Patent Application: US-8423906-A

Abstract:
a method for quantitatively and qualitatively determining the presence of a macromolecule comprises providing nanoparticles in a buffered solution , adding a test sample to the buffered nanoparticle solution , and measuring the difference between the buffered nanoparticles in the presence and absence of the test sample . the nanoparticles are preferably less than 100 nm in size .

Description:
living cell ( 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 ). 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 . chemical or physical linking — bond via covalent , noncovalent , hydrophobic , hydrophilic , electrostatic , van der waals , hydrogen bonding , magnetic or electromagnetic interactions . we have found quantum dots ( qds ) to be very useful in a number of applications including use 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 . 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 . the invention will be more clearly understood by the following examples thereof . 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 ( clo 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 am 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 (& lt ; 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 . thus green ( with photoluminescence maximum at 563 μm ) 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 . we have utilised water - soluble thioglycolic capped cdte nanoparticles of varying sizes for selective nuclear and nucleolar localisation of green cdte qds and cytoplasmic compartmentalisation of red qds , dependent on size and surface chemistry . the cdte nanoparticles showed limited cytotoxicity and proved to be suitable for biological systems . other non limiting examples of nanoparticles which can be used in relation to the invention may comprise semi conductor nanoparticles . ii - vi semiconductor nanoparticles : zno , zns , znse , znte , cds , cdse , cdte , hgs , hgse , hgte . iii - v semiconductor nanoparticles : aln , alp , alas , alsb , gan , gap , gaas , gasb , inn , inp , inas , insb . other possible 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 . two aqueous colloidal solutions of cdte nanocrystals of 2 - 5 nm mean size were used for studies in biological systems . fluorescence - emitting semiconductor nanocrystals ( quantum dots , qds ) 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 . for biomedical purposes in particular , 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 . the fulfillment of the above requirements commonly involves creation of core / shell structures , selection of adequate stabilisers and incorporation of adaptor or linker molecules enabling qds to be physically conjugated with the functionalising molecules of interest . this inevitably leads to the generation of structures typically in the order of 15 - 25 nm in diameter for the stabilised qds per se or larger , depending on the type of molecules used for subsequent conjugation with qds surface . such structures , although possessing a number of valuable characteristics for specific utilization , have intrinsic limitations for penetration into compartments smaller than their physical size thereby precluding their use for targeting more spatially confined environments , e . g . intracellular organelles and nucleus . small qds as represented by cdte nanoparticles ( devoid of enlarging shell ) 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 . a significant number of biological assays designed to characterise intracellular transport and functional responses , such as for example , signalling cascades largely exploit molecular translocation events occurring both within the cytoplasm and between cytosol and the nucleus . therefore there is a growing demand in research institutions and pharmaceutical companies worldwide for the availability of multi - colour photo - stable dyes discriminating between cytosolic and nuclear compartments . contemporary systems of high content screening , in particular are capable of analysing multiplexed experimental setups utilizing a variety of fluorescence - emitting reporters . however , the choice of reliably performing dyes currently on the market is limited to organic dyes with a limited choice of emission wavelengths , e . g . green - emitting calcein and fluorescein derivatives for cytosolic visualisation , blue dapi and hoechst range and far - red - emitting draq - 5 for nuclear imaging . these either commonly overlap in their emission spectra with a multitude of other popular labels ( alexa , fitc , tritc etc .) or represent dna - binding agents emitting in a short - range spectrum ( uv ) causing significant bleaching of other labels in multi - colour systems and photodamage in live cell studies . we applied a panel of cdte quantum dots of different sizes to the routinely maintained macrophage - like cell line tbp - 1 and epithelial cell line ags in a 96 - well plate format suitable for testing on the high content screening cellomics ® kineticscan workstation . the system enables user - independent evaluation of the uptake and intracellular distribution of a large variety of fluorescent labels in the cells at individual and population level . the system performs an automated analysis of the registered events storing both the images of each individual cell and providing the fill quantitative analysis of the overall population dynamics . similar systems are currently in use for carrying out large - scale screening of potential therapeutic compounds . a typical experimental acquisition screen of the kineticscan view optimised for the work with qds in cell systems is given on fig1 . experiments were carried out using fixed and fixed / permeabilised cells thus largely eliminating the influence of other qds localisation factors apart from size selectivity , ( such as pino - and phagocytic vesicle formation , nuclear pore activity and cytoskeleton - dependent transport mechanisms ). fig2 shows an example carried out in cultured epithelial cells . as seen from the fig2 ( a ) , the average nuc / cyt fluorescence ratios were significantly higher in permeabilised cells ( small dashed line ) compared to non - permeabilised ones ( large dashed line δ ) when the qds of apparent particle size of less than 4 nm were used ( points 2 - 4 ). the use of qds with the estimated size of near 4 nm and over ( emitting orange / red and red fluorescence ) eliminated this difference ( points 5 and 6 on the graph ). these results demonstrate the possibility of utilization of cdte qds for a selective multi - colour dyes permitting to visualise discrete subcellular compartments . in this case , fluorescent lifetime imaging method ( slim ) 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 ix71 inverted microscope . the samples were excited by 480 nm picosecond pulses generated by a picoquant , ldh - 480 laser head controlled 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 . in the case when fluorophores are embedded in nonuniform environments , it has been shown that luminescence decays can be best understood by a model of continuous distributions of decay times . ( eftink 1991 ) therefore , to gain a better insight into spatial distribution of lifetimes , the pl kinetics were evaluated from 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 ). fig1 . fluorescence lifetime image of cells . the image was collected at 300 × 300 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 μm × 27 . 2 μm . every pixel in the lifetime image ( a ) gives the lifetime at that particular position in space ( x , y ). the lifetime image ( fig1 ) clearly demonstrates distribution of emitting species over cell cytoplasm , showing the longest pl decay time at the rim of cell ( fig1 , region a ) as compared to the region of nucleus ( fig1 , region a ). fig1 . pl lifetime histograms obtained from regions a ( a ) and b ( b ) of fig1 respectively . in both cases 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 fig1 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 . ( bawendi , carroll et al ., 1992 ; klimov , mcbranch et al . 1999 ) although origin of the longer component of pl lifetime is still in question , recent investigations strongly imply the involvement of surface states in the recombination process in colloidal quantum dots . ( wang , qu et al . 2003 ). faster decays observed in the region a implies that lifetimes of the emitting states are strongly reduced validating the presence of highly - efficient nonradiative energy transfer . in contrast , in region b the quenching - caused nonradiative pathways are no longer competing with the radiative pathways , resulting in 2 - fold enhancement of lifetimes . it is noteworthy that intracellular accommodation of quantum dots in region a is accompanied by modification of only long - lived component , whereas the shorter component of pl lifetime is the same in both regions . this fact confirms strong involvement of surface states in the intracellular quenching mechanism . 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 . these 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 . two cell lines were used : hep2 epithelial cell line , grown in minimum essential medium ( eagle ) with earles salts , 10 % foetal calf serum , 2 mm l - glutamine ; and thp1 monocytic cell line ( ecacc , salisbury , england ) grown in supplemented ( 10 % foetal bovine serum ; 2 mm / l l - glutamine ; 100 μg / ml penicillin ; 100 mg / ml streptomycin ) rpmi 1640 media they were seeded out onto 96 well microtitre plates and onto coverslip slides at a concentration 2 × 10 5 cells / nl . the hep - 2 cells were incubated for 24 hrs , and the thp1 cells , cultured with 100 ng / ml pma ( to enable monocyte to macrophage differentiation ), for 72 hours , both in controlled atmospheric conditions of 37 ° c ., 5 % co 2 . prefixed cells were washed twice in pbs , treated with 3 % paraformaldehyde for 30 minutes , washed again and then permeabilised with 1 . 5 % triton x100 for 15 minutes . the plates were washed twice with pbs and 200 μl 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 ; thp1 cells seeded into a 96 well plate and hep - 2 cells into an 8 chamber coverslip slide ( labtech ). two sets of quantum dots ( qd ) were used in this assay . one had a variation of size as measured in emission wavelength ( 521 , 534 , 542 . 5 , 550 , 562 , 572 , 582 , and 592 nm ). all these were cdse / zns - dlcys with the exception of qds 521 nm and 572 nm ( cdte — coo —). the other set ( cdse ) were of the same size ( 534 nm ) but varied in charge determined by the concentration of the conjugated amino group ( 5 %, 10 %, 20 % nh3 +) or carboxyl group ( 20 %, 50 %, 100 % coo —) and hydroxyl group ( 100 % oh ). all dots were diluted to a concentration of 0 . 2 mg / ml in growth medium . the pbs was replaced by 100 μl of media , and 100 μl of diluted qds were added . the cells were incubated for one hour , washed in media , stained with 1 μg / μl hoescht for 3 minutes . washed with media and analysed using a cellomics kineticscan ®. half of the media ( 100 μl / well from the 96 well plate ; 200 μl / well from the 8 well plate ) was replaced with the diluted qds ( charged particles only ) and incubated for 1 to 3 hrs . the thp - 1 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 coverslips were examined using fluorescent and confocal microscopy . the images from the microtitre plates were acquired using the cellomics kineticscan ® and analysed later on the cellomics toolbox scan ® with the compartmental analysis ® bioapplications ( ca ). using hoescht staining to identify the nucleus which is also defined as the object in channel 1 ( ex 360 ( 50 ); em 515 ( 20 ); blue ), compartmental analysis can give information on the intensity of staining within the cytoplasm ( ring ) and nucleus ( circ = object ) of the cells , as well as organelles within both the nucleus ( cfrcspot ) and cytoplasm ( ringspot ) in channels 2 ( ex 475 ( 40 ); em 515 ( 20 ); green ); and 3 ( ex 560 ( 15 ); em 600 ( 25 ); red ) ( fig1 ). as described in previous studies , 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 tritox100 . the smaller sized particles went into the nuclei and emitted within the green wavelengths ( λ542 . 5 nm ), while the larger particles remained in the cytoplasm and emitted within the red wavelength ( λ562 nm , λ572 nm , λ582 nm ). the exception to these were the particles λ521 nm and λ572 nm , 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 ( fig1 and fig1 ). of interest , the particle λ550 nm 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 ( fig1 ). there were three negatively charged , three positively charge and one neutrally charged nanoparticles , which were tested with both prefixed and live cells . in the prefixed cells both tp - 1 and hep - 2 , all the particles tested positive in the green channel only . while in the hep 2 cells the distribution was equally in the both the nucleus and the cytoplasm ( fig1 ), in the thp1 cells , the nuclei picked up the dots more than the cytoplasm ( fig2 ). the 100 % neg qds located to the nucleoli in both cell lines . in the hep - 2 cells , the 5 %+ qds appeared to have a more filamentous pattern , especially close to the nuclear rim . examination of the qds progress in the live cells was carried out using fluorescence microscopy ( nikon eclipse te 300 ) and on the ultraview live cell imager confocal microscopy workstation ( perkin - elmer life sciences , warrington , england )( nikon eclipse te 2000 - u ). in the hep - 2 cells , the 5 %+ qds located onto what appeared to be the endoplasmic reticulum forming a meshwork surrounding the nucleus giving out fluorescence in the red spectrum . apart from faint cytoplasmic fluorescence in the green channel with the 100 %+ qds none of the other charged dots stained the hep - 2 cells . it was noted that even after 3 hours with the qds the hep - 2 cells still seemed to be very healthy . the 5 %+ qds stained the nucleoli . ( red channel ) in the thp1 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 - 1 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 ( fig2 ), especially the 5 %+ qds ( fig2 ). no staining was noted with the negatively charged qds . however it was noted that the negative control had some autofluorescence caused by fixing with gluteraldehyde ( fig2 ). therefore , it was decided to repeat the assay using 3 % paraformaldehyde as the fixative . while fixation with gluteraldehyde led to fluorescence in the red channel ( fig2 ), the green channel came to the fore with fixation with gluteraldehyde ( fig2 ) with the 5 %+ qds being the only dots to fluoresce in both channels ( fig2 ). unfortunately , the nucleoli appeared to stain in only a few cells with the 5 %+ qds and the 20 %+ qds . however , the nuclear rim stained quite strongly ( fig2 ). the negatively charged particles showed a weak speckled cytoplasmic pattern . we have confirmed that 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 . we have designed a system for protein quantification which exploits the specific destabilization of qds solutions in the presence of physiological buffers . in the system , 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 . three samples of cdte qds with distinctive fluorescence emission spectra ( 541 , 560 and 590 nm , emitting closely to green , orange and red , respectively ) were exposed to the increasing concentrations of purified bovine serum albumin solutions ( 0 - 0 . 01 - 0 . 05 - 0 . 1 - 0 . 5 - 1 - 2 mg / ml ) in the presence of either de - ionised water , standard physiological phosphate buffer ( pbs ), pbs without ca and mg ions or routine culture medium ( co2 - independent equivalent of medium rpmi 1640 ). following a 20 - min incubation at ambient temperature on a rotary shaker , the results of reaction were evaluated visually and using a spectroscopic methods . as seen from the fig3 ( a - b ), there is a detectable decrease of fluorescence intensity signal in the rows from b to g as a function of the decreasing protein concentration in the sample ( 2 to 0 . 01 mg / ml ). row a ( containing no protein ) yielded the poorest stability of the qds solutions . this observation is further supported by time - dependent photoluminescence ( pl ) intensity decays of cdte solutions ( fig4 to 7 ) and by analysis of dependence of integrated pl intensity and values of averaged lifetimes on concentration of bsa protein ( fig8 to 11 ). pl decays were measured using time - correlated single photon counting ( time - harp , microtime 2000 , picoquant ). the samples were excited by 480 nm picosecond pulses generated by picoquant . ldh - 480 laser head controlled by pdl - 800b driver . the set - up was operated at a 20 mhz repetition rate with an overall time resolution of 150 psec . 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 α i were taken into account by normalisation of the initial point in the decay to unity . the quality of fit was judged in terms of χ 2 value ( with a criteria of less than 1 . 1 for an acceptable fit ) and weighted residuals ( fig2 - 5 ( b ) ( c )) the τ i and α i parameters were used then to calculate the average lifetime the results indicate that there is a dose - dependent effect of bsa protein concentration in the sample on the stability and therefore light - emitting properties of qds solutions . none of the existing methods of protein determination offers a practically unlimited range of emission wavelengths which can be utilized for this purpose . 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 . we investigated the particular biopolymers to which the qd &# 39 ; s are possibly binding / interacting with in the cell as the smaller green emitting qd &# 39 ; s have been seen to go deep into the cell and have a distinctive sub - cellular distribution around the nucleoli 3 . the main biopolymers associated with this region of the cell are dna , rna and histones , for this reason their interaction with the qd &# 39 ; s is investigated . each of these biopolymers was investigated separately . for the purposes of this study , green emitting cadmium telluride ( cdte ) quantum dots capped with tga were used . they are ˜ 2 nm in size and of a highly stable nature with a quantum yield of 15 %. the dna , rna , and nuclear lysate used were extracted from hut78 t - cells . core histones were bought in from medical supply company and the bsa was purchased from sigma . the nitrocellulose used was purchased in from millipore . fluorescent lifetime data was collected with the flim system ( microtime200 timeresolved confocal microscope system , picoquant ) equipped with olympus ix71 inverted microscope . the samples were excited by 480 nm picosecond pulses generated by a picoquant , ldh - 480 laser head controlled 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 ). there are a range of excitation ( 275 nm / 360 nm / 485 nm / 590 nm ) and emission ( 460 nm / 465 nm / 535 nm / 595 nm ) filters available . for the purpose of this research the 360 nm excitation and 595 nm emission filters were used . ultra violet images were collected using a sony transilluminator . 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 1 mg / ml using de - ionised water . 2 ug of each sample was then placed on the nitrocellulose ( fig2 , fig2 ). 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 ° c . for ˜ 45 mins . the nitrocellulose was then washed rigorously twice using deionised water and kept moist at all times thereafter as the qd &# 39 ; s deteriorate when they are allowed to dry out . the nitrocellulose was then imaged using the trans - illuminator . table x below illustrates the different fluorescent lifetime decays obtained for the qd &# 39 ; s when mixed with the core histones or dna at various concentrations . for example at a concentration of 0 . 1 mg / ml , the qd &# 39 ; s have lifetime 9 . 6 ns longer than that of the qd &# 39 ; s in the histones . lifetime decay results for qd &# 39 ; s mixed with core histones , dna or rna . there is a significant reduction in the lifetime of qd &# 39 ; s with the histones as there is a reduction in concentration , however , there is no significant change in the lifetime of the qd &# 39 ; s mixed with the dna . rna also showed only to have an impact on the luminescence of the qd &# 39 ; 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 &# 39 ; s in the nucleus and nucleolus . the quenching effect of the rna at high concentrations observed above may be a contributing factor . higher rfu of quantum dots mixed with core histones was observed when compared to that of qd &# 39 ; s mixed with dna . the whole methodology of using nitrocellulose to bind the biopolymers was used to establish whether or not the qd &# 39 ; s would then bind to the biopolymers . no nonspecific binding of the qd &# 39 ; s to the nitrocellulose was observed . fig2 and fig2 clearly illustrate the cdte green emitting quantum dots bind to the histones . there is no binding to the bsa , dna or rna observed . a possible explanation for this could be attributed to the fact that the qd &# 39 ; s are negatively charged and the histones are positively charged , thus there is an attractive force between them , whereas the dna and rna are negatively charged which results in a net negative force between them . this can be used for selective histone - mediated targeting of qds to nuclei and nucleoli . 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 calorimetrically 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 . in this system , 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 . fluorescence lifetime images were collected with the flim system ( microtime200 time - resolved confocal microscope system , picoquant ) equipped with olympus ix71 inverted microscope . the samples were excited by 480 nm picosecond pulses generated by a picoquant , ldh - 480 laser head controlled by a pdl - 800b driver . the setup was operated at a 20 - mb repetition rate with an overall time resolution of ˜ 150 psec . plate read outs were carried out using the spectrafluor plus system ( tecan ). there are a range of excitation ( 275 nm / 360 nm / 485 nm / 590 nm ) and emission ( 460 nm / 465 nm / 535 nm / 595 nm ) filters available . for the purpose of this research the 360 nm excitation and 595 nm emission filters were used . the rfu and degree of polarisation of cdte quantum dots was measured ( table z ), relative to the polarisation of qd &# 39 ; s in water , using a tecan ultra evolution . clearly the tris borate has a quenching effect on the qd &# 39 ; s which would imply there are molecular interactions occurring , this is confirmed by the change in the degree of polarisation when compared to that of the qd &# 39 ; s in water and other buffers . fig2 to fig3 show the effect that varying the protein concentration has on the qd &# 39 ; s . in fig3 to fig3 there is an increase in the rfu observed 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 &# 39 ; s used . the fluorescent lifetime of the qd &# 39 ; s is a measure of the average lifetime that the qd remains in an excited state before returning to the ground state . for the purpose of this research the fluorescent lifetime decay ( τ1 / e ), was calculated using the following equations :( see fig3 also ). the shortest lifetime of the qd is in tris borate (˜ 3 ns ), hepes , tris and elisa share similar lifetimes of ˜ 15 ns , and pbs has a lifetime of ˜ 11 ns . the effect of varying protein ( bovine serum albumin ( bsa )), concentration on the fluorescent lifetime of two different types of qd &# 39 ; s are shown in table s . it is clear to see that the decay times for both qd &# 39 ; s reach a plateau at protein concentrations of 0 . 02 mg / ml to 0 . 005 mg / ml . this could be potentially due to qd concentration , which reaches saturation levels for given protein concentrations . 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 ). 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 . 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 . we found that cdte particles of particular size were able to align / orientate themselves in a particular geometry permitting electrical stimulation and conductivity . we developed a technique to make cdte nano - wires in physiological buffers ( volkov y , et al ) traditionally 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 . we can grow nano - wires of different composition ( qd size ) to varying lengths in physiological buffers . we found that 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 nanowires 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 ). the compounds currently used in inflammation research and treatment ( npx - peg - nh 2 , interferon α - 2a ) were used for conjugation with cdte qds and the efficiency of conjugation confirmed by biochemical methods as described below . tga ( tbioglycolic acid ) stabilised quantum dots were prepared according to the published procedure ( gaponik 2002 ). the concentration of purified tga - qd &# 39 ; s solution were determined by mean of uv - absorption and pl emission as described in yu , w . w . et al . in a typical procedure , x ml of a purified tga - qd &# 39 ; s solution were dissolved in deionized water and mixed with x ml of an edc ( 1 - ethyl - 3 -( 3 - dimethylaminopropyl )- carbodiimide ) solution in order to obtain a concentration ratio ( r = qd &# 39 ; s / edc ) from 2e − 3 to 1 . the reaction mixture is stirred at 0 ° c . or room temperature for 1 hour . then , a desired amount of drug in solution ( npx - peg - nh 2 , interferon α - 2a ) is added to the reaction mixture in accordance to the desired ratio ( qd &# 39 ; s )/( drug ). the mixture is then stirred for 3 hours at rt . after completion of the coupling reaction , precipitated formulations are purified by centrifugation and removal of supernatant . the operation is repeated until disappearance of free drugs in supernatant confirmed by uv - vis absorption . the conjugates are suspended in a basic ( ph = 9 ) phosphate buffer . 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 μm 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 . finally , agarose gel electrophoresis experiment ( fig1 and fig1 ) is performed . in a typical procedure , purified formulations ( 60 - 100 μl per well ) are run in a 1 % agarose gel in tris - hcl buffer ( ph = 8 . 1 ) for 1 h30 , 76v - 100 ma . gels are revealed under uv lamp . the invention is not limited to the embodiments hereinbefore described which may be varied in detail . akerman m e , chan w c , laakkonen p , bhatia s n , ruoslahti e nanocrystal targeting in vivo . proc . natl . acad . sci . usa . 2002 , 99 , 12617 . bruchez m jr , moronne m , gin p , weiss s , alivisatos a p semiconductor nanocrystals as fluorescent biological labels . science . 1998 , 281 , 2013 . chan w c , nie s . quantum dot bioconjugates for ultrasensitive nonisotopic detection . science . 1998 , 281 , 2016 . chan w c , maxwell d j , gao x , bailey r e , han m , nie s . luminescent quantum dots for multiplexed biological detection and imaging . curr . opin biotechnol ., 2002 , 13 , 40 . dubertret b , skourides p , norris d j , noireaux v , brivanlou a h , libchaber a in vivo imaging of quantum dots encapsulated in phospholipid micelles . science . 2002 , 298 , 1759 . gaponik n , radtchenko i l , gerstenberger m r , fedutik y a , sukhorukov g b , rogach a l . labeling of biocompatible polymer microcapsules with near - infrared emitting nanocrystals . nano lett . 2003 , 3 , 369 . gaponik , n . ; 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