Patent Application
20080153085
This invention relates to an analytical method and device for the determination of the presence and/or the concentration of an analyte in a liquid medium. More specifically, the present invention concerns a fast and sensitive optical method for the detection of cancer cells, DNA analyte or single base mutations.
The following references are considered to be pertinent for the purpose of understanding the background of the present invention.
1. Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016.
2. Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142-12150.
3. Mao, C.; Sun, W.; Seeman, N. C. Nature 1999, 397, 144-146.
4. Yurke, B.; Turberfield, A. J.; Mills, Jr. A. P.; Simmel, F. C; Neumann, J. L. Nature 2000, 406, 605-608.
5. Tan, W.; Fang, X.; Li, J.; Liu, X. Chem. Eur. J. 2000, 6, 1107-1111.
6. Dubertret, M.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365-370.
7. Patolsky, F.; Lichtenstein, A.; Kotler, M.; Willner, I. Angew. Chem. Int. Ed. 2001, 40, 2261-2264.
8. Patolsky, F.; Weizmann, Y; Willner, I. J. Am. Chem. Soc. 2002, 124, 770-772.
9. Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016.
10. Willner, I.; Patolsky, F.; Wasserman, J. Angew. Chem. Int. Ed. 2001, 40, 1861-1864.
11. Kan, S. et al, Nature Mater., 2003, 2, 155.
Hybrid systems consisting of semiconductor quantum dots (QDs) coupled to biomaterials find growing interest in the developing research area of nanobiotechnology (1, 2). Photochemically-induced fluorescence resonance energy transfer (FRET) between molecular fluorophores (3-5) or the quenching of excited chromophores by metal nanoparticles (6) was reported to probe DNA hybridization processes, and, specifically, the formation and dissociation of hairpin structures. The replication of DNA on bulk surfaces was recently applied for the amplified bioelectronic detection of DNA (7), and the incorporation of redox-active units into the replicated DNA has enabled the electrochemical probing of the dynamics of replication (8).
The unique photophysical properties of semiconductor nanoparticles establish the possibility of applying semiconductor nanoparticles as efficient fluorescence labels (9) or as photoelectrochemical probes (10). Semiconductor nanocrystals have several advantages as FRET donors. The nanocrystals may be tailored, via control of size, composition and shape (11) to provide exceptional spectral coverage with symmetric emission profiles, enabling optimization of donor-acceptor spectral overlap. Additionally, due to their continuous absorption band they may be excited efficiently at shorter wavelength. Finally, the nanocrystals are significantly more stable emitters compared to the conventional dye molecules and as mentioned above, this is a critical feature for a feasible FRET microscopy scheme.
The present invention provides an optical method and device for the determination of an analyte in an assayed sample. The method and device of the invention are based on the use of semiconductor nanoparticles that carry a recognition agent, thus forming a hybrid system. With this system, in the presence of an analyte and under assay conditions, a reaction occurs causing the immobilization of an acceptor to the recognition agent, either directly or through a reaction product of the recognition agent that is formed in the presence of the analyte.
The nanoparticles provide an active media with respect to electromagnetic radiation. The term “active media” is meant to denote a media capable of interacting with electromagnetic radiation resulting in absorption of the radiation followed by transfer of the energy to an acceptor.
Thus, the detection in the present invention is based on energy transfer between active media (donors) and acceptors. The acceptors accept energy from the donor, which stimulates electronic transitions to higher energy levels so that when they return to lower energy levels they emit energy, e.g. photonic radiation. More specifically, the detection is based on fluorescence resonance energy transfer (FRET) between semiconductor nanoparticle donors, which are excited with electromagnetic radiation, and acceptors in the form of dye-labeled agents, preferably dye-labeled nucleic acids or dye-labeled oligonucleotide sequences, nanoparticles-labeled agents such as nanoparticles-labeled nucleic acids or nanoparticles-labeled oligonucleotide sequences.
The term “donor” denotes a chemical entity having absorption and emission spectra. Typical donors in the present invention are semiconductor nanoparticles. The term “acceptor” denotes a chemical entity where a portion of its absorption spectrum is overlapping a portion of the emission spectrum of the donor such that the acceptor is capable of accepting energy from said donor. Typical acceptor molecules used in the present invention are dye molecules. Non limiting examples of dyes are Rhodamine based dyes, fluoresceines, cyanines, Texas red and other available dyes such as transition metal complexes acting as energy acceptors. Alternatively, the acceptor molecules are nanoparticles with acceptor spectral properties, e.g. InAs acceptors.
The donor nanoparticles are excited using a wavelength wherein absorption of the acceptor is negligible compared to that of the semiconductor nanoparticle. Efficient resonance energy transfer between the donor semiconductor nanoparticles and the acceptors would occur only when the acceptor agents are immobilized to the hybrid system comprising the nanoparticles and the recognition agent. Such attachment may happen only under appropriate reaction conditions and in the presence of the analyte. As used herein the terms “immobilize”, “attach ”and “bind”, at appropriate departures (e.g. attached, attaching, immobilized, immobilization, bound, binding, etc.) denote chemical binding (i.e. chemisorption, covalent linkage e.g. hydrogen bonds, Van der Waals bonds, etc.) or electrostatic linkage, e.g. ionic bonds, etc.
The term “reaction” is used to denote one or more reactions or interactions carried out at once or in sequence, to attach the acceptor, directly or indirectly, to the recognition agent. The “signal” in the context of the present invention is emission of light at a given wavelength corresponding to the emission wavelength of the acceptor. Accordingly, the term “assay conditions” encompasses all the conditions, substances or actions necessary or useful for the appropriate reaction to take place, including sequences of varying conditions or actions.
The recognition agent bound to the semiconductor nanoparticles may react with the analyte to form a complex. The complex may then be the attachment site for the acceptor, possibly by a catalyst. Alternatively, the analyte may be a catalyst that can induce a reaction in which the recognition agent is converted into a product. In this alternative, the signal would be present only if the catalyst converted the recognition agent.
The invention permits the qualitative detection of an analyte, namely to get a Yes/No answer whether the analyte exists in the assayed sample, as well as the quantitative detection, namely determine the presence as well as the level/amount of the analyte in the sample. In the following, the term “determination” or “determining” or “detection” will be used to refer collectively to both qualitative and quantitative assay of the analyte in the assayed sample.
Likewise, the present invention is not limited to the nature of the recognition agent and the analyte, the nature of the reaction or the assay conditions. Nevertheless, it is appreciated that the present invention is especially useful in the determination of DNA or RNA analytes, DNA polymerase or telomerase analytes, cancer cells through telomerase activity and single-base mismatches. In such cases the recognition agent is a single-stranded oligonucleotide.
Thus, according to one aspect of the invention, there is provided a method for determining an analyte in an assayed sample, comprising:
whereby the signal is indicative of the presence and/or the amount of said analyte in the sample.
When the method is designed for quantitative detection, the detection comprises comparing the detected signal with a calibration curve showing the relation between the signal and known and varying amounts of analyte under said assay conditions, thereby determining the amount of said analyte in the sample.
The semiconductor nanoparticles are preferably nanocrystals having various shapes such as for example nanospheres, nanorods, quantum dots, branched structures such as tripods and tetrapods, tubes and wires.
Preferably, the semiconductor material is selected from Group II-VI semiconductors, such as for example CdS, CdSe, CdTe, ZnS, ZnSe, ZnO and alloys (e.g. CdZnSe); Group III-V semiconductors such as InAs, InP, GaAs, GaP, InN, GaN, InSb, GaSb and alloys (e.g., InAsP); Group IV-VI semiconductors such as PbSe and PbS and alloys; and Group IV semiconductors such as Si and Ge and alloys. Additionally, combinations of the above in composite structures consisting of sections with different semiconductor materials, for example CdSe/CdS or any other combinations, as well as core/shell structures of different semiconductors such as for example CdSe/ZnS core/shell nanoparticles or nanorods, are also within the scope of the present invention.
In a preferred embodiment the nanoparticles are in the form of quantum dots and even more preferably the nanoparticles are in the form of core-shell layered quantum dots (abbreviated “QD”). The signal generated is preferably emission of light.
The excitation of the semiconductor nanoparticles with electromagnetic radiation may be carried out at diverse wavelengths, preferably in the UV visible or IR range, depending on the sort of semi-conducting material used and on its form, e.g. nanoparticles, quantum dots, etc.
According to a preferred embodiment, the recognition agent and the analyte form a recognition couple and the detection of the analyte is based on the use of a reagent that binds to the formed couple. A specific example of such embodiment are cases where the analyte is a DNA analyte. In such cases, the assay conditions may comprise DNA polymerase and nucleotide bases, at least one of said nucleotide bases being bound to an acceptor. Experimental results show that the acceptor absorbs a certain wavelength of the energy emitted from the semiconductor nanoparticles upon irradiation of said nanoparticles with electromagnetic radiation, and emits light at a different wavelength.
The method of the invention may also provide the determination of at least one base mismatch. For example, the mutant sequence may have a single known base (the mutant base) replacing another base (being any one of the other three nucleotides) in a known position of the normal gene sequence. The recognition agent is a nucleic acid sequence that is shorter than the mutant sequence and the normal gene sequence, and is complementary to both of them up to one base prior to the mutation site, where said recognition agent terminates. Thus, hybridization of nanoparticle bound recognition agent with of any one of said DNAs leaves a single strand portion of said DNAs, beginning at said known position of the mutation.
The assay conditions in this example comprise a DNA polymerase and a dye-bound nucleotide that is complementary to the known base of the mutant sequence. Accordingly, the dye label would bind, through the DNA complex, to the nanoparticles only in complexes comprising the mutant sequence. Subsequently, irradiation of said nanoparticles with electromagnetic radiation would produce the dye specific signal only in case where the mutant analyte was present in the assayed sample This labeling may be intensified by application of thermal dissociation/annealing/labeling cycles, as part of the assay conditions.
A method for the detection of the presence of a specific base in a single stranded DNA analyte according to the present invention comprises the following steps:
The specific base may be a single-base mutation. Alternatively the specific base may be a part of a longer sequence to be identified in the DNA analyte, and the presence of the base may serve as an indicator to the presence of the longer sequence.
The specific base may also serve to detect a specific SNP (single-base-polymorphism), that naturally occurs between individuals, for forensic reasons, for example for identifying suspects according to blood, semen, hair etc. left in a crime scene.
The method of the invention requires that the nanoparticles be excited in a region of wavelengths where absorption of the acceptor is negligible compared to that of the excited nanoparticles and under conditions where the emission of the nanoparticles overlap with the acceptor.
According to another embodiment, the analyte to be detected is a catalyst that can induce a reaction in which the recognition agent is converted into a product. A specific example of catalysts are enzymes, e.g. telomerase. The detection of telomerase activity is indicative of the presence of cancer cells. A method for detecting telomerase activity according to the present invention comprises:
An alternative method for detecting telomerase activity according to the present invention comprises:
In a further alternative method for detecting telomerase activity, a single-stranded DNA recognition agent, that serves as a primer for telomerase reaction, is provided in solution. In such method the recognition agent is contacted with an assay sample comprising cellular extract from one or more cells suspected of comprising telomerase and nucleotide bases, thereby enabling telomerase-catalyzed DNA elongation reaction and binding of telomere repeat units to said recognition agent. In the next step the product of the elongation reaction is contacted with a nucleotide sequence being complementary to the telomere repeat units such that under assay conditions a hybridization reaction occurs. Each nucleotide sequence carries both donor and acceptor, where the distance between these two moieties is such that under irradiation conditions there is no energy transfer between the donors and the acceptors carried by the same sequence. In other words, energy transfer upon irradiation would occur only between the donor of one sequence and the acceptor of a neighboring sequence. Thus, irradiation of the system causes excitation of the donor nanoparticles of one sequence, transfer of resonance energy from said donor to the acceptor and generation of a signal. It has to be mentioned that transfer of resonance energy would not take place in the absence of said hybridization reaction. The detection of a signal is indicative of the presence and/or amount of telomerase in the sample.
According to another aspect, the present invention provides a sensing device for determining an analyte in an assayed sample, the device comprising an assay unit comprising a system of semiconductor nanoparticles carrying recognition agent and acceptor capable of immobilization, in the presence of the analyte and under assay conditions, to the recognition agent.
In a preferred embodiment, the above sensing device further comprises:
In another aspect, the present invention provides a sensing device for determining the presence of two or more different analytes in an assayed sample, the system comprising (a1) a plurality of assay units, each unit for determining a specific analyte of the two or more different analytes, and having at least one unit for each of said different analytes, each of said units comprising (i) a system of semiconductor nanoparticles carrying recognition agents and (ii) acceptor capable of immobilization, in the presence of the analyte and under assay conditions, to the recognition agent. Each unit, e.g. spot produced by arrayer or spotter, may be found on different locations or regions of a single device.
In a preferred embodiment, the above device further comprises:
The measuring utility in the above system is such that may identify which of the devices or regions in the system gave rise to the signal (and accordingly assist in the identification of the analyte(s) in the assayed sample) or measure the intensity of the signal, or both.
The above device, comprising an array of separate sensing devices is especially useful as a DNA array detection. The separate sensing devices are bound to a substrate by any surface modification technique. Thus, for example, if the substrate is made of gold or glass, the nanoparticles may be attached thereto through di-thiol linkers. The signals generated by the system may be read out by surface imaging techniques, where the emission of photons from areas bound to an analyte, is different from the emission from areas which did not bind an analyte.
In still another aspect, the present invention provides a kit for the detection of the presence or the amount of an analyte in an assayed sample comprising:
In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
A preferred embodiment of the method of the present invention is schematically illustrated in
Control experiments revealed that excitation of the nucleotide mixture that includes the dye modified dUTP in the absence of the CdSe—ZnS QDs at λ=400 nm does not lead to an observable emission at λ=560 nm. However, excitation of the system at λ=550 nm lead to a high fluorescence signal characteristic to the dye. As the emission of the CdSe—ZnS QDs at λmax=560 nm coincides with the absorbency band of the dye (2), the emission observed upon telomerization is attributed to fluorescence resonance energy transfer (FRET) from the nanoparticles to the dye molecules incorporated into the telomeric units by telomerase.
Atomic Force Microscopy (AFM) images of the CdSe—ZnS QDs prior to the telomerization,
The CdSe—ZnS quantum dots enabled also the probing of the dynamics of polymerase replication of DNA, as showed schematically in
The method and device of the invention may be applied for the fast and sensitive detection of cancer cells and as an amplification route for analyzing DNA on chip arrays. The polymerase-induced replication of DNA on the surface of luminescent QDs represents an “AND” gate where FRET readout occurs only if hybridization and replication proceed.
Another preferred embodiment of the invention is schematically showed in
In a further alternative embodiment, schematically showed in
Synthesis of CdSe—ZnS nanoparticles: trioctylphospine oxide (TOPO) protected nanoparticles, soluble in organic solvents were reacted in toluene (1 ml) with 100 μl of mercaptopropionic acid overnight. (Protected from light with aluminum foil). After this incubation time the nanoparticles were transferred to water by adding 1M KOH solution (1 ml), the two phase mixture was separated and this process repeated a second time with clean toluene (1 ml). The water-soluble CdSe—ZnS nano-particles were separated from the excess of mercaptopropionic acid by 3 repeated cycles of precipitation of the nanoparticles with acetone and followed by the re-dissolution of the nanoparticles in a phosphate buffer solution pH 7.4, 10 mM.
Preparation of DNA-modified CdSe—ZnS nanoparticles: The mercaptopropionic acid functionlized CdSe—ZnS nanoparticles (0.1 O.D) were reacted with freshly reduced and purified thiolated oligonucleotides ((1) or (3)) 6-10 O.D) overnight in order to allow the exchange of the thiol group associated with the nanoparticles with thiolated oligonucleotide. The DNA modified nanoparticles were separated from the free oligonucleotides by the use of micro-spin filter (Millipore, 30 KDa) or by acetone precipitation and re-dissolution in phosphate buffer solution. The nucleic acid-modified nanoparticles were obtained with a 70% yield. The fluorescence quantum yield of the nucleic acid-functionalized nanoparticles is ca. 15%.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IL04/00692 | 7/28/2004 | WO | 00 | 1/30/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60490259 | Jul 2003 | US |
