Patent Application: US-90327901-A

Abstract:
a functionalized active - nucleus complex sensor that selectively associates with one or more target species , and a method for assaying and screening for one or a plurality of target species utilizing one or a plurality of functionalized active - nucleus complexes with at least two of the functionalized active - nucleus complexes having an attraction affinity to different corresponding target species . the functionalized active - nucleus complex has an active - nucleus and a targeting carrier . the method involves functionalizing an active - nucleus , for each functionalized active - nucleus complex , by incorporating the active - nucleus into a macromolucular or molecular complex that is capable of binding one of the target species and then bringing the macromolecular or molecular complexes into contact with the target species and detecting the occurrence of or change in a nuclear magnetic resonance signal from each of the active - nuclei in each of the functionalized active - nucleus complexes .

Description:
there are several preferred embodiments of the subject invention disclosed in the specification and depicted in fig1 - 15 . in the subject invention &# 39 ; s most basic configuration the subject invention comprises an active - nucleus ( nmr or mri detectable nuclei , preferably hyperpolarized xenon or hyperpolarized helium , however , 19 f is useful if present at sufficient levels ) and a target carrier that associates with both the active - nucleus and a desired target to produce an detectable characteristic signal ( typically a chemical shift or relaxation time for nmr or a contrast capability for mri ). fig1 depicts the basic biosensor ( the functionalized active - nucleus complex ) 5 configuration and fig2 shows the basic functionalized biosensor 5 bound to a target species / substrate / molecule / analyte 25 . an active - nucleus 10 is bound in a targeting carrier . the targeting carrier comprises , at least , a first binding region 15 , binding the active - nucleus , and a second binding region 20 , wherein the second binding region 20 has a binding affinity for the target species / substrate / molecule / analyte 25 ( the dashed line indicating the binding domain in the target ). the detectable signal generated from the bound complex 5 in fig2 is distinguishable from the detectable signal produced from the unbound complex 5 in fig1 ( see fig1 for an equivalent signal shift ). as indicated above , one extremely useful characteristic of the subject invention is that the signal produced from the subject sensor is highly dependent upon its immediate environment and that signals created from similar , but not identical , sensors can be distinguished and utilized to detect multiple target species / substrates / molecules / analytes within the same sample . for example , fig3 depicts a functionalized active - nucleus complex or subject biosensor 5 ′ that has a varied second binding region 21 , relative to the second binding region 20 seen in fig1 and 2 . thus , biosensor 5 ′ would bind to a different target species / substrate / molecule / analyte or a different location on the original target species / substrate / molecule / analyte 25 . additionally , fig4 illustrates a functionalized active - nucleus complex or subject biosensor 5 ″ that has a varied first binding region 16 , relative to the first binding region 15 seen in fig1 and 2 . thus , biosensor 5 ″ would generate a different signal than the signal produced by biosensor 5 . also , both the first 16 and second binding regions 21 could be varied , relative to the biosensor seen in fig1 and 2 , within the same subject biosensor to form biosensor 5 ′″, as seen in fig5 . as indicated , the subject invention allows a huge array of possible target species / substrates / molecules / analytes to be assayed / screened for in a parallel or multiplexing detection style within a single sample . [ 0048 ] fig6 illustrates a subject biosensor that has several different second binding regions 20 , 21 , 22 , and 23 attached to a first binding region producing sensor 5 ″″. sensor 5 ″″ may bind to one or more targets via the presented second binding regions . as indicated , several possible active - nuclei gases exist for any target species , preferable hyperpolarized xenon , hyperpolarized helium , and sulfur hexafluoride , however , 19 f , in sufficient concentration , is also contemplated for organic / biological targets . with fluorine atoms , an exemplary functionalized sensor comprises a target carrier having multiple fluorines such as a polyfluorinated dendrimer that selectively binds an organic / biological target species / substrate / molecule / analyte , as seen in fig7 . the polyfluorinated first binding region 35 may be a dendrimer or other suitable structure , including , but not limited to natural and synthetic polymers and the like . additionally , sufficient fluorine to produce an acceptable signal may be in the form of fluorine in sulfur hexafluoride and similar compounds . fig8 illustrates sulfur hexafluoride 45 trapped / bound within a functionalized ( target specific binding ) enclosing structure 40 such as in “ bubble ” or “ microbubble ” environment as exemplified by a liposome , micelle , vesicle , bucky - ball type structures , natural and synthetic polymeric cages , and the like . a second binding region 20 is coupled to the enclosing structure 40 and binds the target . conformational changes or alterations in the effective pressure on the “ bubble ” or “ microbubble ” would induce detectable signal variations from the active - nucleus in a subject biosensor . variations in the immediate vicinity of the biosensor should be detectable and include such changes as : ion concentrations , oxygen levels , neuron activity , and the like . it is noted that hyperpolarized xenon and hyperpolarized helium will also function as signal reporting active - nuclei within similar functionalized “ bubble ” or “ microbubble ” structures . it is noted that the subject targeting carrier comprises the first binding region ( 15 and 16 in fig1 - 6 ) that interacts / associates / binds with the active - nucleus . this first binding region includes structures such as monoclonal antibodies , dendrimers , self - assembled lipid complexes , liposomes , cyclodextrins , cryptands , cryptophanes , carcerands , microbubbles , micelles , vesicles , molecular tennis balls , fullerenes , many general cage - like structures , and the like . as long as structure or chemical nature of the first binding region permits effective signal producing interactions with the active - nucleus and binding to the target is not negated , a wide range of acceptable structures exists for this portion of the subject biosensor ( the chemical shifts or relaxation times for the active - nucleus need to maintained as detectable ). further , it is stressed that the second binding region ( 20 , 21 , 22 , and 23 in fig1 - 6 ) in the targeting carrier comprises that portion of the subject biosensor that interacts with the target species / substrate / molecule / analyte . the first and second binding regions may be essentially identical , overlapping , or coextensive or separated by a plurality of atoms . clearly , the embodiments structures depicted in fig1 - 8 for the basic subject biosensor may contain additional useful components / structures . as seen in fig9 a and 9b , one , or more . “ tether ” or “ linker ” or “ spacer ” regions 50 may be included in the biosensor 6 . specifically , fig9 a shows a biosensor comprising a first binding region 15 for the active - nucleus , a bound active - nucleus 10 , a second binding region 20 for the target , and a tether 50 . the tether 50 serves to separate the first 15 and second 20 binding regions and may serve as a site where chemical modification can occur . fig9 b illustrates the binding of the second binding group 20 with a target 25 . the chemical nature of the tether may be varied and includes polymethylenes , homo and heteropolymers , polyethers , amides , various functional group combinations , amino acids , carbohydrates , and the like . if desired , a plurality of tethered second binding groups may be bound to a first binding region , with each tether and / or second binding group the same or different . the tether may be derivatized to include a solubilizing region or other desired chemical feature such as additional binding sites and the like . the solubilizing region aids in solubilizing the biosensor in either a hydrophilic or hydrophobic environment . it is noted that a solubilizing region may also be included , either in addition to or separately , in the first and / or second binding regions . a water solubilizing region may include generally hydrophilic groups such as peptides , carbohydrates , alcohols , amines , and the like ( for a specific example see fig1 and 12 ). [ 0054 ] fig1 illustrates a subject biosensor in which the signal is enhanced by a rapid chemical exchange of the active - nuclei . free active - nuclei 11 rapidly exchange with the active - nucleus 10 bound in the first binding region 15 to produce an overall increase in sensitivity by enhancing the signal . more specifically , a functionalized active - nucleus biosensor is disclosed that capitalizes on the enhanced signal to noise , spectral simplicity , and chemical shift sensitivity of a hyperpolarized xenon to detect specific biomolecules at the level of tens of nanomoles . optical pumping ( 6 ) has enhanced the use of xenon as a sensitive probe of its molecular environment ( 7 , 8 ). laser - polarized xenon has been utilized as a diagnostic agent for medical magnetic resonance imaging ( mri ) ( 9 ) and spectroscopy ( 10 ), and as a probe for the investigation of surfaces and cavities in porous materials and biological systems . as indicated for an active - nucleus , xenon provides information both through direct observation of its nmr spectrum ( 11 - 17 ) and by the transfer of its enhanced polarization to surrounding spins ( 18 , 19 ). in a protein solution , weak xenon - protein interactions render the chemical shift of xenon dependent on the accessible protein surface , and even allow the monitoring of the protein conformation ( 20 ). in order to utilize xenon as a specific sensor of target molecules the xenon was functionalized for the purpose of reporting specific interactions with the molecular target . specifically , a laser polarized xenon was “ functionalized ” by a biotin - modified supramolecular cage to detect biotin - avidin binding , thus , the specific target is avidin . although , as previously indicated , the first binding region that holds the active - nucleus may be one of many possible structures , one suitable first binding region or cage is a member of the cryptophane family . cryptophane has the following structure : [ 0058 ] fig1 ( showing formula ii ) depicts a specific targeting carrier in which the first binding region cryptophane - a 15 is covalently attached to a tether 50 , having a solubilizing region 55 , and biotin as the second binding region 20 ( see the example # 1 below for synthesis details ). the solubilizing region comprises a short peptide chain ( cys - arg - lys - arg ) having positively charged groups at physiological ph values . [ 0059 ] fig1 ( showing formula iii ) shows the functionalized active - nucleus biosensor when the xenon 10 is bound within the first binding region cryptophane - a 15 cage . by way of example and not by way of limitation , one embodiment of the subject invention comprises a functionalized system that exhibits molecular target recognition . fig1 ( without xenon ) and 12 ( with xenon ) show a biosensor molecule designed to bind both xenon and protein . analogous to the general schematic diagrams seen in fig9 a and 9b , the specifically synthesized subject biosensor molecule consists of three parts : the cage 15 , which contains the xenon 10 ; the ligand 20 , which directs the functionalized xenon 10 to a specific protein ; and the tether 50 , which links the ligand 20 and the cage 15 . in this molecule , it is expected that the binding of the ligand 20 to the target protein ( as in analogous fig9 b ) will be reflected in a change of the xenon nmr spectrum . the biotin ( ligand second binding region 20 ) and avidin ( target species ) couple was chosen because of its high association constant (˜ 10 15 m − 1 ) ( 21 ) and the extensive literature characterizing binding properties of modified avidin or biotin ( 22 ). the cage 15 chosen for this embodiment was a cryptophane - a molecule ( 23 ) with a polar peptide chain ( solubilizing region 55 ) attached in order to make the cryptophane - a water - soluble . the cryptophane - a - based biosensor molecule was synthesized by a modified template directed procedure ( 23 ). starting from 3 , 4 - dihydroxybenxaldehyde and using allyl bromide to reversibly protect the meta - hydroxyl group ( 24 ), one of the 6 methoxyl groups in cryptophane - a was regioselectively replaced with a free hydroxyl group . upon reacting with methyl bromoacetate followed by hydrolysis ( 25 ), the hydroxyl group in the modified cryptophane - a was converted to a carboxylic acid , which was subsequently coupled ( using hobt / hbtu / diea activation method ) to the amino - terminus of a protected short peptide cysarglysarg on rink amide resin . the resulting cryptophane - a - peptide conjugate was deprotected and cleaved off the resin using “ reagent k ” ( 26 ), followed by purification with rp - hplc ( microsorbtm 80210c5 , rp - c18 column , flow 4 . 5 ml / min , buffer a : 0 . 1 % tfa inh 2 o , buffer b : 0 . 1 % tfa in ch 3 cn , linear gradient from 40 % to 80 % buffer b in 30 min ). the purified conjugate was reacted with ez - link tmpeo - maleimide activated biotin ( pierce ) to give the desired functionalized water - soluble cryptophane - a , which was further purified by rp - hplc ( same conditions ). the last two peptide conjugated - products were is verified by matrix - assisted laser desorption / ionization ( maldi )- time of flight -( tof ) mass spectrometry . all other intermediates were confirmed by 1 h nmr and maldi - fourier transform mass spectrometry ( ftms ). cryptophane - a has been shown to bind xenon with a binding constant k ≈ 10 3 m − 1 in organic solvents ( 15 ) but the affinity is likely to increase in aqueous solution because of the hydrophobic nature of xenon . the characteristic chemical shift for xenon inside a cryptophane - a molecule is very unusual for xenon dissolved in solution , approximately 130 ppm upfield from that of xenon in water . the only background xenon signal in the sample arises from xenon free in solvent , so the signal from the functionalized xenon is easily distinguishable . in the design of a xenon biosensor , a separate peak corresponding to xenon encapsulated by the cage is necessary , requiring both strong binding and a large difference between the xenon chemical shifts in the cage and solvent environments . the spin - lattice relaxation time for the functionalized xenon described herein was measured to be greater than 40 s , sufficient time for the required transfer , mixing , and detection of the polarized xenon . the biosensor solution was prepared by dissolving ˜ 0 . 5 mg of the cryptophane derivative ( m . w .= 2008 g mol − 1 ) in 700 μl of d 2 o , yielding a concentration of ˜ 300 μm . this concentration was consistent with absorbance measurements at 284 nm ( ε 284 = 36 , 000 m − 1 cm − 1 , determined for unmodified cryptophane - a by successive dilutions of a solution of known concentration ). approximately 80 nmol of affinity purified egg white avidin ( sigma ) was used without further purification . only half of the sample was located inside the detection region , so spectra actually reflect detection of ˜ 40 nmol avidin monomer . natural abundance xenon ( isotec ) was polarized and introduced to the sample using previously described methods ( 16 ), showing ˜ 5 % polarization for the spectra shown in fig1 and 14 . all nmr spectra displayed were obtained in single acquisition experiments at a nominal 129 xe frequency of 82 . 981 mhz . [ 0065 ] fig1 shows the full 129 xe nmr spectrum of the functionalized xenon in the absence of protein ( the trace running near the bottom axis and having a far left peak and far right peaks ). the far left peak at 193 ppm corresponds to xenon free in water while the far right peaks around 70 ppm are associated with xenon - bound cryptophane - a . the far right peaks are shown expanded in the center of fig1 , where the more intense , upfield peak (˜ 70 . 7 ppm ) corresponds to functionalized xenon and has a linewidth of 0 . 15 ppm ( shown by the generalized schematic model , as seen in fig9 a ). a smaller , middle peak (˜ 71 . 5 ppm ) approximately 1 ppm downfield of the functionalized xenon peak is attributed to xenon bound to a bare cage , without linker and ligand . as the unfunctionalized caged xenon does not interact specifically with the protein , it serves as a useful reference for the chemical shift and signal intensity of the functionalized xenon in the binding event . upon addition of ˜ 80 nmol of avidin monomer , a third peak (˜ 73 ppm ) appears approximately 2 . 3 ppm downfield of the functionalized xenon peak , attributable to functionalized xenon bound to the protein . correspondingly , the peak assigned to free functionalized xenon decreases in intensity relative to the reference peak while its position remains unchanged . the peak (˜ 73 ppm ) observed upon the addition of avidin is an unambiguous identifier of biotin - avidin binding , and hence the presence of avidin in solution . the mechanism of the chemical shift change upon binding may result from actual contact between the cryptophane cage and the protein , leading to cage deformation and distortion of the xenon electron cloud . changes in the rotational and vibrational motions of the cryptophane cage caused by binding to the protein could also affect the xenon chemical shift . indeed , the sensitivity of xenon to perturbations of the first binding region cage is so great that deuteration of one methyl group results in a readily discernible change in the bound xenon chemical shift ( 17 ). the subject methodology described herein offers the capability of multiplexing by attaching different second binding regions ligands to different first binding region cages , forming xenon sensors associated with distinct , resolved chemical shifts . as an example of this feature of the subject invention , fig1 shows the changes in bound xenon chemical shift caused by using two different first binding region cages . the top spectrum a is that of xenon bound to cryptophane - a ( n = 2 in formula 1 above ) in a tetrachloroethane solution and the lower spectrum b is that of xenon bound to cryptophane - e ( n = 3 in formula 1 above ), similar to cryptophane - a , but with an additional methylene group added to each of the bridges between the caps . the resulting bound xenon chemical shift is ˜ 30 ppm upfield from that of xenon bound to cryptophane - a . the linewidths for cryptophanes a and e are broadened by the exchange of xenon between the cage and tetrachloroethane , the organic solvent used . the diagram in fig1 indicates schematically a multiplexing system ( multiple functionalized xenon biosensors ) for protein assay or screening procedures . the binding event assay / screening procedures would be distributed over a large chemical shift range by attaching each second binding region ligand to a different first binding region cage . in the absence of the targeted proteins , the spectrum , depicted in fig1 , would consist of three resolved xenon resonances because of the effect on the xenon chemical shift caused by cage modifications . upon binding each of the targeted proteins , the xenon peaks should shift “ independently ,” signaling each binding event and reporting the existence of and amount of protein present . as long as the differences in shift between xenon in the different cages exceed the shift change upon binding , it should be possible to monitor and assign multiple binding events . in fig1 , the top spectrum shows the three distinct functionalized xenon peaks , corresponding to different cages linked to three ligands . the bottom spectrum shows the effect of adding the functionalized xenon to an unknown solution . upon addition to the unknown solution , the leftmost peak shifts entirely , representing the case in which all functionalized xenon is bound to its corresponding protein . the central peak decreases in intensity and a peak corresponding to the protein - bound functionalized xenon appears . the rightmost peak remains unaffected , indicating the absence of the corresponding protein target . thus , enabling experimental data for the subject functionalized active - nucleus biosensor has been disclosed that exploits the chemical shift of functionalized xenon upon binding to a target species / substrate / molecule / analyte . the approach has several critical advantages over aspects of current biosensors , in that multiplexing assays and both heterogeneous and homogenous assays are possible . furthermore , this methodology can be performed in biological materials in vitro or in vivo by combining the spatial encoding capabilities of mri with the biosensing nmr capabilities of the functionalized xenon sensor . as indicated above , potential targets include , are not limited to , metabolites , proteins , toxins , nucleic acids , and protein plaques . it must be stated that , given the basic information presented herein , refinements of the subject functionalized detector molecules / sensors and the nmr procedures disclosed herein should further enhance the presented sensitivity by orders of magnitude , relative to the experimental example described herein and are within the realm of this disclosure . the invention has now been explained with reference to specific embodiments . other embodiments will be suggested to those of ordinary skill in the appropriate art upon review of the present specification . although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding , it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims . all of the following references are herein incorporated by reference . in particular , reference 16 ( s . m . rubin , m . m . spence , b . m . goodson , d . e . wemmer , a . pines , proceedings of the national academy of sciences of the united states of america 97 , 9472 - 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