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WO2007049264A2 - Essais biologiques contenant des particules - Google Patents

Essais biologiques contenant des particules Download PDF

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Publication number
WO2007049264A2
WO2007049264A2 PCT/IE2006/000123 IE2006000123W WO2007049264A2 WO 2007049264 A2 WO2007049264 A2 WO 2007049264A2 IE 2006000123 W IE2006000123 W IE 2006000123W WO 2007049264 A2 WO2007049264 A2 WO 2007049264A2
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Prior art keywords
nanoparticles
qds
buffered
fluorescence
size
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WO2007049264A3 (fr
Inventor
Yuri Volkov
Yury Rakovich
Iouri Kuzmich Gounko
John Dongegan
Dermot Kelleher
Siobhan Mitchell
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College of the Holy and Undivided Trinity of Queen Elizabeth near Dublin
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College of the Holy and Undivided Trinity of Queen Elizabeth near Dublin
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Priority to US12/084,239 priority Critical patent/US20090197291A1/en
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Publication of WO2007049264A3 publication Critical patent/WO2007049264A3/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to the use of nanoparticles in detection and quantification assays.
  • QDs quantum dots
  • colloidal semiconductor nanocrystals are robust particles of size and composition tunable emission. They exhibit wide absorption profiles allowing excitation of various QDs simultaneously, narrow emission spectra and excellent photo stability (Mattoussi et ah, 2002; Michalet et ah, 2001; Chan et al., 2002), making them potentially readily traceable in the cells and tissues of the living organisms.
  • QDs display dimensional similarities to biomolecules permitting their bioconjuagtion and use as sensors.
  • QD studies have been performed primarily using CdSe particles.
  • Early attempts at labelling cells included adding transferrin-QD bioconjugates to HeLa cells thereby allowing receptor-mediated endocytosis (Chan et al., 1998).
  • the avidin-biotin system was employed to label F actin filaments where biotinylated CdSe nanocrystals were used to label fibroblasts incubated in phalloidin-biotin and streptavidin (Bruchez et al., 1998).
  • CdSe-CdS nanocrystals coated with trimethoxysilylpropyl urea and acetate were found to bind with high affinity in the cell nucleus (Bruchez et ah, 1998).
  • CdSe QDs have also been used in metastatic assessment as markers for phagokinetic tracks (Parak et ah, 2002). The first reports of in vivo use show QD-peptide conjugates targeting tumor vasculature (Akerman et al., 2002). Later studies using ZnS coated CdSe QDs encapsulated in PEG micelles show DNA binding and successful microinjection into Xenopus embryos (Dubertret et al., 2002).
  • Radioactive markers such as nucleic acids labelled with 32 P or 35 S and proteins labelled with 35 S or 125 I to detect biological molecules. These labels are effective because of the high degree of sensitivity for the detection of radioactivity.
  • radioisotopes many basic difficulties exist with the use of radioisotopes.
  • compounds such as ethidium bromide, propidium iodide, Hoechst dyes (e.g. benzoxanthene yellow) interact with DNA and fluoresce to visualize DNA.
  • Other biological components can be visualized by fluorescence using such techniques as immunofluorescent microscopy, which utilizes antibodies labelled with a fluorescent tag and recognizing particular cellular target.
  • immunofluorescent microscopy which utilizes antibodies labelled with a fluorescent tag and recognizing particular cellular target.
  • "secondary" polyclonal (rabbit- or goat-anti-mouse) antibodies tagged with fluorescein or rhodamine enable one to visualize "primary" monoclonal antibodies (typically raised in mice or respective hybridoma cells) bound to specific cellular targets.
  • the invention is directed to providing a solution to this problem.
  • Fluorescent dyes also have applications in non-cellular biological systems. For example, the advent of fluorescently-labelled nucleotides has facilitated the development of new methods of high-throughput DNA sequencing and DNA fragment analysis (ABI system; Perkin-Elmer, Norwalk, Conn.).
  • ABSI system Perkin-Elmer, Norwalk, Conn.
  • organic fluorescent dyes One of these limitations is the variation of excitation wavelengths of different coloured dyes.
  • simultaneously using two or more fluorescent tags with different excitation wavelengths requires multiple excitation light sources. This requirement thus adds to the cost and complexity of methods utilising multiple fluorescent dyes.
  • Another drawback when using organic dyes is the deterioration of fluorescence intensity upon prolonged exposure to excitation light.
  • CdSe/ZnS nanocrystals as fluorescent labels for multiphoton microscopy was recently demonstrated by Larson et al (2003). Although the authors visualized quantum dots dynamically through the skin of living mice, this method is of limited usefulness because high pumping intensity is a critical requirement to achieve efficient multiphonon assisted excitation of nanocrystal luminescence.
  • a direct method for conjugating protein molecules to luminescent CdSe-ZnS core-shell nanocrystals was described by Mattoussi et al (2000) and later by Goldman et al (2002). These bioconjugates have been proposed as bioactive fluorescent probes in sensing, imaging, immunoassay and other diagnostic applications.
  • bioconjugates are of relatively large size (30-45 run in diameter) and had a quite limited solubility in water. As result these nanocomposites have only limited capability to penetrate through the cell membrane and can not be used very effectively for intracellular diagnostics.
  • water-soluble CdTe, Cd x Hg (1-X) Te and HgTe nanocrystals have been proposed for biolabeling of biocompatible polymers. In this work the nanocrystals were encapsulated into the polymer with the formation of microcapsules, which have been suggested as potential materials for monitoring the drug delivery process (Gaponik et al, 2003).
  • the initial CdTe or HgTe nanocrystals demonstrated good water solubility and were of small size (4-6 nm) the final composites with the biopolymer were of several micron sizes and were too large to be used for intracellular drug delivery and diagnostics.
  • the invention is directed towards solving at least some of the problems with known systems.
  • the nanoparticles are less than 100 nm in size.
  • the buffered solution comprises an inorganic buffer solution.
  • the buffered solution comprises a phosphate-based buffer solution. In another embodiment the buffered solution comprises a tris-borate based buffer solution.
  • the buffered solution is prepared in water.
  • the macromolecule may be a protein, glycoprotein a peptide.
  • the difference between the buffered nanoparticles in the presence and absence of the test sample is measured by fluorescence.
  • the difference between the buffered nanoparticles in the presence and absence of the test sample is measured by fluorescence intensity and fluorescence life time imaging (FLIM).
  • the difference between the buffered nanoparticles in the presence and absence of the test sample is measured by estimation of the turbidity of the solution containing the nanoparticles.
  • the test sample may be selected from any one or more of blood, sputum, urine, lavage fluid, biopsy material, tissue sample, cultured or primary isolated cells.
  • the nanoparticles comprise a chemically attached entity.
  • the nanoparticles comprise an entity which has been chemically modified.
  • the invention also provides a method for promoting electrical stimulation or conductivity comprising:- providing nanoparticles in the form of nano-wires;
  • the nanoparticles are less than 20nm in size.
  • the difference in conductivity may be measured using fluorescence imaging.
  • the invention provides a method for identifying a target compound useful in the preparation of a medicament for the treatment and/or prophylaxis of a disease state which involves a loss or change in electrical conductivity.
  • the disease state is selected from any one or more of spinal cord injuries, neuron and nerve damage, multiple sclerosis or any other neurodegenerative disease.
  • the invention further provides a method for determining intracellular transport and functional response in a cell comprising the steps of:-
  • the nanoparticles are less than 20nm in size. In another embodiment of the invention the nanoparticle is associated with a biologically active entity.
  • the nanoparticle comprises a chemically attached entity.
  • the method discriminates between the cytosolic and nuclear compartments of a cell.
  • the nanoparticles are up to 20nm in size.
  • the nanoparticles may be up to lOnm in size up to 5nm in size or up to 3nm in size.
  • the nanoparticles are water soluble.
  • the nanoparticles are lipid soluble.
  • the nanoparticles comprises II- VI colloidal nanoparticles.
  • the nanoparticles are CdTe nanoparticles. In another embodiment the nanoparticles are CdSe nanoparticles.
  • Fig. 1 is an example of the Cellomics Kineticscan View screen showing Nuclear to Cytoplasmic fluorescence intensity ratio in AGS cells accumulating CdTe QDs (CircRingAvglntenRatio) in the Compartmental Analysis Bioapplication.
  • Upper window Nuc/Cyt intensity ratio in each individual cell in the well; middle panel, fluorescence detected in blue, green, red channels and composite image (left to right).
  • Lower panel numerical data from cells and outlined nuclear and cytoplasmic areas of the cell included in analysis.
  • Fig. 2 is a graph showing the Nuc/Cyt fluorescence distribution in fixed and f ⁇ xed/permeabilised cultured epithelial cells (small dashed and large dashed lines, respectively). QDs size is increasing from left to right (experimental points 2-6).
  • Fig. 2A Fig. 2B and 2C show the intracellular fluorescence of green emitting QDs in permeabilised (B) and non-permeabilised cells (C).
  • Fig. 3 is a 96-well plate containing solutions of CdTe nanocrystals in water, PBS, PBS without Ca and Mg ions or culture medium with different amounts of bovine serum albumin (BSA) protein.
  • the protein concentration of A is Omg/ml BSA
  • B is 2mg/ml
  • C is lmg/ml
  • E is O.lmg/ml
  • F is 0.05mg/ml
  • G is 0.01mg/ml.
  • Fig. 3A shows the 96-well plate illuminated with light illumination
  • Fig. 3B shows the 96-well plate with UV lamp illumination.
  • Fig. 4 shows the PL intensity decays of a solution of CdTe in water with two different amount of BSA, Omg/ml (a) and 2mg/ml (B). Results of three- exponential analysis of decay curves are shown by the thick black lines with corresponding residuals (b) and (c). Insets show images of luminescence lifetime distribution obtained by FLIM technique scanning the sample across the square of 80x80 ⁇ m size.
  • Fig. 5 shows the PL intensity decays of a solution of CdTe in PBS with two different amount of BSA, Omg/ml (a) and 2mg/ml (B). Results of three- exponential analysis of decay curves are shown by the thick black lines with corresponding residuals (b) and (c). The insets are as described in Fig. 4.
  • Fig. 6 shows the PL intensity decays of a solution of CdTe in PBS-without Ca and Mg ions, with two different amount of BSA, Omg/ml (a) and 2mg/ml (B). Results of three-exponential analysis of decay curves are shown by the thick black lines with corresponding residuals (b) and (c). The insets are as described in Fig. 4.
  • Fig. 7 shows the PL intensity decays of a solution of CdTe in medium with two different amounts of BSA, Omg/ml (a) and 2mg/ml (B). Results of three- exponential analysis of decay curves are shown by the thick black lines with corresponding residuals (b) and (c). The insets are as described in Fig. 4.
  • Fig. 8 is a bar chart showing the dependence of integrated PL intensity (upper panels) and values of averaged lifetimes ⁇ av (bottom panels) on concentration of BSA protein.
  • the CdTe are in water.
  • Fig. 9 is a bar chart showing the dependence of integrated PL intensity (upper panels) and values of averaged lifetimes ⁇ av (bottom panels) on concentration of BSA protein.
  • the CdTe are in PBS.
  • Fig. 10 is a bar chart showing the dependence of integrated PL intensity (upper panels) and values of averaged lifetimes ⁇ av (bottom panels) on concentration of BSA protein.
  • the CdTe are in PBS without Ca and Mg ions.
  • Fig. 11 is a bar chart showing the dependence of integrated PL intensity (upper panels) and values of averaged lifetimes ⁇ av (bottom panels) on concentration of BSA protein.
  • the CdTe are in medium.
  • Fig. 12 shows the synthesis of NPX-PEG-NH 2
  • Fig. 13 shows an agarose gel electrophoresis of TGA QD's (sb 105-2(1)) mixed with EDC (lane 2) and increasing amounts of EDC and a 2 fold excess Of NPX-PEG-NH 2 (lane 3, 4 and 5). UV filter at 516nm and 12s exposure time;
  • Fig. 14 is a FLIM lifetime image of cells
  • Figs. 15a and 15b are PL lifetime histograms obtained from regions A(a) and B(b) of Fig. 1 respectively;
  • Fig. 16 are images of a compartmental analysis
  • Fig. 17 are fluorescent intensities registered by high content screening method in THP-I cells fixed with para-formaldehyde (PFA) permebilised with TritonX 100 and exposed to QDs of increasing sizes.
  • PFA para-formaldehyde
  • Fig. 18 are fluorescent intensities registered by high content screening method in Hep2 cell line fixed with para-formaldehyde (PFA) permebilised with TritonX 100 and exposed to QDs of increasing sizes.
  • PFA para-formaldehyde
  • Fig. 19 illustrates cellular distribution in prefixed Hep2 cells
  • Fig. 20 illustrates cellular distribution in prefixed THP-I cells
  • Fig. 21 illustrates distribution of fluorescent intensities in glutaraldehyde THP-I cells exposed to QDs with different surface charge.
  • Fig.22 illustrates high power magnification images of THP-I of fixed with glutaraldehyde cells and exposed to QDs with 5% positive surface charge.
  • Fig. 23 illustrates background fluorescence levels in THP-I cells after glutaraldehyde fixation registered in different emission channels.
  • Fig. 24 illustrates cellular distribution in live THP-I cells exposed to QDs with different charge.
  • Fig. 25 illustrates cellular distribution in live THP-I cells exposed to QDs with 5 % positive charge.
  • Fig. 26a to 26d illustrates high power magnification images of live THP-I cells exposed to QDs with 5 % positive (a), 100% negative (b), 50% negative (c) and 10% (d) positive charge.
  • Fig 27 is an image of a dot blot illustrating CdTe QDs binding to BSA, DNA, RNA, purified histones and nuclear extract (A-E, respectively). 4 ⁇ l amounts of QDs were applied on the nitrocellulose membranes with pre- bound nucleic acids and proteins.
  • Fig. 28 is an image of a dot blot illustrating CdTe QDs binding to BSA, DNA, RNA, purified histones and nuclear extract (A-E, respectively). 8 ⁇ l amounts of QDs were applied on the nitrocellulose membranes with pre- bound nucleic acids and proteins.
  • Figs 29 to 34 are graphs illustrating the effect of varying protein concentration on quantum dots.
  • Fig. 35 is a fluorescent lifetime decay curve of quantum dots in tris-borate. Detailed description of the invention
  • Living cell refers to the self-replicating biological structure enclosed by an outer membrane and containing cytoplasm, organelles and nucleic acids (i.e. viruses, prokaryotic bacterial cells, protozoa and eukaryotic cells of higher species and multicellular organisms).
  • nucleic acids i.e. viruses, prokaryotic bacterial cells, protozoa and eukaryotic cells of higher species and multicellular organisms.
  • II-VI colloidal quantum dots - are semiconductor nanoparticles of II- VI compounds prepared as a colloidal solution with size-dependent optical and electronic properties.
  • Optical illuminators/emitters any source of ultraviolet, visible or infrared light and combinations thereof
  • QDs Quantum Dots
  • the QDs of the invention offer a method a quantification using an unlimited range of emission wavelengths. This ability has been exploited over a range of applications.
  • the QDs used in the invention are as described in detail in PCT/IE2005/000047 the entire contents of which are herein incorporated by reference.
  • CdTe nanocrystals capped with thioglycolic acid used in the experiments were synthesized in aqueous medium as reported earlier (Gaponik et al, 2002). Briefly, demineralised aqueous solutions containing Cd(C10 4 ) 2 *6H 2 O and a stabilizer (thioglycolic acid , TGA) at pH 11.8 were treated by H 2 Te gas, which was generated by the reaction of Al 2 Te 3 lumps with 0.5 M H 2 SO 4 under nitrogen. The mixture of was then heated under reflux under open-air conditions. This method enabled us to prepare good quality CdTe nanocrystals with a narrow ( ⁇ 10 %) size distribution.
  • Variation of the temperature and the duration of the heating during the preparation of CdTe nanocrystals determines the final size of the nanocrystals and as a result the colour and luminescence maximum of the solution.
  • green (with photoluminescence maximum at 563 nm) CdTe nanoparticles were produced after 15 min of heating under reflux, while red (with photoluminescence maximum at 602 nm) CdTe colloid solution were produced after 24 hours of heating.
  • nanoparticles which can be used in relation to the invention may comprise semi conductor nanoparticles.
  • III- VI semiconductor nanoparticles ZnO, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS,
  • III-V semiconductor nanoparticles AlN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb.
  • Group IV semiconductor nanoparticles Si, Ge, Si 1-x Ge x
  • nanoparticles include SiO 2 (silica), any transition metal oxide (e.g. TiO 2 , ZrO 2 , HfO 2 , MoO 2 , Fe 2 O 3 , Fe 3 O 4 , Co 3 O 4 , ferrites), siloxane nanoparticles, dendrimers (dendritic polymers) and organic polymer nanoparticles.
  • transition metal oxide e.g. TiO 2 , ZrO 2 , HfO 2 , MoO 2 , Fe 2 O 3 , Fe 3 O 4 , Co 3 O 4 , ferrites
  • siloxane nanoparticles e.g. TiO 2 , ZrO 2 , HfO 2 , MoO 2 , Fe 2 O 3 , Fe 3 O 4 , Co 3 O 4 , ferrites
  • siloxane nanoparticles e.g. TiO 2 , ZrO 2 , HfO 2 , MoO 2 , Fe 2 O 3 , Fe 3 O 4 , Co 3
  • Fluorescence-emitting semiconductor nanocrystals have currently become a target of intensive efforts of scientists worldwide as promising material permitting generation of multi-colour labels suitable for industrial and biomedical applications.
  • QDs quantum dots
  • the critical requirements which need to be met are water-solubility, biocompatibility and selective functionalisation of nanoparticles (addition of the desired chemical groups, peptides, proteins or complex molecules) enabling the adaptation of QDs for specific uses.
  • Small QDs as represented by CdTe nanoparticles possess several unique features making them usable for a variety of biomedical purposes. These include QDs application as dyes for multi-colour intracellular contrasting imaging, fluorescent detector systems responding to changes in protein-rich environment and QDs ability to serve as building blocks for formation of complex lattices of two- and three- dimensional nature. Conjugates of medicinal drugs with small non-shell coated nanoparticles can be utilised for improved targeted compound delivery into cells.
  • Fig. 2 shows an example carried out in cultured epithelial cells. As seen from the Fig. 2(A), the average Nuc/Cyt fluorescence ratios were significantly higher in permeabilised cells (small dashed line ) compared to non-permeabilised ones (large dashed line ⁇ ) when the QDs of apparent particle size of less than 4 nm were used (points 2-4).
  • Fluorescent Lifetime imaging method enables to detect interaction of QDs with target structures by registering changes in fluorescence emitting properties of QDs. Significant changes in fluorescence lifetimes may serve as an indicator of strong interaction of QDs with certain molecules or subcellular structures.
  • Fluorescence lifetime images were collected with the FLIM system (Microtime200 time-resolved confocal microscope system, PicoQuant) equipped with Olympus LX71 inverted microscope.
  • the samples were excited by 480 nm picosecond pulses generated by a PicoQuant, LDH-480 laser head contolled by a PDL-800B driver.
  • the setup was operated at a 20-MHz repetition rate with an overall time resolution of - 150 psec.
  • Lifetime maps were calculated on a pixel-by-pixel basis by fitting the lifetime of each pixel to the logarithm of the intensity and the FLIM system response was negligible compared with typical lifetimes of the quantum dots.
  • FLIM images maps of two-dimensional in-plane variations of the PL decay times measured in micro-PL setup. In this case every pixel in the lifetime image gives the lifetime at particular position in space (x,y).
  • FIG. 14 Fluorescence lifetime image of cells. The image was collected at 300x300 pixel resolution with 4096 time channels; 2 ms acquisition time was provided per pixel and total recording time was 8.95 min. Image size: 27.2 ⁇ m x 27.2 ⁇ m. Every pixel in the lifetime image (a) gives the lifetime at that particular position in space b ⁇
  • the lifetime image clearly demonstrates distribution of emitting species over cell cytoplasm, showing the longest PL decay time at the rim of cell (Fig.14, region A) as compared to the region of nucleus (Fig.14, region A).
  • lifetime distributions consist of two maximums centered at 0.8 and 2.4 ns for region A and 0.8 and 4.5 ns in the region B. Comparing lifetime histogram obtained from different intracellular regions it is amply clear that drastic reduction of long-lived component is observed in region A.
  • Two-peak structure shown in Figure 15 implies that at least two different decay processes are involved in nonradiative decay. The shorter lifetime can be attributed to the intrinsic recombination of initially populated electronic states in the core of quantum dots.
  • the High Content screening and analysis systems enable to perform user- independent evaluation of the uptake and intracellular distribution of a large variety of fluorescent labels in the cells at individual and population level in multi-well format at a speed of up to several plates per hour. Thsee systems perform an automated analysis of the registered events storing both the images of each individual cell and providing the full quantitative analysis of the overall population dynamics, including below-average responses.
  • Appropriately designed fluorescent QDs with selective specificity and emission colour can be suitable for targeted visualisation of cellular organelles and multi-parametric analysis of cell population responses by means of high content analysis.
  • HEp-2 epithelial cell line grown in minimum essential medium (Eagle) with Earles Salts, 10% Foetal Calf Serum, 2mM L-glutamine
  • Thpl monocytic cell line ECACC, Salisbury, England
  • ECACC fetal bovine serum
  • lOO ⁇ g/mL penicillin lOOmg/mL streptomycin
  • RPMI 1640 media RPMI 1640 media They were seeded out onto 96 well microtitre plates and onto coverslip slides at a concentration 2x105 cells/mL.
  • the HEp-2 cells were incubated for 24 hrs, and the Thpl cells, cultured with lOOng/mL PMA (to enable monocyte to macrophage differentiation), for 72 hours, both in controlled atmospheric conditions of 37°C, 5% CO2.
  • Prefixed cells were washed twice in PBS, treated with 3% paraformaldehyde for 30 minutes, washed again and then permeabilised with 1.5% Triton XlOO for 15minutes. The plates were washed twice with PBS and 200 ⁇ L of PBS added. The plates were then sealed with parafilm and kept @ 4°C until required. Cells were also prepared for live analyses as above; Thpl cells seeded into a 96 well plate and HEp- 2 cells into an 8 chamber coverslip slide (LabTech). Preparation of Quantum Dots
  • QD quantum dots
  • the PBS was replaced by lOO ⁇ L of media, and lOO ⁇ L of diluted QDs were added.
  • the cells were incubated for one hour, washed in media, stained with l ⁇ g/ ⁇ L
  • Half of the media (lOO ⁇ L/well from the 96 well plate; 200 ⁇ L/well from the 8 well plate) was replaced with the diluted QDs (charged particles only) and incubated for 1 to 3hrs.
  • the THP-I cells were examined under the fluorescence microscope at 30 minutes, 1 hour and 3 hours. At 1 hour and at 3 hours, the cells were counterstained with Hoescht and fixed with 1% gluteraldehyde. This part of the experiment had to be repeated, with 3% paraformaldehyde used as a fixative instead of 1% gluteraldehyde.
  • the size of the nanoparticle relates not only to where it locates within the cell but also at what wavelength it fluoresces.
  • the particles added to cells that had been previously fixed in paraformaldehyde and permeabilised with TritonXlOO.
  • the smaller sized particles went into the nuclei and emitted within the green wavelengths ( ⁇ 542.5nm), while the larger particles remained in the cytoplasm and emitted within the red wavelength ( ⁇ 562nm, ⁇ 572nm, ⁇ 582nm).
  • the exception to these were the particles ⁇ 521nm and ⁇ 572nm, these showed no affinity for the cells at all. This was probably due to the modifications of these particular particles which also were negatively charged (Fig. 17 and Fig. 18).
  • the particle ⁇ 550nm showed strong fluorescence in both channels but at different locations, the rim of the epitheliod cell line (HEp-2 cell) staining red while the cytoplasm stained green (Fig. 18).
  • the 5%+QDs stained the nucleoli (red channel) in the Thpl cells and also seem to accumulate at the nuclear rim (green channel). When examined under UV light there was also QDs in the cytoplasm. After fixing the THP-I cells with 1% gluteraldehyde and counterstained with Hoescht, they were analysed on the Cellomics KineticScan. The 5%+QDs stained twice as strongly as the other dots in both channel 2 and channel 3. Interestingly, all the positively charged dots and the neutral QDs show fluoresecence in the nucleoli and at the nuclear rim (Fig. 21), especially the 5%+QDs (Fig. 22). No staining was noted with the negatively charged QDs.
  • QDs size of QDs affects where they locate within the cells. It is also important for the emitting wavelength. Conditions within the cells also affect the wavelength as can be seen when one part of the cell fluoresces green, yet another part fluoresces red.
  • the QDs charge affects also how easily the cell will actively take up the QDs, the positively charged cells being more "appetising" than the negatively charged QDs.
  • the positively charged QDs also seemed to be aiming for the nucleus, and getting into the nucleoli. Fixation is an important aspect of QD staining. We have shown that 1% gluteraldehyde enhanced the pattern already seen in the live cells.
  • Quantitative protein determination in complex solutions represents a routine task of most biochemical, immunological and general cell biology laboratories. To date, the choice of these methods is limited to traditional Bradford, Lowry methods or similar and their modifications, all of which are largely based of the formation of protein/reagent complexes providing a colorimetrically detectable reaction product.
  • the readout is subsequently performed as light absorption measurement at a specific wavelength.
  • PL decays were measured using time-correlated single photon counting (Time-Harp, Microtime 2000, Picoquant). The samples were excited by 480nm picosecond pulses generated by Picoquant. LDH-480 laser head controlled by PDL-800B driver. The set-up was operated at a 20MHz repetition rate with an overall time resolution of ⁇ 150psec. Decays were measured at 60000-80000 counts in the peak and reconvoluted using non-linear least squares analysis (FluoFit, PicoQuant) using an equation of the form:
  • the pre-exponential factors ⁇ were taken into account by normalisation of the initial point in the decay to unity.
  • the quality of fit was judged in terms of ⁇ 2 value (with a criteria of less than 1.1 for an acceptable fit) and weighted residuals (Fig 2-5 (b) (c))
  • the Tj and cq parameters were used then to calculate the average lifetime
  • the method may be used for the quantitative determination of other molecules possessing QDs-stabilizing properties in solutions using specifically chemically modified QDs.
  • the method may also be used for quantitative evaluation of the presence of proteins with different properties using QDs with targeted chemical modifications.
  • CdTe green emitting cadmium telluride
  • TGA green emitting cadmium telluride
  • Fluorescent lifetime data was collected with the FLIM system (Microtime200 timeresolved confocal microscope system, PicoQuant) equipped with Olympus 1X71 inverted microscope.
  • the samples were excited by 480 nm picosecond pulses generated by a PicoQuant, LDH-480 laser head contolled by a PDL-800B driver.
  • the setup was operated at a 20-MHz repetition rate with an overall time resolution of - 150 psec. Plate read outs were carried out using the SPECTRAFluor Plus system (Tecan).
  • SPECTRAFluor Plus system Tecan
  • Nitrocellulose was used to bind the biopolymer samples, BSA, DNA, RNA, core histones and the nuclear lysate samples. Each sample was diluted to a concentration of lmg/ml using de-ionised water. 2ug of each sample was then placed on the nitrocellulose ( Figure 27, Figure 28). The quantum dots were used as received and diluted to one in a hundred using deionised water. The nitrocellulose was then "flooded" with the QD solution and incubated at 37 0 C for ⁇ 45mins. The nitrocellulose was then washed rigorously twice using deionised water and kept moist at all times thereafter as the QD 's deteriorate when they are allowed to dry out. The nitrocellulose was then imaged using the trans-illuminator.
  • Table X illustrates the different fluorescent lifetime decays obtained for the QD' s when mixed with the core histones or DNA at various concentrations. For example at a concentration of O.lmg/ml, the QD's have lifetime 9.6ns longer than that of the QD's in the histones.
  • RNA also showed only to have an impact on the luminescence of the QD's at the very highest concentration, where a quenching effect was observed. [FLIM of whole cells shows a dramatic reduction in the lifetime of the QD's in the nucleus and nucleolus. The quenching effect of the RNA at high concentrations observed above may be a contributing factor. Plate Reader Results
  • Quantitative protein determination in complex solutions represents a routine task of most biochemical, immunological and general cell biology laboratories. To date, the choice of these methods is limited to traditional Bradford, Lowry methods or similar and their modifications, all of which are largely based of the formation of protein/reagent complexes providing a colorimetrically detectable reaction product. The readout is subsequently performed as light absorption measurement at a specific wavelength.
  • We hereby suggest a system for protein quantification which is based on a different principle, exploiting specific destabilization of QDs solutions in the presence of physiological buffers.
  • protein plays the role of a stabilising agent, maintaining QDs in fluorescence-emitting suspension. The higher the concentration of protein, the higher is the stability of the solution and hence the intensity of the fluorescent signal.
  • the principle of quantitative stabilisation of QDs by protein solutions in the presence of opposite-acting destabilizing buffer holds true to the wide spectrum of CdTe quantum dots and therefore could be used in the fluorimetric systems working in a desired wavelength interval..
  • Figure 29 to Figure 34 show the effect that varying the protein concentration has on the QD' s.
  • FIGs 31 to figures 34 there is an increase in the RFU onbserved for all of the buffers with the exception of the sharp peaks with ELISA and HEPES. This work is still under investigation and is to be repeated a number of times. From here it is expected to then concentrate on a particular buffer and protein concentration and vary the type of Qd's used.
  • the fluorescent lifetime of the QD's is a measure of the average lifetime that the QD remains in an excited state before returning to the ground state.
  • the fluorescent lifetime decay ( ⁇ l/e), was calculated using the following equations:(See Figure 35 also).
  • the shortest lifetime of the QD is in tris borate ( ⁇ 3ns), hepes, tris and elisa share similar lifetimes of ⁇ 15ns, and PBS has a lifetime of ⁇ 1 Ins.
  • the nervous system in the human body is made up of billions of nerve cells, or neurons, organized in various networks. The majority of these neurons are located in the brain, brain stem and spinal cord, which constitute the so-called central nervous system (CNS).
  • CNS central nervous system
  • This network of interconnected neurons distributes messages as electrical impulses between the body and the brain. Messages that are received by the brain include sensory impulses that inform the brain about, for example, heat, pain or location of a part of the body. Conversely, messages are also sent by the brain to different parts of the body in order to elicit a muscle contraction that, for example, moves the hand from a burning flame.
  • the synaptic cleft Between adjacent neurons, there is a microscopic gap called the synaptic cleft. However small, the electrical signal carrying a message cannot bridge the synaptic cleft as it is. The solution to this is the synapse, an elegant way of bridging the gap chemically.
  • the electrical impulse triggers the release of certain chemical substances into the gap. These substances are called neurotransmitters and are carried over the small synaptic cleft by diffusion. Once on the other side of the cleft, the neurotransmitters bind to certain proteins, called receptors, that are attached to the cell surface of the receiving cell. The binding of the transmitter to the receptor leads to the generation of a new electrical impulse.
  • the intensity and strength of the electrical impulse will decide which neurotransmitter to be released.
  • Several medical disorders are caused by the dysfunction of neurotransmission in the central nervous system such as spinal cord injuries, neuron and nerve damage.
  • nano-wires are produced in cell-damaging toxic reagents.
  • the ability to grow straight and branching nano-wires in a physiological solution is an advantage.
  • Their use as conductors in this complex cell system using a network of nano-wires as a multi dimensional signalling structure may be of therapeutic value as electrical conductivity is a familiar feature for example, of multiple sclerosis.
  • nano-wires have an inherent ability to conduct electricity. Once a protein or a matrix or a firing neuron is present the ability to conduct along the wire is different. The conductivity of the nano wires are examined by patterning a surface with a matrix and then analysing the conductivity/ fluorescence intensity along the wire (between two electrodes or measuring life time fluorescence imaging). Drag conjugates
  • TGA (thioglycolic acid) stabilised quantum dots were prepared according to the published procedure (Gaponik 2002). The concentration of purified TGA-QD 's solution were determined by mean of UV-absorption and PL emission as described in Yu, W.W. et al.
  • EDC l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide
  • precipitated formulations are purified by centrifugation and removal of supernatant. The operation is repeated until disappearance of free drags in supernatant confirmed by UV-vis absorption.
  • Non-precipitated formulations are purified by gel exclusion chromatography over a G-25 column equilibrated in deionized water or phosphate buffer. All formulations are finally filtered over 0.2 ⁇ m filters.
  • the drug coating on the nanoparticles is assayed by various techniques.
  • UV-PL spectra of conjugates may show a shift in absorption or emission peak.
  • the lifetime of the conjugated nanoparticles is compared with starting nanocrystal material.
  • Goldman ER Anderson GP, Tran PT, Mattoussi H, Charles PT and Mauro JM. Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunosays, Anal. Chem.2002, 74, 841. Jaiswal JK, Mattoussi H, Mauro JM, Simon SM. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nature Biotechnol. 2003, 21, 47.
  • Peng ZA Peng X. Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor. J. Am. Chem. Soc. 2001, 123, 183.

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Abstract

L'invention concerne un procédé pour déterminer de manière quantitative et qualitative la présence d'une macromolécule comprenant la création de nanoparticules dans une solution tamponnée, l'ajout d'un échantillon test à la solution de nanoparticules tamponnées, et la mesure de la différence entre les nanoparticules tamponnées en présence ou en l'absence de l'échantillon test. Les nanoparticules présentent une taille inférieure à 100 nm.
PCT/IE2006/000123 2005-10-27 2006-10-27 Essais biologiques contenant des particules Ceased WO2007049264A2 (fr)

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