EP2300807A1

EP2300807A1 - A method, an apparatus, chemical kits and a program for analyzing the distribution of different types of nanostructures and/or sub-nanostructures in a sample

Info

Publication number: EP2300807A1
Application number: EP08773771A
Authority: EP
Inventors: Ashraf Ahmad; Kannan Balasubramanian; Klaus Kern
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 2008-06-30
Filing date: 2008-06-30
Publication date: 2011-03-30
Also published as: WO2010000277A1

Abstract

A method for analyzing the distribution of different types of nanostructures and/or sub-nanostructures in a sample, comprising the steps of - adding at least first and second fluorescent agents to the sample - irradiating the sample and the agents with light - measuring the emission spectra of the sample and agents over a range of wavelengths characteristic for the different types of said structures and agents and - analyzing the spectra to identify the distribution from the spectral responses. Also disclosed and claimed are corresponding apparatuses, a kit for establishing a distribution of different types of nanostructures and/ or sub-nanostructures in a sample, a laboratory workstation and a program carrier with related program.

Description

A method, an apparatus, chemical kits and a program for analyzing the distribution of different types of nano structures and/or sub-nanostructures in a sample

The invention relates to a method of analyzing the distribution of different types of nanostructures and/or sub-nanostructures in a sample, to an apparatus for analyzing the distribution of different types of nanostructures and/ or sub-nanostructures in a sample and to a related chemical kit, a related laboratory workstation and a related analysis program.

There is considerable interest in nanostructures and sub-nanostructures at the present time due to their unique chemical and physical features; therefore, such structures are widely utilized in the micro and nano fabrications and technologies. Nanostructures and sub-nanostructures are commercially available or can be locally synthesized in a laboratory, such structures can be, but are not limited to, one to several atom(s) and/ or compound(s) in thickness, length and/or diameter, can be of organic and/ or inorganic chemical nature and can be in polymer, oligomer and/ or monomer (supramolecular structures) format, some examples of such structures are: carbon nanotubes (single or multi wall), nanowires, gra- phene, fullerene, nano-particles, quantum dots.

All nanostructures and sub-nanostructures have in common the ability to absorb, (re-)emit and/ or scatter the light. Here, carbon nanotubes were chosen as an example of said structures. Carbon nanotubes (CNTs) are emerging as prospective candidates for a variety of applications. In spite of the huge promise, very few applications have been realized in the form of products. One of the major hurdles for widespread deployment of CNTs is the inability to differentially quantify the concentration of the different chiralities of nanotubes in a solution. The chirality of a CNT is expressed as a vector (n, m) which defines how the graphene sheet (nanotubes ori- gin) is rolled up. By way of example, to produce a (7, 5) nanotube, the graphene sheet is rolled up so that the atom labeled (0, 0) is superimposed on the one labeled (7, 5). Carbon nanotubes can, for example, be produced by an arc evaporation method in which a current of about 50 amps is passed between two graphite electrodes in an atmosphere of helium. The graphite vaporizes and condenses in the apparatus including on the cathode where the nanotubes are to be found. Another manufacturing process is the HiPCO process (High- Pressure pyrolysis of CO Process) and yet another is the CoMoCAT process (used by the University of Oaklahoma). As made the carbon nanotubes are typically present in many different chiral forms and can also have different constitutions such as single wall nanotubes "SWNTs" and double wall nanotubes "DWNTs".

As with any chemical, the estimation of CNT concentration is vital for further processing and for obtaining reproducible functionalization. It is known that there are significant differences between, for example, HiPCO and CoMoCAT carbon nanotube products and there is certainly also no guarantee that one batch of material is identical to the next. Thus most of the commercially available raw materials contain a mixture of plural types of CNTs.

There is currently no reliable standard method available for performing a quantitative analysis of the numbers of different chiral forms present in a sample of carbon nanotubes, i.e. the population of each chiral form in the sample, and this is a serious problem. For example, in the absence of such a method, it is not possible to check the properties or quality of as purchased material and it is difficult to select desired chiral forms for specific applications.

In US 2006/0141634 a method is described in which a photolumines- cence intensity of a solution is determined, a sample of carbon nanotubes of unknown concentration is mixed with the solution and a photolumines- cence intensity of the mixture of the sample of carbon nanotubes and the solution is determined. Then the concentration of carbon nanotubes is determined on the basis of the determined photo luminesence intensity of the mixture and of the solution. The solution is typically a dye solution such as a TAMRA dye solution or a Rhodamine 6G dye solution. The mixture is typically held in a microfluidic channel and this is part of an optical trap which can be illuminated with laser light to concentrate the carbon nan- tubes in the optical trap. By switching the laser on and off the photolumi- nesence measurements can be carried out for the solution largely without nanotubes (laser off) and for the mixture with carbon nanotubes (laser on). The measurement itself is made by exploiting the property of fluorescence quenching. That is to say the sample (with or without the optically trapped carbon nanotubes) is exposed to light from a light source such as a mer- cury lamp. The spectral intensity of the fluoresence is measured using a photoluminesence detector for example a charge-coupled device (CCD) or spectrometer. The carbon nanotubes mixed with the solution exhibit fluorescence quenching. Thus a SWNT sample functionalized with DNA oligomers (CNT-DNA) and DNA-Tamra solution has been observed to have a decreased photo luminescent intensity as compared to that of the TAMRA dye solution alone.

However, the method is complicated, expensive, time consuming and unreliable. Moreover, the use of an optical trap to concentrate the CNTs means that the CNT concentration is falsified and the system is not sufficiently sensitive to the different chiral forms present in the sample.

The proposal discussed above has severe limitations due to the very high background signal and the absence of a single wavelength at which all nanotubes selectively absorb. Furthermore, the sensitivity of this method is very poor.

Another proposal is to be found in US 2008/0014654. That reference, which lists numerous sources of information on the manufacture of CNTs and techniques of suspending and chemically functionalizing them, basically provides a method comprising the steps of:

- (a) dispersing a sample in a solvent, wherein the sample comprises sin- gle-wall carbon nanotubes of undetermined composition, and wherein at least some of the single-wall carbon nanotubes are in a disaggregated state as a result of said dispersing;

- (b) irradiating the sample so as to effect fluorescence of the SWNTs;

- (c) detecting and analyzing the emitted near-infrared fluorescence with an emission spectrometer; and

- (d) performing a compositional analysis on the sample by comparing the fluorescence of the sample to at least one database of theoretically predetermined fluorescence profiles corresponding to specific SWNT compositions and abundances so as to be determinative of the composition of the SWNTs in the sample.

- (e) determining the sample's near- infrared emission spectrum, wherein a comparison and combination of emission and absorption spectra, for at least two laser wavelengths, provides a measure of the extent of fluorescence in the sample. A corresponding device for analyzing SWNTs comprises:

- (a) at least one light source effective for inducing fluorescence in SWNTs;

- (b) an emission spectrometer (e.g., a spectrograph or an interferometer- based device) effective for detecting fluorescent emission in the near- infrared region of the electromagnetic spectrum;

- (c) a sample holder for a sample comprising SWNTs of undetermined composition, wherein the sample holder permits the passage of light corresponding to excitation and emission wavelengths involved in fluorescence of the SWNTs in the sample; and - (d) a computer program for performing a compositional analysis of the sample based on a comparison of the fluorescence of the sample to database of pre-determined fluorescence profiles corresponding to specific SWNT compositions and abundances so as to be determinative of the composition of the SWNTs in the sample.

That invention exploits knowledge about the spectroscopic properties of SWNTs to provide specialized methods and apparatus for efficient fluori- metric analysis of bulk SWNT samples. In some embodiments, such analysis is predicated on a recognition that visible light, at a single well- chosen wavelength, can induce near-infrared fluorescence emission from a wide variety of distinct semiconducting SWNT species. Thus, by using a detector that registers all of these characteristic emission wavelengths in parallel, an information-rich emission spectrum can be acquired from a bulk sample in approximately one second. The spectrum can then be rap- idly computer- simulated as a combination of peaks from specific nanotube species whose spectral signatures are known from theoretically calculated values. It is said that rapid compositional analyses of bulk SWNT samples can be achieved by combining this data reduction approach with a customized apparatus. In some embodiments of that invention a solid SWNT sample is ultrasoni- cally dispersed in a D2O or H2O solution of a surfactant such as sodium dodecylsulfate (SDS) or sodium dodecyl benzene sulfonate (SDBS). The resulting suspension is then transferred to a spectrofluorimetric cuvette. When the sample cuvette is placed into the fluorimetric analyzer, it is irradiated with laser light at a specific wavelength capable of inducing near- infrared fluorescent emission from only certain limited chiral forms of disaggregated semiconducting SWNT in the sample that are supposed to be in resonance with the laser wavelength used. This emission is collected, directed into a spectrograph, and measured with a multichannel detector array. Then the excitation light source is blocked and the sample is illuminated with a broadband light source in the near-infrared. Transmission of this light through the cuvette is measured by the spectrograph and detector array to obtain the sample's near-infrared absorption spectrum. Both emission and absorption spectra are automatically transferred to a computer and evaluated to determine the SWNT species giving the fluorescent emission, their relative abundances, and the approximate fraction of absorbing species that fluoresce. This compositional analysis is based on prior assignments of optical transitions to various SWNT species, desig- nated by (n, m). See Bachilo et al., Science, 2002, 298, 2361-2366; and Weisman et al., Nano Lett., 2003, 3, 1235-1238. The compositional analysis is presented in the form of an index of the sample's fluorescent quality, an inventory of specific nanotube structures and abundances, and /or as a distribution of nanotube diameters and chiral angles.

This method is thus based on photoluminescence excitation (PLE) spectroscopy, wherein the band-gap fluorescence of semiconducting CNTs in solution is mapped as a function of the incident laser wavelength that is continuously tuned over the entire visible range. To achieve PLE of semi- conducting CNTs it is unavoidable to use a highly coherent and intense excitation light source. This is guaranteed mainly by lasers. In order to obtain a complete and precise distribution of different chiralities of semiconducting SWNTs a tunable continuous wave (CW) laser is required. Apart from being intolerably expensive, this method has the disadvantage that only semiconducting CNTs can be identified. Metallic nanotubes, impurities etc. cannot be detected using this method at all. In order to overcome the cost problem, the proposal demonstrates the use of 2 or 3 individual lasers as excitation sources. However this requires tedious and time-consuming fitting procedures to theoretical simulations, which should be optimized individually for the kind of dispersing agent and the type of raw material used. In addition, this procedure has even lower sensitivity coupled with the inability to reliably deploy it for functionalized CNTs.

The object of the present invention is to propose a relatively simple and inexpensive, sensitive, efficient and fast method, apparatus and chemical kits for establishing a differential quantative distribution of different types of nanostructures and/or sub-nanostructures in a sample.

In order to satisfy this object there is provided, in accordance with the present invention, a method of establishing a distribution of different types of nanostructures and/or sub nanostructures in a sample, comprising the steps of:

- adding at least first and second fluorescent agents (chemical compounds that absorb and emit light, e.g., fluorescent dye, quantum dots and/ or fluorescent nano- or sub-nano-particles) to the sample

- irradiating the sample and the agents with light that is characteristic of or in resonance with the said agents

- measuring the absorption and emission spectra (ultraviolet, visible and/ or infrared) for the combination of sample and fluorescent agent(s), measuring the absorption spectra of the sample without fluorescent agent and

- analyzing the spectra to identify the relative qualitative and quantitative distribution of said structures and measure the unit of each said struc- ture from the spectral responses.

The program for analysis includes but is not limited to

- identifying the composition of said structures based on the emitted light from said structures that were excited by resonantly absorbing the emit- ted light from said fluorescent agents

- obtaining an inverse relative ratio of the emission spectrum of said structures with the agents with respect to known calibrated emission spectrum of the agents, in different wavelength ranges characteristic of the agents, with or without integration.

A corresponding apparatus for establishing a distribution of different types of nanostructures and/or sub nanostructures in a sample, comprises:

- a means for adding at least first and second fluorescent agents to the sample

- at least one light source for irradiating the sample and the agents with light

- a spectrometer for measuring the absorption and emission spectra of the sample and agents over a range of wavelengths characteristic for the dif- ferent types of said structures and agents and

- a computer analyzer for analyzing the spectra to identify the distribution from the spectral responses. In some embodiments, the entire apparatus can be realized using micro or nanofluidics as a lab-on-a-chip system with an associated scanner/reader.

This method and apparatus for differential quantitative and/ or qualitative identification of said structures can be performed in different formats: liquid, solid, gas or plasma formats. The liquid format in which said structures are suspended in aqueous and/or organic solvent with or without organic or non-organic solubility enhancer (surfactant, detergent, chemi- cal functionality, oligo and/or poly nucleotide, peptide and/or saccharine), this format can be achieved in pico, nano, micro and/ or millifluidic format, it can be performed in cuvette, test tube or capillary format. In the solid format said structures can be deposited, smeared, dried, grown and /or electrically trapped on a substrate surface.

The above recited method and apparatus have the advantage that they provide high sensitivity and rapid data gathering at a relatively low cost. Moreover, they allow full quantification of the samples, i.e. the identification of the different nanostructures or sub-nanostructures present and the concentration of each of the different structures in the sample. The method and the apparatus also allow a fully automated system to be designed. The fluorescent agents are typically chemical compounds that absorb and emit light (e.g., fluorescent dyes, quantum dots, fluorescent nano- or sub-nano-particles) that can be used as free (non-connected) sin- gle or multiple agent(s). Each agent is selected for a specific one of said structures. Alternatively, at least two agents can be chemically connected, but with each agent being selected for a specific one of said structures. It is also possible for the connected agents to be in resonance and act as FRET (Free Resonance Energy Transfer) agents so that the connected agents act as one agent for a specific one of said structure. The first and second fluorescent agents can be added simultaneously or in mixed form to the sample. Alternatively, the first and second fluorescent agents can be added sequentially to the sample. It is also possible to pro- vide a plurality of like samples, for example by dividing the unknown sample into a plurality of like samples of equal volume, on the reasonable assumption that the statistical distribution of the different components (chiralities) of the structures is the same in each sample. In this case, the method can be realised by adding the first and second fluorescent agents to respective like samples.

The added fluorescent agent to the sample is excited at a specific wavelength, as a result the fluorescent agent emits light that is in resonance with specific said structure(s) (chirality(ies)). The resonance energy trans- fer typically results in emission spectra of the specific said structures that are used to identify such specific said structure(s)(chirality(ies)).

In a preferred embodiment of the method the emission spectrum is measured in a first wavelength range for each said agent and in a second wave- length range characteristic for the emission spectra from a specific said structure (chirality) of the nano or sub-nanostructures in the sample.

This has the advantage that the quenching of light from the fluorescent spectrum results in a modified spectrum in one range of wavelength and the resonant energy transfer results in a spectral response in a second different range of wavelengths. Thus, by measuring both spectra, more information is available leading to more accurate and faster identification of the populations of the different components (chiralities) of the said structures in the sample. The sample can be irradiated with first and seconds light sources respectively matched to said first and second agents. The irradiation of the or each sample to produce fluorescent excitation preferably takes place in ultraviolet or visible light ranges. This facilitates the resonant energy transfer to produce lower energy radiation in the near infrared spectrum.

This arrangement has the significant advantage that broad band radiation is used to excite fluorescence in selected wavelength ranges, i.e. the fluorescent response of the quantum dots, which is in relatively narrow wave- length ranges which can be readily and freely selected, by selecting the precise sizes and compositions of the quantum dots, to harmonize with the responses of the different components (chiralities) of the said structures, as will be explained later in more detail.

Alternatively, the sample can be sequentially irradiated with different wavelengths, i.e. wavelengths selected to harmonize with the responses of the different components (chiralities) of the said structures

The sample can also be simultaneously irradiated with different wave- lengths, since the spectrometer that is used, or the spectral analysis that is carried out, can readily identify the different components (chiralities) of the said structures. Thus, in practice, the spectral responses as measured with a spectrometer are associated with different components (chiralities) of the said structures.

The present invention has the further advantage that the sample can be observed with a microscope when irradiated to visualize and/ or characterize and/or measure the said structures and/or in a positioning method to localize a sample or a particular said structure in the sample. The preferred design of the apparatus is set out in claims 18 to 26.

The present invention also makes it possible to supply kits to users of the method or apparatus of the invention for establishing a distribution of dif- ferent types of nanostructures and/or sub nanostructures in a sample, the kit comprising at least some of the following items:

- at least one substrate for carrying the sample

- a medium for fixing the sample to the substrate

- at least first and second fluorescent agents, e.g. in the form of fiuores- cent dyes

- a washing medium

- an enhancer

- instructions for using the kit and

- a software program for evaluation of the measurements.

The apparatus can also be provided in the form of a laboratory work station comprising the apparatus set forth above and also optionally at least one said kit and a positioning system for positioning the sample on the substrate at the desired location.

Finally, the invention extends to a program carrier including a software program adapted to carry out the above described methods and/ or to operate the above described apparatus and/ or to evaluate the measurements made by an associated spectrometer.

The invention will now be described in more detail with reference to embodiments and the accompanying drawings in which are shown:

Figs IA and IB Excitation and emission spectra for carbon nanotubes, Figs. 2 A to 2 D a schematic diagram to explain the differential identification of carbon nanotubes,

Figs. 3 A and 3B a schematic diagram to explain the use of multiplex dyes for the differential identification of nanotubes,

Figs. 4A and 4B a schematic diagram explaining the use of dyes in resonance which exploit FRET to increase sensitivity,

Figs. 5A to 5C diagrams showing the different absorption spectra for

HiPCO and CoMoCAT nanotubes,

Fig. 6 a diagram to illustrate the enhancement of infrared emission by HiPCO non-oxidized nanotubes,

Fig. 7 a further diagram to illustrate the enhancement of infrared emission by HiPCO oxidized nanotubes,

Fig. 8 a further diagram to illustrate the enhancement of infra- red emission by CoMoCAT nanotubes,

Figs. 9A, B, C images illustrating the visualization of HiPCO nanotubes with a confocal microscope,

Fig. 10 a schematic diagram for the layout of a laboratory workstation for carrying out the method of the present invention,

Fig. 11 a schematic drawing illustrating a laboratory work- station conceived in accordance with Fig. 10, Fig. 12 a schematic drawing of one version of a reading unit as provided in Fig. 11 and

Fig. 13 a diagram showing the absorption and emission characteristics typical for a quantum dot.

Turning now to Figs. IA and IB there are shown two diagrams illustrating the chiral forms (n,m) and spectral responses (excitation and emission) of different carbon nanotubes.

In Fig. IA it can be seen that this illustrates the different chiral forms of carbon nanotubes with reference to their identifying vectors, such as (7, 5), (10, 0) or (9, 1) and shows the excitation wavelength E22 and the emis- sion wavelength Eu for each chiral form. For example, for the chiral form (7, 5), E22 is 640 nm and Eu is slightly less than 1020 nm.

Thus, Fig. IA shows that each nanotube is excited (absorbs light) at a specific wavelength in the visible or ultraviolet range and emits light at a spe- cific wavelength in the infrared range, so at least one of these wavelengths is distinguishable for a specific nanotube. The longitudinal shaded area is for metallic nanotubes at which they absorb light but do not emit it, while crossed shaded area is for non-nanotube structures such as amorphous carbon (sub-nanostructures) and nanoparticles. It should be noted that:Fig. IA shows many of the commonly found carbon nanotubes chiralities, but is not exhaustive, i.e. many other chiralities exist and can be analyzed using same principle in this patent, and while all nano structures and sub-nanostructures have in common the ability to absorb light and re-emit or scatter the light, carbon nanotubes were chosen to be an example of said structures. Fig IB shows that fluorescent dyes can be used to identify the type of nanotubes in a sample because nanotubes are excited by absorbing the emitted light from fluorescent dyes; therefore, the absorbed light as well as the emitted light from nanotubes can be used to identify the type of nano- tube and other nanostructures in a sample. Thus, the partly overlapping spectral ranges 10, 12, 14, 16 and 18 are the spectral ranges of respective fluorescent dyes (quinine, fluorescein, rhodamine B, nile blue and atto 725 respectively). It can be seen, for example, that the fluorescent dye with the spectral range 14 emits light that is in resonance with (6,5), (8,4), (9,2) and (11,1) nanotube chiralities, as a result this light acts as an excitation source and as a result a differential emission peaks can be detected, i.e. (6,5) chirality emits at 980nm, (8,4) chirality emits at 11 IOnm, (9,2) chirality emits at 1140nm and (11, 1) chirality emits at 1270nm, but no emission is detected at 1500nm from (14, 1) chirality which is not in resonance with fluorescent agent 14 but in resonance with fluorescent agent 18. At the same time because (8, 1), (7, 0), (7,3) and (10,0) chiralities are rarely found in nanotubes sample; therefore, the reduction in the emission of fluorescent agent 10 corresponds to the presence of metallic nanotubes, while the reduction in the emission of fluorescent agent 12 corresponds to the presence of non-nanotubes structures such as amorphous carbon (sub-nanostructures) and nanoparticles in the sample.

Figs. 2A to 2D illustrate the concept of the differential identification of nanotubes.

Fig 2 A shows a fluorescent dye I (FDI) which is excited by light 1 {hvl) and emits hυ2. Fig. 2B shows that hv2 emitted by FDI is in resonance with nanotube I (NTI) but not NTII; therefore, NTI absorbs hv2 and emits hv3, as a result FDI can be used to identify the presence of NTI but not NTII. On the other hand, Figs. 2C and 2D show that emitted light hv5 from FDII is in resonance with NTII not NTI; therefore, if FDII is mixed with a sample and the emitted hv5 is not reduced significantly (quenched) and hv6 is not detected then precisely NTII is not in the sample. If the emitted hv5 is at least partly reduced (quenched) and hv6 is detected, then NTII is present in the sample; therefore, the reduction in the emitted hv5 and the amplitude of detected hv6 allow the differential identification of nanotube chiralities and the relative concentration in an unknown sample composition.

Fig. 3 illustrates the use of "multiplex dyes" for the differential identification of nanotubes. In accordance with the present teaching it is necessary to use more than one dye in a mixture in many cases in order to identify different chiralities of nanotubes in one test. Some fluorescent agents (dyes) interact with each other and the use of a mixture of the dyes mentioned in Fig. 2 is not possible unless a specific spacer or linker is used as illustrated at 20 in Fig. 3. This linker keeps a specific distance between the two dyes to prevent physical (light transfer) or chemical interaction between them.

Thus, in Fig. 3A, the FDl is excited by light of wavelength hυl and emits light of wavelength hv2. FDII is excited by light hv4 and emits light of wavelength hv5. hυl and hv4 can be supplied by broad band illumination or by selective illumination, e.g. from two separate laser sources or by fil- ters selectively filtering broad band or spectral radiation to yield primarily hυl and hυ4.

Fig. 3B shows that the hυ2 which is emitted from FDI is in resonance with NTI; therefore, NTI is excited by hυ2 to emit hv3 whereas emitted hυ5 from FDII excites NTII to emit light at hυβ. Thus, the population of NTI in the sample can be identified by the amplitude of the reduction of hv2 emission and the amplitude detection of hv3, whereas that of NTII can be identified differentially with reference to the amplitude of the reduction of hv5 emission and the amplitude of hv6 detection.

Turning now to Figs. 4A and 4B there can be seen diagrams which illustrate the use of FRET dyes.

Fig 4A shows that FDI and FDIII are in resonance (FRET dyes), FRET dyes are the most specific way that can be used in fluorescent study, because there is no overlap between the excitation light source used to excite fluorescent dyes and the detected emitted light from the dye. Usually, in the case of using a single dye, hvl excites FDI and hv2 is emitted, the main disadvantage in this aspect is the overlap between both wavelengths, and in this case the nanotubes will be excited not only from the light emitted from FDI (hv2) but also from the light source (hvl). In the example of Fig. 4A the FRET dyes FDI and FDIII are in resonance and permit energy transfer (hv2) from FDI to FDIII, i.e., emitted hv2 from FDI excites FDIII to emit hv5 which excites NTII to emit hv6 because hv2 and NTII are in reso- nance. Therefore, highest specificity will be achieved because hvl is not interfering with hv6, as a result the emitted hv6 only arises upon hv5 excitation.

It is noted that the hv2 is only detectable when the individual fluorophores FDI and FDIII are free and no longer connected. If they are connected as in this case, then it will be below the noise level. Even if there were a considerable intensity from hv2, this will not affect the detection of the signal at hv5 and hv6. Turning now to Fig. 5A this figure shows the different absorption spectra for HiPCO and CoMoCAT nanotubes, the three lines show the maximum emission wavelengths for the fluorescent dyes used in B and C, fluorescein ( ), rhodamine B ( ) and quinine ( ). The dominant nanotubes in the CoMoCAT sample are in resonance with rhodamine B (Fig. 5A) and this is the reason why the quenching pattern of rhodamine B is typical (Fig. 5B, ), but it is not the case with quinine because Co-

MoCAT has no tubes in resonance with it (Fig. 5B, ). Instead the

HiPCO sample has predominant metallic nanotubes in resonance with quinine; therefore, it showed a saturation pattern in addition to the quenching one (Fig. 5C, ), at the same time HiPCO showed a low level rhodamine B quenching pattern because it has some tubes in resonance with it. As mentioned previously, in the range of fluorescein (490- 530nm) there are no nanotubes to absorb light but instead impurities do (Fig. IA), therefore the only structures that absorb the emitted light from fluorescein are the amorphous carbon and nanoparticles impurities which are much more in CoMoCAT than in HiPCO sample according to supplier information; therefore, CoMoCAT sample showed saturation in addition to the quenching pattern which is not the case for HiPCO (Fig. 5B and Fig. 5C, ).

It should be noted that the vertical axis in Figs. 5B and 5C is labelled FIO /FI. This is the standard way to express quenching data because in this way all data is normalized according to the absence of quencher. Fluorescent dyes usually have some inherent quenching effect, especially when the concentration is high, and to remove this systematic or technical variation the quantity FIO/ FI is used, where FIO is the fluorescent intensity without quencher and FI is the fluorescent intensity with a certain amount of quencher. In this case an inverse relative value will be used. Another reason for this is that some devices read fluorescent intensity up to 1000, others up to 100 and some devices are more sensitive than others. In this case using FI for one concentration will vary from one device to another, but using FIO/ FI the same results will be generated. The use of FIO/ FI is a standard method known as a Stern-Volmer plot that is usually employed to illustrate quenching mechanisms.

All results shown in Fig. 5 fit completely with theoretical data. It is easily concluded that the CoMoCAT sample has a large quantity of impurities, a very low number of metallic nanotubes and a high quantity of nanotube forms that absorb rhodamine emitted light (565-585nm), in addition it is concluded that the HiPCO sample is more pure with a higher quantity of metallic nanotubes than that in resonance with quinine. As a result, it is possible to identify the components of an unknown sample, for nano and/or sub-nanostructures. Up-to-date there is no method and/or device to identify the compositional distribution, the relative individual quantity and /or the total quantity and the purity of nano and /or sub- nanostructures in an unknown sample. Both US applications (US 2006/0141634 and US 2008/0014654) lack the ability to fully characterize an unknown sample, e.g., metallic nanotubes, sub-nanostructures (amorphous carbon) and nanoparticles can not be detected by near infrared emission detection method (US 2008/0014654); therefore, the purity of the sample can not be determined at all. The US 2006/0141634 application has no method and/ or explanation to determine the purity and/ or how to differentially quantify and/ or identify the nanotube composition of a sample. At the same time a scientifically wrong quenching equation and pattern have been shown, a linear equation has been explained which does not fit at all with quenching mechanisms.

Fig. 6 illustrates the enhancement of nanotube infrared emission by the incorporation of a fluorescent agent. The near infrared emission spectra is measured for HiPCO non-oxidized nanotubes solution with ( ) and without ( ) a fluorescent dye (nile blue). A laser light source at 638nm was used for excitation. It is clear that the emission peaks of some nanotubes are detected upon dye addition (peak 1 stands for (9, 1) chirality, 2 for (8,3), 3 for (6,5) or (7,3)) and others peaks are improved (4 for (7,5) and 5 for (10,2) or (8, 1)). Without nile blue the acquired maximum signal was 18101 counts, the power was 0.006097 nW and the emission efficiency was 0.004 nW x cm, while upon nile blue incorporation the acquired maximum signal was improved to 18851 counts, the power was improved to 0.066099 nW and the emission efficiency was improved to 0.044 nW x cm.

Fig. 7 illustrates the enhancement of nanotubes infrared emission by the incorporation of fluorescent agent. Here, HiPCO oxidized nanotubes solu- tion is again excited with laser light 638 nm with ( ) and without ( ) fluorescent dye (nile blue) . It is clear that the emission of some peaks ( 1 stands for (9, 1) chirality, 2 for (7,5)) are detected upon dye addition, and others are improved (3 for (7,6) and 4 for (10,0) or (8,6)).

Fig. 8 illustrates the enhancement of nanotubes infrared emission by the incorporation of a fluorescent agent. CoMoCAT nanotube solution is excited with laser light 638nm with ( ) and without ( ) fluorescent dye

(nile blue). It is clear that the emission of peak 1 (stands for (8,3) chirality) is detected upon dye addition, and others are improved (2 for (7,3) and 3 for (7,5)). It is clear from Figs. 6, 7 and 8 that adding a fluorescent dye which is in resonance with specific nanotubes will excite those nanotubes in resonance; therefore, detecting the emitted light from nanotubes upon FRET (between dye and nanotubes) will enable complete characterization of the nanotube sample, i.e., absorbing the emitted light from fluorescent dyes and detecting the emitted light from nanotubes will enable complete characterization of nano and sub-nanostructures. The application US 2008/0014654 depends mainly on characterizing nanotubes by detecting the emitted light upon laser excitation and that is an extremely severe disadvantage of the method and device described there because it requires a tunable CW laser which is highly expensive. Using fluorescent dyes is much easier and more efficient. Fluorescent dyes are in close proximity to nanotubes (in a solution or on a dry surface) and this is the ideal condition for energy transfer (excitation) and as a result quantum yield will be increased. In addition a regular light source can be used instead of laser source and as a result a continuous excitation (whole range) can be easily achieved.

Fig. 9 shows diagrams and images relating to the visualizing of HiPCO nanotubes with a confocal microscope by using fluorescent dyes. Fig. 9A shows an AFM image for nanotubes such as 30 between electrodes 32, 34 and a diagram for a cross-section of nanotube. In Fig. 9B a thin film of fluorescein covers the nanotube 30 and the surface 36 of the substrate and is visualized by a confocal microscope. The nanotubes absorb the emitted light (quench) from the fluorescent dye covering the nanotubes; therefore, the nanotube 30 appears black while the surface 36 of the substrate is shining (Fig. 9B). In Fig. 9C a thick film of fluorescein covers the nanotube 30 and the substrate 36, but in this case the nanotubes can not absorb all the emitted light (quench) from all fluorescein molecules, i.e., the nanotubes will absorb the light from the close fluorescent molecules as in Fig. 9B, but the outer layer of fluorescent molecules will not be affected, because the distance for optimal energy transfer is a few nm (more or less than this distance no energy transfer takes place); therefore, the nanotube 30 is shining (Fig. 9C). Thus, the present invention provides a simple and effective method based mainly, but not only, on FRET between a fluorescent agent and nano or sub-nanostructures, i.e., fluorescent agent is excited and the emitted light is used to identify the components of a sample by determining how much the nano and/ or sub-nanostructures are absorbing the emitted light from the fluorescent agent and by detecting the emitted light from the nano and/or sub-nanostructures when said structures absorb the emitted light from the fluorescent agent. This method of differential quantitative identification can be performed in different forms; it can be performed in liquid, solid, gas or plasma formats. In the liquid format said structures are suspended in an aqueous and /or organic solvent with or without an organic or non-organic solubility enhancer (surfactant, detergent, chemical functionality, oligo and /or poly nucleotide, peptide and /or saccharine). This can be done in a pico, nano, micro and/ or millifluidic format, for example it can be performed in a cuvette, test tube or capillary. In the solid format said structures can be deposited, smeared, dried, grown and/or electrically trapped on a substrate surface.

Thus, the present invention provides a simple and effective method based on fluorescence quenching and related emission spectra of different chiral forms of nanotubes to quantitatively estimate the amount of CNTs in solutions. The method is based on the addition of the CNT suspension to a solution containing at least first and second conventional low-cost fluorescent dyes (e.g. such as Rhodamine B -RB), preferably a plurality of dyes is used (not just two different ones). Excitation (e.g. 530 nm for RB) of the solution at the right wavelength (using either a lamp or a lamp and a mono-chromator or a diode laser) results in a quenching of the fluorescence (e.g. 540-700 nm for RB) intensity by the CNTs. By comparing the intensity of emitted light (at 574 nm for RB) with and without CNTs, the concentration of CNTs can be estimated with a high degree of accuracy and reproducibility. The quenching process relies on the mechanism of Fδrster Resonance Energy Transfer (FRET), whereby the excited dye transfers the absorbed energy to the CNTs reducing the intensity of the emitted light from the fluorophore. The dyes used can be chosen in such a manner that their emission spectra cover the absorption range of most of the CNTs ensuring a fairly constant excitation of tubes with varying chiralities. In addition to measuring the fluorescent quenching a second spectrum is measured in a different wavelength range and this is the emission spectrum for the carbon nanotube itself.

The measurements made to date show that both metallic and semiconducting CNTs can be quantified using this procedure. Moreover, an extremely high sensitivity is obtained that is at least two orders of magnitude better than the other two methods mentioned above. Finally, only minor corrections are necessary when the type of dispersing agent is varied or when a different tube raw material is used or when the tubes are (bio) -chemically functionalized. A direct consequence is the possibility to determine the chirality of tube species in solution by utilizing a combination of dyes with differing emission spectra through a differential quench- ing technique.

Thus, a cost-effective and versatile system based on this principle can be realized by incorporating a lamp, monochromators, filters and a detector - all of them operating in the visible region.

Turning now to Fig. 10 there is shown a block diagram of a possible layout for a nano and sub-nano autoanalyzer that is able to at least differentially identify and quantify the nano and/or sub-nanostructure components of a sample (the method of the present application), i.e. Fig. 10 shows in schematic form an apparatus for carrying out the present invention, with the apparatus being shown in a very schematic form in Fig. 11. It should be stressed that the layout of Fig. 10 and the schematic diagram of Fig. 11 are just one basic outline of how the apparatus of the invention could be realized and should not be considered restrictive of the present invention. Referring now to Figs. 10 and 11, the reference numeral 40 refers to a sample input unit basically comprising a rack 42 for receiving a plurality of special tubes, schematic illustrated as test tubes 44. Above the rack there is a dispensing unit 46 which is able to dispense nanotubes either in loose bulk form or already supported in a liquid into individual ones of the tubes 44.

Both the rack 42 and the dispensing unit 46 are connected via respective leads 48, 50 to a tower 52 of a control unit generally designated by 54 which basically consists of at least a computer and a printer with specific software at least for: handling, controlling and arranging all processes and units of the said autoanalyzer, data collection, storage, fitting and analysis with printing facility to print protocol, data, results in hard or soft format. Generally the control unit comprises (at least) the computer tower 52 embodying the usual and/or unusual (if necessary) microprocessors, memory facilities as well as a disc input 56 for receiving a computer disc 58 burned with said software, a monitor 60, a keyboard 62 and a mouse 64. The monitor 60, the keyboard 62 and the mouse 64 are again connected by respective leads 66, 68 and 70 to the computer tower 52. Data relating to the particular experiment and the sample used can be entered into the computer at the keyboard 62 with the aid of the mouse 64 and these can also be used to call up historic data as required.

The computer system 54 is able to control the rack 42, more specifically a linear actuator associated with the rack 42, to move in accordance with the double file 72 to position the sample holders 44 beneath the dispens- ing nozzle 76 of the dispensing unit 46. Furthermore, the computer tower can control the dispensing unit 46 via the lead 50 to deposit a predetermined quantity of the sample from a supply into respective ones of the tubes 44.

The reference numeral 78 shows a manipulating arm belonging to a robot (not shown) controlled via the computer system 54 via the lead 80. The robot arm 78 can, for example, move the sample tube 44 into a position between the outlet nozzles 82, 84, 86 and 88 of the pipetting and dispens- ing facilities 90, 92, 94, 96 which are connected via respective leads 98, 100, 102, 104 to the computer tower 52 (only a part of the leads is shown in Fig. 11). In this way the sample in the tube 54 can be prepared, for example by the addition of a liquid carrier from the dispensing unit 82, by the addition of a first fluorescent dye from the dispensing nozzle 84, of a second fluorescent dye from the dispensing nozzle 86 and optionally a diluent or a dye enhancer from the dispensing nozzle 88. The robot arm 78 can be adapted to shake and mix the respective tube 44 components at each dispensing facility 90, 92, 94, 96 to thoroughly mix the sample with the added liquid. The quantity added is controlled via the computer through the respective lines 98 to 104. Once the sample has been prepared in this way in the sample processing and preparation unit as just described and identified generally by the reference numeral 106, the tube can then be positioned in an incubation unit 108 (also belonging to the sample processing and preparation unit 106) where it can be conditioned for effecting the actual measurement. The incubation unit 108 has, for example, a cooling coil 110, a heating coil 112, a thermometer 114 and a timer 116 which are connected to the computer via respective lines 118, 120, 122 and 124. The cooler 110 can, for example, be a liquid cooling system controlled by the computer via the lead 118 which, for example, operates a pump for pumping cold liquid through the coolant circuit. The heater 112 could be a resistance heater with the energy supplied to the resistance heater being supplied via power leads under the control of the computer 52 via the lead 120. The thermometer 114 can be connected by lead 122 to the computer and enables the computer to control or balance the heating and cooling supplied to the incubation chamber 108 to bring the sample to a desired temperature. The timer 116 allows the duration of heating and/ or cooling phases in the incubation chamber 108 to be controlled. Once the sample has been brought to the desired temperature, it is then placed into the reading unit schematically illustrated by the refer- ence numeral 126. The reading unit 126 may have a special mixture holder, such as a cuvette, a substrate or a capillary, onto which the sample contained in the sample tube 44 is placed or, as schematically illustrated here, the sample tube 44 can itself be placed in the reading unit. Apart from the holder for the mixture, i.e. for the sample, the reading unit 126 comprises an excitation unit 128 which is a light source, for example a lamp with or without a monochromator combination and/ or a filter, or a laser light source for directing a beam of light onto the sample in the sample holder. The reading unit also comprises a detection unit, typically including an ultraviolet, visible and infrared detector, which may be realized as a camera or as a diode array or generally take the form of a spectrometer. One specific but not exclusive form of the reading unit is schematically illustrated in Fig. 12 and shows how a switchable mirror can be used to view the ultra violet and visible spectrum with one detector and the infrared spectrum with a second detector.

The reference numerals 136 and 138 show lenses for concentrating the light from the excitation unit 128 onto the sample and for concentrating light from the sample onto the detection unit 130. The excitation unit 128 and the detection unit 130 are connected to the computer tower 52 via respective leads 132 and 134. After the sample has been measured and analyzed, the software provided with the computer is able to analyze the emission spectral data to determine the relative quantity of sample components of said structures, and final results can be visualized on the monitor 60 and printed and/ or saved in a hard or soft format. For said structures in solid format (deposited, dried, trapped, smeared or grown on a substrate surface), the device setup can be modified in order to scan the substrate surface to generate an image (in a classical, confocal or near- field mode) and/ or generate a curve showing the differential quantitative and/or qualitative distribution of said structures. In order to generate such an image of the surface, the provided lenses can be automatically or manually adjustable.

Once the sample has been analyzed, then the sample tube is taken to the regeneration, washing and disposing unit identified generally by the refer- ence numeral 140. The unit 140 contains a plurality of receptacles into which the contents of the sample tube can be placed and in which they can be treated in one way or another for disposal or reuse. For example the content of the sample holder could be poured through a filter in unit 142 to separate the carbon nanotubes from the liquid carrier and fluores- cent dyes. The separated dyes can then be transferred as schematically illustrated by the arrow 144 to a unit 146, since there is no reason why the same combination of dyes should not be used with further nanotube samples. The sample holder 44 would then be placed in the unit 148 which might, for example, contain a washing system for washing the sam- pie holder for reuse and for collecting the waste remnants of the fluorescent dyes for disposal. Alternatively, the sample tube 44 could be a disposal tube which is simply binned in the receptacle 150 for disposal as general industrial waste.

Claims

1. A method of establishing a quantitative and /or qualitative identification of the distribution of different types of nano structures and /or sub nanostructures in a sample, comprising the steps of

- adding at least first and second fluorescent agents to the sample

- irradiating the agents with light

- said structures absorb emitted light from the agent and emit new light - measuring the emission spectra of the sample and agents over a range of wavelengths characteristic for the different types of said structures and agents and

- analyzing the spectra to identify the distribution from the spectral responses.

2. A method in accordance with claim 1, wherein said nanostructures and sub-nanostructures can be one to several atom(s) and/ or compound^) in thickness, length and/or diameter, can be of organic and /or inorganic chemical nature and can be in polymer, oligomer and/ or monomer (supramolecular) format, for a non-exclusive example, such structures can be: carbon nanotubes (single or multi wall), nanowires, graphene, fullerene, nano-particles, quantum dots.

3. A method in accordance with claim 1 or claim 2, wherein said agents are chemical compounds that absorb and emit light, for a non-exclusive example, such agents can be fluorescent dyes, quantum dots and /or fluorescent nano- or sub-nano-particles.

4. A method in accordance with claim 1, wherein the first and second fluorescent agents - are added simultaneously or in mixed form to the sample

- are added sequentially to the sample.

- are added to respective like samples.

- can be chemically linked with and /or without energy transfer be- tween agents; therefore, either both agents act as one agent or both agents act as two agents.

5. A method in accordance with any one of the preceding claims, wherein the emission spectrum is measured in a first wavelength range for each said agent and in a second wavelength range characteristic for the emission spectra from the chiral forms of said structures in the sample.

6. A method in accordance with claim 5, wherein the second wave- length range is in the (near) infrared.

7. A method in accordance with any one of the preceding claims, wherein the sample is irradiated with first and second wavelengths respectively matched to said first and second agents.

8. A method in accordance with any one of the preceding claims, wherein the irradiation of the or each sample to produce fluorescent excitation takes place in ultraviolet or visible light ranges

9. A method in accordance with any one of the preceding claims, wherein the fluorescent agents are quantum dots selected to produce radiation at selected different wavelengths for inducing resonant energy transfer (Fόrster resonance energy transfer) to respective chiral forms of carbon nanotubes.

10. A method in accordance with any one of the preceding claims, wherein broad band radiation is used to excite fluorescent agent in selected wavelength ranges.

11. A method in accordance with any one of the preceding claims, wherein the sample is sequentially irradiated with different wavelengths.

12. A method in accordance with any one of the preceding claims 1 to 10, wherein the sample is simultaneously irradiated with different wavelengths.

13. A method in accordance with any one of the preceding claims, wherein peaks of the spectral response as measured with a spec- trometer are associated with different chiral types.

14. A method in accordance with any one of the preceding claims, wherein a plurality of fluorophores (FRET or non FRET format) are connected chemically.

15. A method in accordance with any one of the preceding claims, wherein the sample is observed with a microscope when irradiated to visualize and/or characterize and/or measure the said structures and/ or in a positioning method to localize a sample or a particular nanostructure or CNT in the sample.

16. An apparatus for establishing a distribution of different types of nanostructures and/or sub nanostructures in a sample, the apparatus comprising: - a means for adding at least first and second fluorescent agents to the sample

- at least one light source for irradiating the sample and the agents with light - a spectrometer for measuring the emission spectra of the sample and dyes over one or more ranges of wavelengths characteristic for the different types of said structures and agents and

- a computer analyzer for analyzing the spectra to identify the distribution from the spectral responses.

17. An apparatus in accordance with claim 16 and having a sample input unit.

18. An apparatus in accordance with claim 17 wherein said sample in- put unit comprises a rack with sample holders such as tubes where said sample is loaded.

19. An apparatus in accordance with any one of claims 16 to 18 and having a sample processing and preparation unit.

20. An apparatus in accordance with claim 19 wherein said sample processing and preparation unit comprises pipetting and dispensing facilities for at least one of the sample, a diluent and the fluorescent agents, a mixing facility for at least the samples and the fluorescent agents and optionally any diluent, and optionally an incubation facility such as at least one of a temperature controller, a timer, a heater and /or a cooler.

21. An apparatus in accordance with one of claims 16 to 20 and having a reading unit.

22. An apparatus in accordance with claim 21 wherein said reading unit comprises a mixture holder such as a cuvette, a substrate or a capillary, any of the foregoing mixture holders being either disposable or permanent, an excitation unit such as a light source a mono- chromator a filter or a condensing lens, and a detection unit such as a condensing lens, a filter a UV and/or IR and/or visible radiation detector, a camera or a diode array or other form of spectrometer.

23. An apparatus in accordance with any one of the claims 16 to 22 and having an output unit.

24. An apparatus in accordance with claim 23 wherein said output unit comprises a computer and associated software and optionally a dis- play and/ or printer.

25. An apparatus in accordance with one of claims 16 to 24 and having a regeneration washing and disposing unit.

26. A kit for establishing a distribution of different types of nanostruc- tures and/ or sub nanostructures in a sample, comprising at least some of the following items

- at least one substrate for carrying the sample

- a medium for fixing the sample to the substrate - at least first and second fluorescent agents, e.g. in the form of fluorescent dyes

- a washing medium

- a dye enhancer

- instructions for using the kit and - a software program for evaluation of the measurements.

27. A laboratory work station comprising the apparatus of any one of claims 16 to 25

- optionally at least one kit in accordance with claim 26 - a positioning system for positioning the sample on the substrate at the desired location.

28. A program carrier including a software program adapted to carry out the method of any one of claims 1 to 16 and/ or to operate the appa- ratus of any one of claims 17 to 26 and/ or to evaluate the measurements made by an associated spectrometer.

29. A program for analysis for use in conjunction with a method in accordance with claim 1 , said program including but not being limited to

- identifying the composition of said structures based on the emitted light from said structures that were excited by resonantly absorbing the emitted light from said fluorescent agents

- obtaining inverse relative ratio of the emission spectrum of said structures with the agents with respect to known calibrated emission spectrum of the agents, in different wavelength ranges characteristic of the agents, with or without integration.

30. A program in accordance with claim 29 including a method of differ- ential quantitative identification which can be performed in different forms in that it can be performed in liquid and non-liquid formats, the liquid format being one in which said structures are suspended in aqueous and/ or organic solvent with or without organic or nonorganic solubility enhancer (surfactant, detergent, chemical func- tionality, oligo and/ or poly nucleotide, peptide and/ or saccharine) for example in a pico, nano, micro and/ or millifluidic format, and for example being performed in a cuvette, test tube or capillary and the non-liquid format being one in which said structures can be smeared, dried, grown and/ or electrically trapped on a substrate surface.