachment may be achieved by covalently binding the protein to the surface of the nanowire with bi- functional molecules such as glutaraldehyde, carbodiimides, biotin-avidin and other molecules with one or more functional groups on each of at least two ends as are well-known to those skilled in the art. Additionally, bi- functional spacer molecules such as N- hydroxysuccinimide derivatized polyethylene glycols may be used to bind sensor molecules such as proteins.
Functionalization of the surface of a nanowire for attaching sensor molecules may also be performed with silyl-alkyl-aldehydes or by deposition of self- assembling monolayers or functionalized polymers such as polyethylenglycol or polysilanes.
In a further embodiment of the present invention the sensor molecules may themselves be modified with detectable markers such as fluorescent groups in order to enhance the optical and/or conductive properties of the nanowires. Typical fluorescent dye include Cy5, Cy3, Texas Red, FITC, Alexa 647 etc.
Sensor molecules may also be labelled with a dye such as Qsy7, Qsy9, Qsy21, Qsy35, all of which are available from Molecular Probes.
The detection principle of the present invention relies on the fact that an analyte molecule within the cell interacts specifically with a corresponding sensor molecule. The energy shift that occurs upon this interaction is then transmitted to the nanowire which is associated with the sensor molecule. This transferred energy shift then
leads to a change in the optical and/or conductive properties of the nanowires which can be recorded by a detection device that is coupled to the nanowire.
If for example a protein in the cell binds to an antibody which is specific for this protein and which is attached to the nanowire, this will lead to a change in the optical properties of a nanowire which can then be detected. If moreover the antibody is e.g. labelled with a fluorescent dye, the energy change and consequently the change in the optical properties of the nanowire will be amplified.
The nature of the analyte molecules that can be detected in a cell in accordance with the present invention is not limited. Thus, analyte sensor molecules include ions, proteins, nucleic acids, lipids, sugar structures, enzymatic cofactors, vitamins, amino acids etc.
The method of the invention can not only be applied to determine the presence of a compound, but also to monitor a cellular process. If e.g. a tubulin- or actin-specifϊc protein or small molecule such as taxol is used and itself labelled with a fluorescent marker, one can measure and visualize dynamics of cytoskeleton arrangements. In view of the dimensions of nanowires and the accurate positioning of the nanowire within a cell, it is possible to record these dynamics at high spatial resolution.
The type of cell that can be analyzed with the methods in accordance with the invention is not subject to any limitations. Thus, one may analyze a prokaryotic cell or eukaryotic cell. One can analyze cells of e.g. mammalian origin, single cells as well as clustered cells.
The type of cells may also differ. Thus, one may analyze keratinocytes, lymphocytes, fibroblast, hepatic cells etc. It is to be noted that the afore-described embodiments of the present invention relate to methods in which a nanowire which may be associated with a sensor molecule or not, is used contact a single cell or a multitude of cells such as a tissue or an organ which are not in direct contact with the human or animal body.
The present invention provides for numerous advantages. Thus, the methods in accordance with the invention can be used for basic scientific purposes such
as e.g. determining the pH or certain ion concentrations in certain distinct locations of e.g. a mammalian cell.
If, for example, a nanowire is used which carries a nucleic acid-based sensor molecule, the methods in accordance with the present invention may be used to establish the concentration of complementary RNAs in different parts of a cell. Such methods will be particularly interesting in e.g. developmental biology. It is known that during development of an organism from an inseminated egg, distribution of distinctive RNA molecules to certain parts of such a cell plays a major role for establishing cell polarity and determination of germline layers. With the present method, these processes can be specifically analyzed at high spatial resolution.
Of course, the methods in accordance with the present invention can also be used for diagnostic purposes. Thus, one may obtain cellular samples from a patient and use a nanowire to detect the presence of e.g. molecular markers that are indicative of a certain disease such as a distinct cancer type. Due to the dimensions of the nanowire it will be possible to perform that type of analysis with a high specificity and spatial resolution. These diagnostic embodiments of the present invention may not only be performed outside the human or animal body, but also directly on the human or animal body.
Thus, the use of nanowires as described by the present invention provides a valuable tool for non-invasive measurements in living cells at high spatial resolution.
Another interesting aspect and benefit of the present invention is that testing with nanowires can be coupled to (non-invasive) electrostimulation which also allows to measure the behaviour of a biological system.
In one of the preferred embodiments invention, the above described methods are performed in a multiplex fashion. This means that numerous nanowire sensors are simultaneously introduced in either the same of different cells. If e.g. the nanowires are modified with different sensor molecule the same cell or the same type of cells can be analyzed at the same time with respect to different properties and analytes.
In the following, examples are provided in order to illustrate some specific embodiments of the present invention. However, these examples are not to be construed as limiting the scope of the invention in any way.
Example 1 - determination of ion concentration within a cell. The surface of a silicon nanowire of dimensions 10 nm in diameter and 5 m in length is functionalized to provide covalent attachment of a Ca-ion indicator. Such a Ca-ion indicator can be a fluorescent dye whose quantum yield is dependent on the presence of Ca-ions in the ambient solution. An example is Fluo-Calcium indicators (available from Invitrogen GmbH, Karlsruhe, Germany).
The nanowire is then mounted to a gold-coated support tip being made of silicon. The support tip is attached to a device which can be used for three-dimensional positioning and optical detection. Such a device may be a typical laser scanning fluorescence microscope that is linked to a nanomanipulator available from e.g. Omicron Nanotechnology GmbH (Taunusstein, Germany).
The functionalized nanowire can then be brought into contact with a cell in a cultured dish as depicted in Fig. 1. By positioning the nanowire, the tip being functionalized with the Ca-ion sensor is pushed through the cell's membrane into the cytosol of the cell.
Upon contacting Ca-ions in the living cell, the tip of the nanowire will become fluorescent and the resulting signal can be recorded by e.g. a CCD camera.
This approach is particularly useful for determining the properties and behaviour of synapses of neuronal cells or of the neuromuscular junction. Example 2 - Use as nanoelectrodes
Nanowires can also be used as a nanoelectrode for measuring the potential levels on the surface of living neuronal cells. As depicted in Fig. 2, conducting nanowires are positioned in a cell in order to sense electroactive species. Furthermore, this approach can be used for electrostimulation of cells in a non-invasive nano-patch clamping technique which allows recordings of e.g. cytoplasmic levels of chemical messengers at high specificity and spatial resolution.