Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

• the present invention refers to optical methods of monitoring and measuring the growth of microorganisms in cultural media, and more particularly, such methods which employ oxygen-quenchable phosphorescent compounds and dendritic polymeric derivatives of such oxygen-quenchable phosphorescent compounds .
• Methods for the detection and accurate measurement of the presence and growth progression of various microorganisms are useful for a variety of purposes, including monitoring yields in the production of microorganisms in industrial fermentation process and the early detection of pathogenic microorganisms .
• the substrate is iron (III) salts mixed with K 3 Fe(CN) 6 , iron (II) salts mixed with K 4 Fe(CN) 6 or sodium tungstate (Na 2 W0 4 )
• the mixture of indicators methylene blue and resazurin is said to demonstrate bacterial growth by changing color from blue to red more rapidly than resazurin alone.
• the method is also said to be improved by the addition of a redox stabilizer such as potassium hexacyanoferrate,
• mixtures of inorganic salts of iron (III) such as NH 4 Fe(S0 4 ) 2 and K 3 Fe(CN) 6 , or iron (II) such as K 4 Fe(CN) 6 , or Na 2 W0 4 by itself are employed in culture media as redox indicators to demonstrate the growth of microorganisms .
• Such a method is not commercially practical, however, as the amounts of redox indicators required to demonstrate microorganism growth are not consistently non- toxic, and/or require an inordinate amount of care to exclude toxic amounts to prevent false negative results .
• Such methods as are all conventional methods, are not sufficiently sensitive to reduce the time required for demonstration of microbial growth from several weeks to a matter of days .
• microorganism growth is rapidly and accurately demonstrated by inoculating, or otherwise contacting a culture medium comprising a solubilized oxygen-quenchable phosphorescent compound with a substrate suspected of carrying or associated with one or more microorganisms, and then detecting microorganism growth and identifying microorganisms by causing the phosphorescent compound to phorphoresce and measuring microorganism presence and growth by oxygen-dependent quenching of phosphorescence.
• a light source means preferably a modulated light source, is employed for excitation of phosphorescence of the soluble phosphor in the microorganism-containing medium and determining both the phosphorescence intensity and delay time between the excitation light intensity and phosphorescence emission. Phosphorescence lifetime from the measured delay and/or intensity is calculated, followed by calculation of oxygen partial pressure (concentration) in the culture medium from oxygen dependence on the phosphorescence lifetime and appropriate calibration constants, i.e., quenching constant, and lifetime in the absence of oxygen.
• FIG.l illustrates an exemplary embodiment for the production of PdTBP and PdTPTPB functionalized derivatives, for initiating divergent dendrimer growth .
• FIG.2 illustrates another exemplary embodiment for the production of PdTBP and PdTPTBP functionalized derivatives for initiating divergent dendrimer growth .
• FIG.3a illustrates the production of dendrimer growth on a core functionalized porphyrin with functional groups located at the para-positions of meso-phenyl rings.
• FIG.3b illustrates the production of dendrimer growth on a core functionalized porphyrin with functional groups located at the meta-positions of meso-phenyl rings.
• FIG.4a illustrates a preferred embodiment of the invention of the production of a functionalized PdTBP with meta- (or psuedo meta-) functional groups by direct nitration of non-substituted TBP into meso-positions to produce (Pd) teranitrotetrabenzoporphyrin (PdTNTBP) .
• FIG.4b further illustrates the preferred embodiment of the functionalized core porphyrin of FIG. 4a by the transformation of (Pd)TNTBP into the corresponding tetraminotetrabenzoporphyrin (TATBP or PdTATBP) .
• FIG.4c further illustrates a preferred embodiment of the invention by additional functionalization of TATBP or PdTATBP in FIG.4b with 1, 3, 5 - benzenetricarboxylic acid to produce (Pd) metacarboxytetra-benzoporphyrin (MCTBP or PdMCTBP)
• FIG.5 illustrates the occurrence of branching in a divergent dendrimer growth mode through amide linkages formed using gluta ic acid.
• FIG.6 illustrates a preferred embodiment of the invention of divergent dendrimer growth through two generations using MCTBP or its derivative PdMCTBP as a core porphyrin and diallylglutamate as a monomeric unit.
• FIG. 7 illustrates a preferred embodiment of the invention of the modification of an outer layer of dendritic porphyrin .
• FIG. 8 illustrates another preferred embodiment of the invention of the modification of an outer layer of dendritic porphyrin.
• the present invention provides a process for the rapid and accurate demonstration of microorganism growth in a culture medium via inclusion in said culture medium of one or more non-toxic, water-soluble and/or otherwise physiological media-soluble phosphorescent compounds which measure oxygen content (partial pressure) by oxygen dependent quenching of phosphorescence in a culture medium.
• One of the most effective methods of growing microorganisms is in a culture medium.
• An accurate measure of the rate of oxygen depletion in the culture medium can be used to determine not only whether growing organisms are present in the culture following inoculation, but also the rate of growth of that organism in the culture.
• a very rapid determination of the presence of organisms such as mycobacteria is provided, as well as early indication of the type of organism present.
• organisms such as mycobacteria
• the presence of growing organisms in a culture medium typically results in consumption of oxygen at rates above the rate of oxygen consumption in a sterile culture medium.
• the presence or growth of organisms in a culture medium can be determined from the relationship of the number of organisms present per unit volume of an incubation medium being proportional to the rate of depletion of oxygen from the medium.
• water- soluble, non-toxic phosphorescent compounds are admixed with or are otherwise solubilized within a culture medium.
• the culture medium can then be inoculated with microorganisms and thereafter exposed to a modulated light source for excitation of the phosphor to phosphorescence to allow determination of both the phosphorescence intensity and delay time between the excitation light intensity and phosphorescence emission.
• the phosphorescence lifetime from the measured delay and/or intensity is calculated as well as that of oxygen partial pressure (concentration) in the culture medium from the oxygen dependence on the phosphorescence lifetime with respect to a quenching constant and lifetime in the absence of oxygen.
• the present inventive method provides an optical method of measuring oxygen concentration in culture media with high accuracy and precision.
• t 0 and t are the phosphorescence lifetimes in the absence of oxygen
• P0 2 is the oxygen pressure for a lifetime of t
• k Q is the quenching constant.
• the constant k Q is related to the frequency of collisions between the excited triplet state molecules and molecular oxygen and the probability of energy transfer occurring when these molecules collide.
• Phosphorescence may be measured by any available means in accordance with the present invention.
• two conventional methods for measuring phosphorescence lifetime (or decay time) are the "pulse method” in the time domain, and the “phase method” in the frequency domain.
• the pulse method a sample is excited by a short pulse of light and the resulting phosphorescence emission in the longer wavelength is an exponentially decaying function with a measurable rate of decline.
• the pulse method is used in most of the existing instruments for oxygen measurement.
• phase method a sample is excited with modulated light, with absorbed light being re-emitted as phosphorescence after a certain delay period.
• phosphorescent emission is also modulated with the same frequency but delayed in time (phase shifted) with respect to the excitation sinusoid. This phase shift, found experimentally, is used to calculate the phosphorescent lifetime.
• phase method is preferably used in the present invention due to the advantages that (i) frequency lock amplification can be used to greatly increase sensitivity and (ii) interference from ambient light is greatly decreased since only singles with the same modulation frequency as the excitation light is amplified, which largely eliminates interference by other ambient light sources.
• phase shift the mathematical relationship between phase shift and phosphorescence lifetime can be described as follows:
• t 0 at 38°C equals 646 ⁇ sec and the lifetime at air saturation is 16 ⁇ sec .
• the physiologically important range of oxygen concentrations extends from zero to approximately 150 Torr (air saturation) . If follows from the Stern-Volmer relationship (1) and equation (2) that to maintain the phase shift of about.35.5° for all oxygen concentrations in the range, it is necessary to be able to vary the modulation frequencies from 100Hz to 2000 Hz.
• the phosphorescence signal is preferably sampled (digitized) at 37.5 kH 2 or greater.
• a preferred instrument for practice of the present invention can be constructed from Analog Devices ADSP-2181 and AD 1847 Stereo Codec with stereo high precision 48kHz, 16 bit, Delta-Sigma ADCs with 64x oversampling.
• a sine wave signal of the desired frequency can be generated by the DSP using a 16 bit DAC and smoothing circuits of the Stereo Codec, and this signal will control the current in the LED or laser diode driving circuit .
• the LED driver circuit is designed to provide a greater than 90% modulation of light output. This is accomplished by adding a DC signal to the sinusoidal signal such that the minimum current is just above the threshold for light emission. Above this threshold, the light output is a nearly linear function of the current through the LED.
• LEDs Light-emitting diodes
• LEDs can be used as excitation sources.
• LEDs provide monochromatic light with a relatively broad bandwidth. This light is preferably passed through an interference filter to block the long wavelength "tail" in the emission of the LED, which otherwise might interfere with the measurements.
• the photodetector can be either a silicon photodiode with a built-in preamp or a photomultiplier .
• the photodetector output is amplified to provide a signal of optimal voltage for digitizing by the ADC.
• the photodiodes with an internal amplifiers are selected for the optimal light sensitive surface area and lowest noise level.
• the OPT202 unit (Burr- Brown) has an appropriate surface area (more than 5 mm2 ) and excellent photosensitivity, about 500 mV/mW for the 600 to 850 nm wavelength range and is preferred for use in the present invention.
• the signal from the photodiode can be further amplified with an AC-coupled operational amplifier.
• the quality of the phase detection depends on the reduction of noise level in the photodiode output signal.
• the photodiode output signal is delivered to the analog multiplexer and then to the input of the 16 bit, 48 kHz Delta-Sigma digitizer, such as a 16 bit analog-to-digital converter (ADC) and digitized.
• the digital signals will be processed to extract the signal strength (magnitude) and phase relative to the excitation light. Calculations of the phosphorescent lifetime and oxygen pressure will follow above- described procedures .
• this invention is based on the measurement of the quenching effect of the partial pressure of oxygen (oxygen concentration) available in a culture medium to determine the presence and amount of microorganism present in the medium.
• the method of this invention is useful in demonstrating the presence and growth of any oxygen depleting microorganism, identifying the microorganisms and testing them for sensitivity to antibiotics by measurement of oxygen partial pressure via phosphorescence emitted by soluble, oxygen-quenchable phosphorescent compounds (phosphors) .
• the microorganisms may be from such sources as urine specimens, matter from wounds and abscesses and blood, tissue and sputum samples, and be present in gels or broths with various substrates along with one or more phosphors.
• Exemplary bacteria include species from the genera Bacillus , Nycobacterium, Actinomyces, Nocardia, Pseudomonas,
• Methanomonas Protaminobacter, Methylococcus , Arthrobacter, Methylomanas , Brevibacterium, Acetobacter, Micrococcus, Rhodopseudomonas, Corynejbacfcerium, Microbacteriuir., Achro obacter , Methylobacter , Methylosinum, Methylocystis , Acinetojacter, and mixtures thereof.
• the rapid detection attributes of the present invention reduce the time typically required for growth demonstration/ identification of, for example, mycobacteria from approximately several weeks to less than a week or a matter of days.
• the inventive method is particularly suited for the rapid growth demonstration of about several days of such slow growing tuberculosis agents as M. tuberculosis and M. bovis and the M. Avium which appears in AIDS patients, all of which require at least eight to ten weeks of incubation for growth demonstration by conventional methods.
• the inventive method is also useful in monitoring the production of microorganisms in fermentation processes which are widely use for a variety of purposes including chemical conversions, protein preparation, chemical reactions/chemical compound production, examples of which are discussed in U.S. Patent No. 4,226,989.
• Water soluble oxygen-quenchable phosphorescent compounds (phosphors) useful in the present invention and which are currently employed in methods for determining tissue oxygen concentration/oxygen partial pressure by measuring the quenching effect of oxygen on molecular phosphorescence of organic compounds are described, for example, in U.S. Patent No. 4,947,850, which is incorporated herein by reference.
• the phosphorescent chromophor e.g., PdPorph and PtPorph is the phosphorescent portion of the phosphor that can be converted to the triplet state (T.) by light absorption, followed by a return to the ground state by light emission, or phosphorescence.
• the phosphors should be non-toxic to microorganisms or of negligible toxicity, and should also be of sufficient solubility in culture media such that oxygen molecules can approach close enough for efficient quenching to provide for reliable and accurate oxygen measurements, and the measurement of microorganism growth.
• a new class of phosphors particularly suitable for oxygen measurement and concomitant microorganism growth identification in accordance with this invention has recently been reported in Vinogradov and Wilson, J " . Chem . Soc . , Perkin Trans . 2: 103-111 (1995), and in U.S. Application Serial No.
• dendritic derivatives of the aforementioned phosphors which are highly efficient and highly soluble phosphorescent compounds which are surrounded by an inert globular structure, an example of which is derivatized PdTBD surrounded by three- dimensional supramolecular structure known as a dendrimer.
• Such compounds are described in U.S. Application Serial No. 08/767,158, filed December 16, 1996, now U.S. Patent No. , the entirety of which is incorporated herein by reference .
• Dendrimer phosphors useful in this invention are three-dimensional supramolecular radial symmetrical molecules comprised as an initiator functionalized core, which in the present invention are oxygen-measuring phosphors, with interior layers attached to the core which are comprised of, for example, three or four arms with each arm being composed of repeating units, and with the layer of repeating units in each arm considered to be a generation of the dendrimer.
• the outermost generation typically contains terminal functional groups, such as a primary amine attached to the outermost generation.
• the size and shape of the dendrimer molecule, and the functional groups present therein can be controlled by the choice of the initiator core, the number of generations, and the nature of the repeating units employed at each generation.
• the chemical functionality of the repeating units in the interior layers can be amidoamines , such as diethylene diimine, and with terminal functionalities, such as, for example, amino groups, hydroxyl groups, carboxylic acid groups, carboxylates and the like.
• amidoamines such as diethylene diimine
• terminal functionalities such as, for example, amino groups, hydroxyl groups, carboxylic acid groups, carboxylates and the like.
• dendrimers are combinations of monomeric units which allow branching at each step of polymerization. As shown, for example, by Blumen et al . , Angewandte Che_ ⁇ ie. Int . , Ed. Eng .
• dendrimers tend to form globular structures with increasing numbers of monomeric units, which eventually will cover the centralized functional entity or compound. See also, for example, Winnik et al . , U.S. Patent No. 5,256,193.
• the more typically used divergent synthetic method employs a reverse order of synthesis which involves an initial reaction of a monomer with an initiator core, followed by successive reaction of the resulting functional groups with a difunctional compound, such as a diamine, to provide the next generation of reactive amino groups such that layers of monomeric units are added to a central core sequentially until the desired degree of branching is achieved.
• a difunctional compound such as a diamine
• one-, two-, and three-layer polyglutamate dendritic cages synthesized divergently around novel derivatized metallo extended porphyrin oxygen-measuring phosphor compounds results in phosphors which are highly water-soluble in a wide pH range and display narrow distribution of phosphorescence lifetimes in deoxygenated water solutions.
• the combination of the novel phosphor derivatives with dendrimers which are used as the phosphor's surrounding environment provides a novel class of phosphorescent probes for accurate and reliable oxygen measurements in culture mediums for reliable and fast culture growth demonstration and identification.
• the dendritic phosphors are prepared from phosphors described in copending U.S. Application Serial No. 08/137,624 and Vinogradov and Wilson, J " . Chem . Soc , Perkin Trans . 2:103- 111 (1995), and preferably are of the following formula:
• R x is hydrogen or substituted or unsubstituted aryl
• R 2 and R 3 are independently hydrogen or are linked together to form substituted or unsubstituted aryl
• M is H 2 or a metal
• M is preferably a metal selected from the group consisting of Lu, Pd, Pt, Zn, Al , Sn, Y and La, and derivatives thereof, with Pd, Pt and Lu being most preferred.
• suitable metal derivatives include, Pd tetrabenzoporphyrin (PdTBP) , Pd tetraphenyltetrabenzoporphyrin (PdTPTBP) , and PtTBP, PtTPTBP, LuTBP and LuTPTBP and naphthaloporphyrins , such as , for example, LuTNP and PdTPTNP, all of which are described in U.S. Serial No. 08/137,624.
• the phosphors are tetrabenzoporphyrin (hereinafter "TBP") compounds, which correspond to the compound of formula I above wherein vicinal R 2 and R 3 groups are linked together to form benzene rings which are fused to the respective pyrrole rings .
• TNP tetranaphthoporphyrin
• TAP tetraanthraporphyrin
• TBP TNP and TAP compounds
• Preferred TBP compounds have the following formula
• TBP metallotetrabenzoporphyrin
• TBP compounds Particularly preferred among the TBP compounds are the compounds of formula IV above where at least one of R x is substituted or unsubstituted phenyl . These compounds are referred to hereinafter as phenyltetrabenzoporphyrin (hereinafter "PhTBP”) compounds. Preferred PhTBP compounds include substituted or unsubstituted tetraphenyltetrabenzoporphyrin (hereinafter "TPTBP”) compounds, including meso-tetraphenyltetrabenzoporphyrin (hereinafter ".m-TPhTBP”) compounds, which have the following formula :
• TPTBP compounds are substituted compounds of formula V where x is an integer from 1 to 3.
• substituent groups are desired which impart such desirable properties to the compounds as solubility in polar solvents, including aprotic solvents, such as dimethylformamide (DMF) , acetone and chloroform (CHC1 3 ) , and protic solvents, such as water.
• polar solvents including aprotic solvents, such as dimethylformamide (DMF) , acetone and chloroform (CHC1 3 ) , and protic solvents, such as water.
• aprotic solvents such as dimethylformamide (DMF) , acetone and chloroform (CHC1 3 )
• protic solvents such as water.
• the degree of substitution and the nature of the substituent groups may be tailored to obtain the desired degree of solubility and in the desired solvent or solvent mixture.
• a preferred synthetic preparation of the phosphors for use in the present invention is now illustrated.
• synthesis of PdTBP derivatives with chemically active functional groups is carried out to allow for further addition of dendritic fragments.
• the actual layer-by-layer divergent growth of the dendrimer polymeric structure around the porphyrin core is accomplished to form the completed probe.
• Functionalizin ⁇ a (Pd)TBP into (Pd) MCTBP TBP and tetraphenyltetrabenzoporphyrins (TPTBP) for use in this invention can be synthesized by the template condensation of potassium phthalimide with phenylacetate in the presence of Zn salts, according to the method reported by Kopranenkov et al . , J. Gen . Chem . (Russ.) 51: 2165-2168 (1981) and Ichimura et al . , Inorg. Chim . Acta . 182: 83-86 (1991).
• Tetratoluyltetrabenzoporphyrin can also be synthesized in approximately 10% yield by using 4-methylphenylacetate as a condensing agent. See, for example, Kopranenkov et al . (1981) .
• functional groups must be added to the formed TBP and TPTBP structures .
• TBP and TPTBP in accordance with this invention include a) electrophilic substitution (chlorosulfation, nitration, etc.) of phenyl rings in TPTBP ' s , and b) electrophilic substitution, such as nitration, of meso-positions of non-substituted TBP followed by reduction and attachment of 1 , 3 , 5 , -tricarboxylic acid fragments .
• PdTPTBP can be readily chlorosulfated and converted into the corresponding sulfonamide with aminopolyethyleneglycols . See Vinogradov and Wilson (1995).
• the employ of phenyl rings substituted with methyl groups will significantly decrease the number of isomers formed in electrophilic substitution due to stearic restrictions, especially when soft electrophiles are used for modification, thereby increasing selectivity.
• the latter is similar to that observed for the porphyrin bound to albumin and is suitable for measurements in vivo .
• molecular modeling shows that if dendrimer growth starts from the meta- positions, globular structures form much faster and only three to five layers of monomers are needed for generation of a fully globular structure (see FIG. 3b) .
• the molecular weight of the probe molecules will be between about 4,000 and 5,000 Daltons, which is a desirable size for good penetration through the kidney filters.
• porphyrin moiety will direct electrophilic substitution to the para- and orth-positions of the phenyl rings .
• another reaction pathway to achieve formation of PdTBP with meta- (or pseudo meta-) functional groups is provided. This reaction is based on the direct nitration of non-substituted TBP into meso-positions, (see FIG. 4a). As shown in FIG. 4a, the arrows indicate the most probable direction for electrophilic attack. Direct nitration of porphyrins is known. See Drach et al., J. Org. Chem . 39: 3282-3284 (1974) and Bonnet et al . , J. Org.
• strong ionic nitrating agents such as, for example, BF 4 N0 2 or highly activated covalent nitrating systems, such as, for example, AcON0 2 /BF 3 ⁇ T 2 0 and RON0 2 /TiCl 4 be employed to increase both overall yield of nitration and the relative yield of tetranitrotetrabenzoporphyrin (TNTBP) .
• Nitration can be carried out at the earliest state of transformation when TBP is present as its Zn complex.
• Zn tetranitrotetra- benzopophyrins can be easily demetallated by using Ac0H/H 3 P0 4 and that the insertion of Pd into TNTBP proceeds faster than into non-substituted TBP, which is due to increased non-planarity of the tetranitrated macrocycle, as confirmed using molecular-mechanics calculations (MacroModel V.3.5, MM2 force field).
• TNTBP or PdTNTBP
• TATBP or PdTATBP tetraaminotetrabenzoporphyrin
• the resulting TATBP can be produced in good yield by preferably employing systems with increasing reducing activity, such as Zn/HCl, SnCl 2 /AcOH, Na/MeOH, NaBH 4 /MeOH, LiAlH 4 /THF.
• systems with increasing reducing activity such as Zn/HCl, SnCl 2 /AcOH, Na/MeOH, NaBH 4 /MeOH, LiAlH 4 /THF.
• TATBP After formation of TATBP, further derivatization can be achieved by any of several methods employing high reactivity of the amino groups .
• a preferred method is amide formation between 1, 3 , 5-benzene-tricarboxylic acid and TATBP (or PdTATBP) carried out in the presence of dicyclohexylcarbodiimide (DCCD) to produce a TBP containing pseudo meso-phenyl substituents with meta-carboxyl groups, or as termed herein, metacarboxytetrabenzoporphyrin (MCTBP) .
• DCCD dicyclohexylcarbodiimide
• MCTBP metacarboxytetrabenzoporphyrin
• MCTBP or its Pd derivative, as shown below can be used as a core for dendritic polymer growth. See FIG. 4c.
• a preferred direct synthesis of functionalized porphyrins is provided which leads directly to substituted TPTBP with chemically active functionalities and suitable as a core for dendritic polymer growth.
• tetrabenzoporphyrins, TBP, and tetraphenyltetrabenzoporphyrins, TPTBP are generally synthesized by template condensation of potassium phthalimide with sodium acetate or sodium phenylacetate in the presence of Zn salts.
• functional groups in either phthalimide or phenylacetic acid fragments usually do not survive.
• PdTBrPTBP meso-p-Br- phenyltetrabenzoporphyrins
• PdTClPTBP meso-p-Cl- phenyltetrabenzoporphyrins
• PdTPhTBP ' s containing Br-substituents can be converted into corresponding carboxyl compounds as follows:
• Dendrimers can be grown from any multi-substituted core, such as a multi-substituted porphyrins , with their different respective properties merging with increase of polymer layers .
• a divergent dendritic growth scheme example in accordance with this invention is conveniently shown as built around that of a functional (Pd) MCTBP core. While a convergent growth scheme is also contemplated, divergent growth is preferred as it appears to allow for more economical use of PdMCTBP and for more convenient measurements of optical and quenching properties on each step of modification. Once the necessary protection of the porphyrin is achieved, as measured by oxygen quenching constant, the addition of extra layers is not necessary; a finished probe molecule having the desired optimal size is easily synthesized.
• any one of several known monomeric units for the formation of divergent dendrimers are useful, such as, for example, as described in U.S. Patent Nos. 4,507,466; 4,631,337; 4,558,120; 4,568,737 and 4 , 587 , 329 , and in Tomalia et al . Angewandte Chemie, Int. Ed. Eng . 29:138-175 (1990) and Tomalia et al . MacroMolecules, 19 : 2466-246 * 5 (1986), the entire disclosures of which are incorporated herein by reference.
• glutamic acid diallyl ester diallylglutamate
• PdMCTBP glutamic acid diallyl ester
• Diallylglutamate has two protected carboxylic groups and one amino group as shown in FIG. 5. Branching and dendritic polymer formation occurs through formation of amide linkages of each step of polymer formation. It is noted that the reaction scheme in FIG. 5 is drawn for simplicity reasons, and only illustrates non-protected glutamic acid, and not diallylglutamate.
• the allylic moiety on the introduced carboxylic groups can be readily removed by treatment of the ester with warm aqueous NaOH . Amide linkages are completely stable under these reaction conditions. Thus, hydrolysis gives porphyrin with twice as many carboxyl groups, which is ready for the addition of a new glutamate layer, or a second generation.
• the two first stages of the overall reaction process are shown in FIG. 6. Step 1 denotes amide linkage formation, while Step 2 denotes base catalyzed hydrolysis of the allyl ester protective groups.
• Purification of the final reaction product can be achieved using membrane filtration, dialysis and size exclusion chromatography, such as successfully employed * for the purification of "caged" Zn porphyrin. See Jin et al . , J Chem . Soc . Chem . Commun . 1260-1262 (1993).
• Phosphor toxicity evaluation for individual candidates in accordance with the invention can be conveniently carried out with the following protocol.
• Phosphor powder Pd-meso-tetra (4-carboxyphenyl) porphyrin with two layers of gultamate dendrimer, was dissolved in five milliliters of distilled, deionized and filter sterilized water through an 0.2 mM filter to provide a solution with a concentration of 8mM and a pH of 7.4 to provide final dilutions with concentrations in the culture medium of 4,8 and 16 micro-molar.
• the three dilutions are made to create stock solutions and to add an equal amount of phosphor solution into each test tube.
• Control tubes are supplied with the same amount of sterile water.
• Each of the final phosphor dilutions (1:500 1:100 and 1:2000) was prepared in duplication. Paired tubes are inoculated with two difference concentrations of Mycobacterium tuberculosis culture: 1,000,000 cells/ml and 10,000 cells/ml. Same bacterial concentrations are inoculated into no-phosphor control tubes.
• three noninoculated tubes are set up with just phosphor dilutions as a negative control.
• excitation light is used which is ulated sinusoidally at a frequency of from 20 to 20,000 Hz.
• This light source can be any of several difference sources and the modulation can be either direct modulation of the light source or passing the light through a modulation device such as a rotating wheel with slots for the light to pass through.
• the light source is a light-emitting diode (LED) or a laser diode, where the latter is a special case of the former.
• solid state light sources can be readily modulated at the desired frequency and are monochormatice, i.e., light emission occurs primarily in either a broad band (up to about 60 nm bandwidth at halfheight for LEDsO or a narrow band of 1 nm or less (for laser diodes) . As a result, little optical filtering is required for optimal application to this type of measurement of phosphorescence lifetimes.

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