CA1134642A - Pheromone detection system - Google Patents
Pheromone detection systemInfo
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- CA1134642A CA1134642A CA000354584A CA354584A CA1134642A CA 1134642 A CA1134642 A CA 1134642A CA 000354584 A CA000354584 A CA 000354584A CA 354584 A CA354584 A CA 354584A CA 1134642 A CA1134642 A CA 1134642A
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- aldehyde
- pheromone
- flavine
- reduced
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- 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
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
ABSTRACT
Insect pheromone long chain aldehydes are assayed in liquid samples, down to extremely small quantities, by a bio-luminescent technique. A sample of reduced flavine mononucleo-tide and bacterial luciferase enzyme is treated with hydroxyl-amine, to scavenge out background aldehyde, and is then reacted with the aldehyde containing liquid sample for assay, in the presence of water and oxygen, to cause oxidation of the reduced flavine mononucleotide, with emission of light. By determination of the maximum light intensity and optionally the decay curve of the emitted light, the amounts of aldehyde in the liquid sample can be accurately assayed, and information can be obtained concerning the chemical identity of the alde-hyde. The process is useful in the monitoring of atmospheric aldehyde concentrations, in association with insect pheromone traps and matins disruption by pheromones.
Insect pheromone long chain aldehydes are assayed in liquid samples, down to extremely small quantities, by a bio-luminescent technique. A sample of reduced flavine mononucleo-tide and bacterial luciferase enzyme is treated with hydroxyl-amine, to scavenge out background aldehyde, and is then reacted with the aldehyde containing liquid sample for assay, in the presence of water and oxygen, to cause oxidation of the reduced flavine mononucleotide, with emission of light. By determination of the maximum light intensity and optionally the decay curve of the emitted light, the amounts of aldehyde in the liquid sample can be accurately assayed, and information can be obtained concerning the chemical identity of the alde-hyde. The process is useful in the monitoring of atmospheric aldehyde concentrations, in association with insect pheromone traps and matins disruption by pheromones.
Description
1~3464~
FIELD OF THE INVENTION
; This invention relates to pheromone monitoring systems, and more particularly to methods of detecting and monitoring the quantity of pheromone substances in a gaseous or liquid environmental sample.
BACKGROUND OF THE INVENTION
It is known that animals secrete substances known as pheromones, to influence the behaviour of other animals of the same species. Insects, for example, secrete sex pheromones, in extremely low levels, to attract mates of the opposite sex.
The mechanism of production and secretion of sex pheromones by insects is still incompletely understood. They appear to be secreted by moths or beetles during mating periods, at a particular daily calling stage. Insect population control systems have recently been proposed, based on the use of the sex pheromones of the insects concerned.
Some insects that cause damage to forestry and farming products have been reported to secrete long chain unsaturated aldehydes that function as sex pheromones, either alone or in combination with other substances. Examples include such major pests as the corn earworm (cotton bollworm), the dermestia beetles, the spruoe budw~rm and the tobacco budworm. Consequently, methods have been developed to monitor or control the population of these pests by use of the appropriate sex pheromone substance, either synthetically or naturally produced. Lures ~a~
- 1134~i4Z
have been made, containing the sex pheromone, optionally in admixture with a solid or liquid carrier, in which the insects are trapped and counted so as to monitor the population.
Infested areas may be sprayed with sufficient sex pheromone to confuse the insects and prevent their mating and reproduction.
The sex pheromone may be included in a pheromone trap, in which insects are collected for killing, e.g. by inclusion of insecticide in the trap.
Further developments and studies of sex pheromones and their potential uses in insect population control are, however, hampered by the lack of a suitable method for deter-mining the concentrations of the pheromones being used.
Effective concentrations of pheromones for this purpose are extremely small, in many cases below the minimal range. Before the efficiency and potential utility of any method of insect population control based upon the use of sex pheromones can be properly assessed and developed, there needs to be a method which can be used in association with the pheromone which will permit the accurate and rapid detection and monitoring of the pheromone substances. Only in this way can efficiency of traps be established, and the necessary effective concentrations of pheromones be established and hence applied.
BRIEF DESCRIPTION OF THE PRIO~ ART
The current practice for monitoring pheromone con-centrations is by means of gas-liquid chromatcgraphy. In such processes, a sample of atmosphere is collected by absorption from the air, extracted with an organic solvent, in some instances derivatized to a form more easily detected on analysis, and then subjected to gas chromatography. Such a procedure is difficult and time-consuming, requiring the conducting of several steps of sample preparation. Furthermore, such methods are generally not sensitive enough to detect the very lcw concentrations of sex pheromones effective in the atmosphere as insect attractants.
It is kncwn that long chain aldehydes will react with reduced flavine mononucleotide, in the presence of oxygen and a bacterial luciferase enzyme, to oxidise the flavine mono-nucleotide substrate and the aldehyde, with the emission of light, at 490 nm. This phenomenom is generally known as bacterial bioluminescence. Descriptions of this process are to be found in the academic literature, for example in a paper by J. W. Hastings, "Annual Review of Biochemistry", 1968, Volume 37, Pages 597-630, and in a paper by J. W. Hastings and K. H. Nealson, "Annual Review of Microbiology", 1977, Volume 31, Pages 549-595. There have also been reports that certain long chain fatty acids, specifically myristic acid, will also cause in vivo bacterial bioluminescence presumably by being converted "in vivo" to the corresponding aldehyde.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that, with suitable modifications, the light emitting reaction of oxidation of a reduced flavine nucleotide catalysed by a 6~Z
luciferase enzyme can be conducted in the presence of a sex pheromone aldehyde, and can be used to monitor very accurately the amount of sex pheromone aldehyde present, down to extremely small quantities. It has been found that the intensity of emitted light is a function of the quantity of sex pheromonal aldehyde present. By the process of the invention, quantities of sex pheromone aldehyde as low as 0.1 picamoles (10 -13moles) or 20 picagrams can be readily determined. Moreover, by analysing the decay characteristics of the emitted light after it reaches its maximum intensity, the identity of the phero-monal aldehyde may be determined.
Thus according to the present invention, there is provided a process of analysing a liquid sample for content of aldehyde of sex pheromonal origin which comprises:
preparing a solution of reduced flavine mononucleotide and luciferase enzyme;
treating said solution with hydroxylamine;
reacting the so-treated solution with said aldehyde-containing liquid sample in the presence of water and oxygen, to cause oxidation of the reduced flavine mononucleotide with emission of light;
and determing the intensity of said light emission, to determine therefrom the content of aldehyde in said liquid sample.
The amount of sex pheromonal aldehyde to be deter-~346 ~Z
mined in the monitoring of pheromonc traps, down to 0.1 picamoles, is so low that special methods of assay and pre-cautions in connection therewith have to be adopted in order to ensure that all of the aldehyde detected is derived from the sex pheromone being monitored. Thus, special precautions have to be taken to remove background aldehyde contaminations.
In accordance with the invention, hydroxylamine is used for this aldehyde scavenging process, and special methods and procedures for reducing the flavine mononucleotide and pre-paring the reaction solutions for the light-emitting lumines-cence reaction are used. The method of the invention is then accurate and sensitive enough, to allow quantitative detection and measurement of sex pheromonal aldehydes down to the necessary low concentrations for insect trap monitoring.
Thus in the process of the invention, the reduced monoflavine nucleotide is prepared in admixture with the enzyme luciferase. This mixture is scavenged for residual aldehyde with hydroxylamine. Then the sample of sex pheromonal aldehyde in water is added. The water contains the required amount of oxygen for the luminescence reaction. Surprisingly, it is found that the sex pheromonal aldehyde in the sample reacts with the reduced monoflavine nucleotide to give light emission of intensity dependent quantitatively on the amount of sex pheromonal aldehyde, despite the presence in the reaction mixture of residual amounts of hydroxylamine, the background 1134~i~Z
aldehyde scavenger. It would have been expected that the residual hydroxylamine would have reactedextensively with the se~
pheromonal aldehyde, to interfere with the reaction between the sex pheromonal aldehyde and the reduced flavine mononucleotide, but surprisingly this does not happen. This feature permits the necessary thorough scavenging of background aldehyde with hydroxylamine to give the required accurate determinations of sex pheromonal aldehyde, but also dictates certain conditions on the order of addition of reagents in the process of the invention. Thus, it is necessary to avoid having the test sample and hydroxylamine present in admixture in the reaction medium in the absence of the reduced flavine mononucleotide and bacterial luciferase enzyme. For example, pre-mixing of the liquid test sample and the enzyme solution, ; followed by addition of the reduced flavine mononucleotide thereto, a procedure which would normally be adopted to give increased sensitivity, cannot in fact be adopted sinoe this precludes scavenging of the enzyme solution with hydroxylamine. Only by the procedure of the invention can the scavenging with hydroxylamine of all the reagent solutions, necessary to give results of sufficient accuracy, be properly conducted.
DESCRIPTION OF THE PREFERRED EMsoDIMENTs Whilst there are several known and acceptable methods for making reduced flavine mononucleotide, the solution of ; reduced flavine mononucleotide is preferably prepared by reduction of the flavine mononucleotide, in the presence of 113~64Z
the luciferase enzyme, with sodium dithionite. This is best conducted shortly before the light-emitting reaction with the aldehyde test sample to be conducted. Sodium dithionite, a solid, powdery material under normal conditions, does not have an adverse effect on the luciferase enzyme, at least over the short term. It is preferred to include enzyme stabilizers such as mercaptoethanol, and phosphate buffers to maintain a pH of 6-8, in the solution, to ensure stability of the enzyme therein. Since the assay method of the invention may require to be conducted under field conditions, it is desirable to use convenient solid powdered reagents, such as sodium dithionite where available, and as many premixed solutions as possible. Other methods for reduction of flavine mono-nucleotide, such as bubbling gaseouS hydrogen through a solution thereof, are known and can be used under certain circumstances. ~lowever, bubbling hydrogen will deactivate the luciferase enzyme, and so this method cannot be used with a premixed flavine mononucleotide-luciferase solution. lt is also satisfactory to form a premixed solution of the luciferase enzyme and appropriate buffers with the hydroxylamine, and then mix this solution with flavine mononucleotide before or after it has been reduced.
This reaction is suitably conducted at or about room temperature or field temperatures, provided that the reagents and solutions are kept in the liquid phase. Slightly elevated 1~34164Z
temperatures may offer certain advantages. The reaction should be conducted at pH at which the enzyme is stable, normally approximately neutral.
After mixing of all of the necessary reagents and sample, light is emitted as a result of the flavine oxidation reaction. The emitted light rapidly rises to a maximum intensity, and then decays in intensity. The aldehyde con-centration in the sample is determinable from the maximum light intensity. By analysis of various features of the light intensity-decay curve, in the absence of interfering impurities, characterization of the aldehyde can be obtained, by comparison with standard curves.
The specific choice of species of luciferase enzyme is made in accordance with the nature of the pheromonal aldehyde to be detected. The light emitting response of the reaction mixture is different, depending upon the chosen luciferase enzyme. It appears that each luciferase enzyme species responds over a relatively narrow range of chain length aldehydes, one luciferase enzyme being preferred over the range C10-C16 chain aldehydes, and another through the range C14-C20.
The bacteria luciferase enzymes which are used in the process of the invention comprise a relatively small, but kncwn and characterised group. They are obtained and purified from marine bacteria, being synthesized as the bacteria grow. Samples of 113~64Z
suitable marine bacterla are available from standard culture collections. The enzymes may be stored for extended periods of time, under cool conditions, in buffered solutions e.g.
aqueous glycerol solutions. They may be prepared for use by dilution into aqueous solution, in the absence of glycerol, suitably buffered. The term "bacterial luciferase enzyme"
refers to an enzyme of bacterial origin/ which will catalyze the reaction of reduced flavine mononucleotide and aldehyde in the presence of oxygen with the emission of light. At least six of these enzymes have been purified and characterized.
The best ones for use in the process of the present invention are those giving high sensitivity, with a low response in the absence of exogeneous aldehyde and a high response in the presence thereof. Routine screening tests of the available enzymes will readily show those best for use in the inven-tion under any specific conditions.
The sex pheromonal aldehydes which have been identi-fied for four major insect pests/ namely the dermestia beetles, ; the corn earworm,the spruce budworm and the tobacco budworm, to which this invention is especially applicable/ all have chain lengths of 14 to 16 carbon atoms, and internal un-saturation. The aldehyde's unsaturation should not be closer to the chain end than the 5-position/ for reaction with the luciferase enzyme in the assay system of the present invention.
1~34~ Z
In another embodiment of the invention, the sex pheromonal substance to be detected is initially secreted by the insect in a form other than aldehyde. It may, for example, be secreted in the form of a long chain alcohol, acid or ester.
Indeed, the biological precursor of the sex pheromonal aldehyde is in many cases believed to be the corresponding alcohol.
The major component of the sex pheromone of the silkworm is a long chain alcohol. In such cases, there is included in the process of the present invention a step of converting the pheromone into an aldehyde ready for assay with reduced flavine mononucleotide and luciferase enzyme. The preferred method of such conversion is by means of an enzyme, e.g.
horse liver alcohol dehydrogenase, when the initial substance is in alcohol form, or a suitable chemical reagent.
In addition to detection of pheromonal aldehyde in atmosphere, by collection thereof in a liquid form, the process according to the present invention is also useful in the determination of pheromonal contents of insect glands, for study of the pheromonal emission cycles of the lnsect. The measurement of pheromone levels in insects is one of the first steps in the study of pheromone biosynthesis and release. Such studies are important for the effective application of pheromones for the control of insect populations and the prevention of damage to forests and farming crops. The assay according to the present invention is sensitive enough to 1~3464Z
measure quantitatively the pheromone levels in the gland of a single insect such as spruce budworm. The pheromone may be obtained from the spruce budworm by obtaining a heptane or hexane extract of the gland, in which the major component of the spruce budworm pheromone (E~ tetradecenal) is soluble, evaporati~g off the organic solvent, re-dissolving the residue in water, and subjecting this solution to the assay method of the invention. The amount of pheromone obtained from a heptane extract of the gland of the single spruce budworm is in the range of 0.4 to 7 0 pmoles. Heptane extracts of the body of the same insect contain little if any pheromone ~0-0.4 pmoles) The chemical reactions which occur on subjection of reduced flavine mononucleotide to aldehyde, oxygen and luciferase enzyme are incompletely understood, but are believed to involve the initial reaction of the mononucleotide with molecular oxygen to form an unstable oxidized interme-diate, followed by reaction with the aldehyde and molecular rearrangement to form a hydroxyl-substituted mononucleotide and a fatty acid. Light is emitted spontaneously at the molecular rearrangement stage, it is believed.
- The methods of collection of sampies for use in the present invention are generally as used in the prior art, with other monitoring methods. Cold traps for condensation of atmos-pheric samples therein can be used, but may be difficult to operate under field conditions. It is preferred to absorb the aldehyde from the il346 ~Z
atmospheric sample onto a suitable absorbent, for example by drawing the atmospheric sample, of known volume, through a gas chromatography column, packed with Porapak Q, then extracting the absorbent with a suitable organic solvent such as hexane or heptane, subsequently evaporating off the solvent and dissolving the residue in water.
The detection and analysis of the emitted light can be conducted using any known suitable light detection system for luminescence studies (i.e. a photo-multiplier tube). When quantitative determinations of aldehydes are the only required measurement, it may be equipped with a digital readout of maximum light intensity.
When it is desired to study other parameters, the detector may be coupled to an automatic plotter to print out a full light intensity emission and decay curve. From this curve, the aldehyde amount can be determined from the intensity maximum. The decay rate and curve should give information concerning the identity of the aldehyde present. Thus with an aldehyde sample of unknown type and amount, by use of various luciferase enzymes and comparison of curve shapes with standard curves, the type and amount of aldehyde can often be elucidated. The decay rate, e.g. from 80% to 40%
of intensity maximum, is especially useful in this regard.
The accompanying figures are graphical representa-tions of decay curves and the like, derived from specific 11346~Z
samples as detailed below in the specific examples, given by way of illustration only.
EXAMPLES
Samples for test were generally obtained by the following procedure.
Air was passed over two micrograms of E-ll-tetradecenal, the major component of the spruce budworm pheromone, for four hours at a flow rate of 70 liters per hour. The air stream was then passed through a column of Porapak Q (0.2 grams). The Porapak Q was extracted with one ml of hexane for two minutes, the hexane evaporated (about 5 minutes) and the aldehyde dissolved in water. The total recovery of pheromone as analysed by the bioluminescent assay was 25~ using this method.
In other cases, the pheromonic aldehyde was recovered by subjecting an atmospheric sample to a cold trap (dry ice acetone, minus 70C) to condense the aldehyde and other residual materials therein, followed by preparation of an aqueous solution thereof. Such methods have given recoveries of the order of 15%.
.
This example is given to demonstrate the general procedures according to the invention, but does not involve the use of hydroxylamine as an aldehyde scavenger.
The aqueous solution sample under test was injected 113~6~;~
into the assay mixture containing luciferase and reduced flavine mononucleotides which have been reduced in situ with sodium dithionite. This resulted in a rapid rise in luminescence to a maximum, dependent upon the amount of luciferase and pheromone aldehyde. Since excess mononucleotide or oxygen is rapidly removed by chemical oxidation or reduction respectively, the light intensity subsequently decays, dependent primarily on the turnover rate of the enzymes intermediate formed at the start of the reaction. Figure 1 illustrates the bioluminescent response of Beneckea Harveyi luciferase - to the isomers (E and Z) of the spruce budworm pheromone, ll-tetradecenal, determined in the procedure generally outlined above. The aldehyde (21 ng) in 1.0 ml of water at 22C was injected into 1.0 ml of 0.05 M of phosphate, 0.05 M
Of mercaptoethanol, pH 7.0, containing luciferase (about 10 micrograms) and S x 10 5M reduced flavine mononucleotide (reduced with about 0.5 milligrams of sodium dithionite). The light was detected with a photomultiplier tube and recorded graphically. One unit of luminescence corresponds to 5.5 x 10 9 quanta per second based on the standard of Hastings and Weber "J. OPT. SOC. AM., Volume 53, pages 1410-1415, (1963)." All aldehyde stocks were prepared in dimethyl formamide and stored at 4C. Luciferase was purified to homogeneity from the bioluminescent bacterium Beneckea harveyi~ The solid line represents the response of E-ll-tetradecenal and the dotted line represents the response of Z-ll-tetradecenal, of the spruce budworm pheromone.
Table I below shows the results of conducting the procedure described above on samples of the three listed aldehydes. Each aldehyde was assayed to give a curve as shown in Fig. l, and the maximum luminescence and the time required for light emission to decay from 80~ to 40% of its maximum of intensity recQrded. The values given in Table I for each aldehyde are the average of between 10 and 14 determinations.
TABLE I
Bioluminescent Response of Luc;ferase to Spruce Bud~Jorm Pheromone Maximum Half-time (sec) for Aldehyde Luminescence (+ s.d.)Luminescent Decay (+ s.d.) Tetradecanal 16 + 3 0.71 + .04 E-ll-Tetradecanal 16 + 2 0.79 + .09 Z~ Tetradecanal 13 + 1 0.99 + .07 .
EXAMPLE II
Two and three day old Eastern spruce budworms, Choristoneura Fumiferana, maintained in continuous light, were excised between 15 and 17 hours, EST, and extracted with microliters of heptane for 10 minutes. The heptane extract was directly transferred to a 20 ml glass vial, evporated under 113~642 low vacuum for 2 minutes to remove the heptane, vortexed for 10 seconds with 10 ml of water, and then assayed 10 minutes later by the assay procedure according to the invention, i.e.
as described in Example 1 but with 0.01 M hydroxylamine present in the assay mixture to lower the endogeneous activity.
The results are given in Figure 2 and Table II, In Figure 2, the maximum luminescence is plotted versus the amount of added E~ tetradecenal. Each point is the average of two assays with the bars representing the range of the experimental data. The abdomen samples in Table II refer to the tissue in immediate proximity to the gland with the mass of material analysed being approximately twice that for the gland. Each budworm sample was analysed at least twice, the average value corrected for background response for reference heptane samples containing no budworm material and then converted into nanograms based on the luminescent response of standards of E-ll-tetradecenal in heptane carried through the identical process.
TABLE II
Pheromone Levels in the Spruce Budworm . Average Amount Number of Per Budworm - Budworms Analyzed nq + s.d. Range (ng) Female Gland 32 2.3 + 1.6 0.2 - 5.7 Female Abdomen 6 0.1 ~ 0.07 0.0 - 0.2 Female Head/Antennae 6 0.2 + 0.07 0.1 - 0.3 I Male Head/Antennae 6 0.1 + 0.06 0.0 - 0.2 The results in ~igure 2 indicate that amounts of pheromone as low as lO0 fentomoles can be measured according to the process of the present in~vention, with the elimination of the background aldehyde as described. Whilst these specific experiments in Table II were conducted on extract from insect budworms, it is clear that essentially similar results are obtained on atmospherically collected samples, of the same order of concentration.
The capability to detect very low amounts of phero-mones using the luminescence assay according to the present invention is of major advantage for studying the syntheses, regulation and release of the pheromone from the female of the species such as the spruce budworm. The assay is sensitive enough quantitatively to measure the pheromone levels in the gland of a single spruce budworm. The results show that the amount of pheromone obtained from a heptane extract of the gland of a single spruce budworm moth is in the range of 1-25 picamoles. In contrast, heptane extracts of the body of the same insect contained little if any pheromone. The measurement of pheromones by the bioluminescent assay process according to the present invention, as applied to pheronome release into the air, has the advantages of rapidity, sensitivity and ease of quantitation. Trapping of the atmospheric pheromones by 1~3~6 ~Z
condensation with water in cold traps can be undertaken, since the presence of water does not interfere wtih the analysis of samples by this method and the pheromones are relatively stable in water.
Whilst the invention has been described in reference to certain specific examples and procedures, it will be appreciated that it is not to be limited in scope to the precise experimental conditions and procedure described.
The scope of the invention is limited only by the scope of the appended claims.
FIELD OF THE INVENTION
; This invention relates to pheromone monitoring systems, and more particularly to methods of detecting and monitoring the quantity of pheromone substances in a gaseous or liquid environmental sample.
BACKGROUND OF THE INVENTION
It is known that animals secrete substances known as pheromones, to influence the behaviour of other animals of the same species. Insects, for example, secrete sex pheromones, in extremely low levels, to attract mates of the opposite sex.
The mechanism of production and secretion of sex pheromones by insects is still incompletely understood. They appear to be secreted by moths or beetles during mating periods, at a particular daily calling stage. Insect population control systems have recently been proposed, based on the use of the sex pheromones of the insects concerned.
Some insects that cause damage to forestry and farming products have been reported to secrete long chain unsaturated aldehydes that function as sex pheromones, either alone or in combination with other substances. Examples include such major pests as the corn earworm (cotton bollworm), the dermestia beetles, the spruoe budw~rm and the tobacco budworm. Consequently, methods have been developed to monitor or control the population of these pests by use of the appropriate sex pheromone substance, either synthetically or naturally produced. Lures ~a~
- 1134~i4Z
have been made, containing the sex pheromone, optionally in admixture with a solid or liquid carrier, in which the insects are trapped and counted so as to monitor the population.
Infested areas may be sprayed with sufficient sex pheromone to confuse the insects and prevent their mating and reproduction.
The sex pheromone may be included in a pheromone trap, in which insects are collected for killing, e.g. by inclusion of insecticide in the trap.
Further developments and studies of sex pheromones and their potential uses in insect population control are, however, hampered by the lack of a suitable method for deter-mining the concentrations of the pheromones being used.
Effective concentrations of pheromones for this purpose are extremely small, in many cases below the minimal range. Before the efficiency and potential utility of any method of insect population control based upon the use of sex pheromones can be properly assessed and developed, there needs to be a method which can be used in association with the pheromone which will permit the accurate and rapid detection and monitoring of the pheromone substances. Only in this way can efficiency of traps be established, and the necessary effective concentrations of pheromones be established and hence applied.
BRIEF DESCRIPTION OF THE PRIO~ ART
The current practice for monitoring pheromone con-centrations is by means of gas-liquid chromatcgraphy. In such processes, a sample of atmosphere is collected by absorption from the air, extracted with an organic solvent, in some instances derivatized to a form more easily detected on analysis, and then subjected to gas chromatography. Such a procedure is difficult and time-consuming, requiring the conducting of several steps of sample preparation. Furthermore, such methods are generally not sensitive enough to detect the very lcw concentrations of sex pheromones effective in the atmosphere as insect attractants.
It is kncwn that long chain aldehydes will react with reduced flavine mononucleotide, in the presence of oxygen and a bacterial luciferase enzyme, to oxidise the flavine mono-nucleotide substrate and the aldehyde, with the emission of light, at 490 nm. This phenomenom is generally known as bacterial bioluminescence. Descriptions of this process are to be found in the academic literature, for example in a paper by J. W. Hastings, "Annual Review of Biochemistry", 1968, Volume 37, Pages 597-630, and in a paper by J. W. Hastings and K. H. Nealson, "Annual Review of Microbiology", 1977, Volume 31, Pages 549-595. There have also been reports that certain long chain fatty acids, specifically myristic acid, will also cause in vivo bacterial bioluminescence presumably by being converted "in vivo" to the corresponding aldehyde.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that, with suitable modifications, the light emitting reaction of oxidation of a reduced flavine nucleotide catalysed by a 6~Z
luciferase enzyme can be conducted in the presence of a sex pheromone aldehyde, and can be used to monitor very accurately the amount of sex pheromone aldehyde present, down to extremely small quantities. It has been found that the intensity of emitted light is a function of the quantity of sex pheromonal aldehyde present. By the process of the invention, quantities of sex pheromone aldehyde as low as 0.1 picamoles (10 -13moles) or 20 picagrams can be readily determined. Moreover, by analysing the decay characteristics of the emitted light after it reaches its maximum intensity, the identity of the phero-monal aldehyde may be determined.
Thus according to the present invention, there is provided a process of analysing a liquid sample for content of aldehyde of sex pheromonal origin which comprises:
preparing a solution of reduced flavine mononucleotide and luciferase enzyme;
treating said solution with hydroxylamine;
reacting the so-treated solution with said aldehyde-containing liquid sample in the presence of water and oxygen, to cause oxidation of the reduced flavine mononucleotide with emission of light;
and determing the intensity of said light emission, to determine therefrom the content of aldehyde in said liquid sample.
The amount of sex pheromonal aldehyde to be deter-~346 ~Z
mined in the monitoring of pheromonc traps, down to 0.1 picamoles, is so low that special methods of assay and pre-cautions in connection therewith have to be adopted in order to ensure that all of the aldehyde detected is derived from the sex pheromone being monitored. Thus, special precautions have to be taken to remove background aldehyde contaminations.
In accordance with the invention, hydroxylamine is used for this aldehyde scavenging process, and special methods and procedures for reducing the flavine mononucleotide and pre-paring the reaction solutions for the light-emitting lumines-cence reaction are used. The method of the invention is then accurate and sensitive enough, to allow quantitative detection and measurement of sex pheromonal aldehydes down to the necessary low concentrations for insect trap monitoring.
Thus in the process of the invention, the reduced monoflavine nucleotide is prepared in admixture with the enzyme luciferase. This mixture is scavenged for residual aldehyde with hydroxylamine. Then the sample of sex pheromonal aldehyde in water is added. The water contains the required amount of oxygen for the luminescence reaction. Surprisingly, it is found that the sex pheromonal aldehyde in the sample reacts with the reduced monoflavine nucleotide to give light emission of intensity dependent quantitatively on the amount of sex pheromonal aldehyde, despite the presence in the reaction mixture of residual amounts of hydroxylamine, the background 1134~i~Z
aldehyde scavenger. It would have been expected that the residual hydroxylamine would have reactedextensively with the se~
pheromonal aldehyde, to interfere with the reaction between the sex pheromonal aldehyde and the reduced flavine mononucleotide, but surprisingly this does not happen. This feature permits the necessary thorough scavenging of background aldehyde with hydroxylamine to give the required accurate determinations of sex pheromonal aldehyde, but also dictates certain conditions on the order of addition of reagents in the process of the invention. Thus, it is necessary to avoid having the test sample and hydroxylamine present in admixture in the reaction medium in the absence of the reduced flavine mononucleotide and bacterial luciferase enzyme. For example, pre-mixing of the liquid test sample and the enzyme solution, ; followed by addition of the reduced flavine mononucleotide thereto, a procedure which would normally be adopted to give increased sensitivity, cannot in fact be adopted sinoe this precludes scavenging of the enzyme solution with hydroxylamine. Only by the procedure of the invention can the scavenging with hydroxylamine of all the reagent solutions, necessary to give results of sufficient accuracy, be properly conducted.
DESCRIPTION OF THE PREFERRED EMsoDIMENTs Whilst there are several known and acceptable methods for making reduced flavine mononucleotide, the solution of ; reduced flavine mononucleotide is preferably prepared by reduction of the flavine mononucleotide, in the presence of 113~64Z
the luciferase enzyme, with sodium dithionite. This is best conducted shortly before the light-emitting reaction with the aldehyde test sample to be conducted. Sodium dithionite, a solid, powdery material under normal conditions, does not have an adverse effect on the luciferase enzyme, at least over the short term. It is preferred to include enzyme stabilizers such as mercaptoethanol, and phosphate buffers to maintain a pH of 6-8, in the solution, to ensure stability of the enzyme therein. Since the assay method of the invention may require to be conducted under field conditions, it is desirable to use convenient solid powdered reagents, such as sodium dithionite where available, and as many premixed solutions as possible. Other methods for reduction of flavine mono-nucleotide, such as bubbling gaseouS hydrogen through a solution thereof, are known and can be used under certain circumstances. ~lowever, bubbling hydrogen will deactivate the luciferase enzyme, and so this method cannot be used with a premixed flavine mononucleotide-luciferase solution. lt is also satisfactory to form a premixed solution of the luciferase enzyme and appropriate buffers with the hydroxylamine, and then mix this solution with flavine mononucleotide before or after it has been reduced.
This reaction is suitably conducted at or about room temperature or field temperatures, provided that the reagents and solutions are kept in the liquid phase. Slightly elevated 1~34164Z
temperatures may offer certain advantages. The reaction should be conducted at pH at which the enzyme is stable, normally approximately neutral.
After mixing of all of the necessary reagents and sample, light is emitted as a result of the flavine oxidation reaction. The emitted light rapidly rises to a maximum intensity, and then decays in intensity. The aldehyde con-centration in the sample is determinable from the maximum light intensity. By analysis of various features of the light intensity-decay curve, in the absence of interfering impurities, characterization of the aldehyde can be obtained, by comparison with standard curves.
The specific choice of species of luciferase enzyme is made in accordance with the nature of the pheromonal aldehyde to be detected. The light emitting response of the reaction mixture is different, depending upon the chosen luciferase enzyme. It appears that each luciferase enzyme species responds over a relatively narrow range of chain length aldehydes, one luciferase enzyme being preferred over the range C10-C16 chain aldehydes, and another through the range C14-C20.
The bacteria luciferase enzymes which are used in the process of the invention comprise a relatively small, but kncwn and characterised group. They are obtained and purified from marine bacteria, being synthesized as the bacteria grow. Samples of 113~64Z
suitable marine bacterla are available from standard culture collections. The enzymes may be stored for extended periods of time, under cool conditions, in buffered solutions e.g.
aqueous glycerol solutions. They may be prepared for use by dilution into aqueous solution, in the absence of glycerol, suitably buffered. The term "bacterial luciferase enzyme"
refers to an enzyme of bacterial origin/ which will catalyze the reaction of reduced flavine mononucleotide and aldehyde in the presence of oxygen with the emission of light. At least six of these enzymes have been purified and characterized.
The best ones for use in the process of the present invention are those giving high sensitivity, with a low response in the absence of exogeneous aldehyde and a high response in the presence thereof. Routine screening tests of the available enzymes will readily show those best for use in the inven-tion under any specific conditions.
The sex pheromonal aldehydes which have been identi-fied for four major insect pests/ namely the dermestia beetles, ; the corn earworm,the spruce budworm and the tobacco budworm, to which this invention is especially applicable/ all have chain lengths of 14 to 16 carbon atoms, and internal un-saturation. The aldehyde's unsaturation should not be closer to the chain end than the 5-position/ for reaction with the luciferase enzyme in the assay system of the present invention.
1~34~ Z
In another embodiment of the invention, the sex pheromonal substance to be detected is initially secreted by the insect in a form other than aldehyde. It may, for example, be secreted in the form of a long chain alcohol, acid or ester.
Indeed, the biological precursor of the sex pheromonal aldehyde is in many cases believed to be the corresponding alcohol.
The major component of the sex pheromone of the silkworm is a long chain alcohol. In such cases, there is included in the process of the present invention a step of converting the pheromone into an aldehyde ready for assay with reduced flavine mononucleotide and luciferase enzyme. The preferred method of such conversion is by means of an enzyme, e.g.
horse liver alcohol dehydrogenase, when the initial substance is in alcohol form, or a suitable chemical reagent.
In addition to detection of pheromonal aldehyde in atmosphere, by collection thereof in a liquid form, the process according to the present invention is also useful in the determination of pheromonal contents of insect glands, for study of the pheromonal emission cycles of the lnsect. The measurement of pheromone levels in insects is one of the first steps in the study of pheromone biosynthesis and release. Such studies are important for the effective application of pheromones for the control of insect populations and the prevention of damage to forests and farming crops. The assay according to the present invention is sensitive enough to 1~3464Z
measure quantitatively the pheromone levels in the gland of a single insect such as spruce budworm. The pheromone may be obtained from the spruce budworm by obtaining a heptane or hexane extract of the gland, in which the major component of the spruce budworm pheromone (E~ tetradecenal) is soluble, evaporati~g off the organic solvent, re-dissolving the residue in water, and subjecting this solution to the assay method of the invention. The amount of pheromone obtained from a heptane extract of the gland of the single spruce budworm is in the range of 0.4 to 7 0 pmoles. Heptane extracts of the body of the same insect contain little if any pheromone ~0-0.4 pmoles) The chemical reactions which occur on subjection of reduced flavine mononucleotide to aldehyde, oxygen and luciferase enzyme are incompletely understood, but are believed to involve the initial reaction of the mononucleotide with molecular oxygen to form an unstable oxidized interme-diate, followed by reaction with the aldehyde and molecular rearrangement to form a hydroxyl-substituted mononucleotide and a fatty acid. Light is emitted spontaneously at the molecular rearrangement stage, it is believed.
- The methods of collection of sampies for use in the present invention are generally as used in the prior art, with other monitoring methods. Cold traps for condensation of atmos-pheric samples therein can be used, but may be difficult to operate under field conditions. It is preferred to absorb the aldehyde from the il346 ~Z
atmospheric sample onto a suitable absorbent, for example by drawing the atmospheric sample, of known volume, through a gas chromatography column, packed with Porapak Q, then extracting the absorbent with a suitable organic solvent such as hexane or heptane, subsequently evaporating off the solvent and dissolving the residue in water.
The detection and analysis of the emitted light can be conducted using any known suitable light detection system for luminescence studies (i.e. a photo-multiplier tube). When quantitative determinations of aldehydes are the only required measurement, it may be equipped with a digital readout of maximum light intensity.
When it is desired to study other parameters, the detector may be coupled to an automatic plotter to print out a full light intensity emission and decay curve. From this curve, the aldehyde amount can be determined from the intensity maximum. The decay rate and curve should give information concerning the identity of the aldehyde present. Thus with an aldehyde sample of unknown type and amount, by use of various luciferase enzymes and comparison of curve shapes with standard curves, the type and amount of aldehyde can often be elucidated. The decay rate, e.g. from 80% to 40%
of intensity maximum, is especially useful in this regard.
The accompanying figures are graphical representa-tions of decay curves and the like, derived from specific 11346~Z
samples as detailed below in the specific examples, given by way of illustration only.
EXAMPLES
Samples for test were generally obtained by the following procedure.
Air was passed over two micrograms of E-ll-tetradecenal, the major component of the spruce budworm pheromone, for four hours at a flow rate of 70 liters per hour. The air stream was then passed through a column of Porapak Q (0.2 grams). The Porapak Q was extracted with one ml of hexane for two minutes, the hexane evaporated (about 5 minutes) and the aldehyde dissolved in water. The total recovery of pheromone as analysed by the bioluminescent assay was 25~ using this method.
In other cases, the pheromonic aldehyde was recovered by subjecting an atmospheric sample to a cold trap (dry ice acetone, minus 70C) to condense the aldehyde and other residual materials therein, followed by preparation of an aqueous solution thereof. Such methods have given recoveries of the order of 15%.
.
This example is given to demonstrate the general procedures according to the invention, but does not involve the use of hydroxylamine as an aldehyde scavenger.
The aqueous solution sample under test was injected 113~6~;~
into the assay mixture containing luciferase and reduced flavine mononucleotides which have been reduced in situ with sodium dithionite. This resulted in a rapid rise in luminescence to a maximum, dependent upon the amount of luciferase and pheromone aldehyde. Since excess mononucleotide or oxygen is rapidly removed by chemical oxidation or reduction respectively, the light intensity subsequently decays, dependent primarily on the turnover rate of the enzymes intermediate formed at the start of the reaction. Figure 1 illustrates the bioluminescent response of Beneckea Harveyi luciferase - to the isomers (E and Z) of the spruce budworm pheromone, ll-tetradecenal, determined in the procedure generally outlined above. The aldehyde (21 ng) in 1.0 ml of water at 22C was injected into 1.0 ml of 0.05 M of phosphate, 0.05 M
Of mercaptoethanol, pH 7.0, containing luciferase (about 10 micrograms) and S x 10 5M reduced flavine mononucleotide (reduced with about 0.5 milligrams of sodium dithionite). The light was detected with a photomultiplier tube and recorded graphically. One unit of luminescence corresponds to 5.5 x 10 9 quanta per second based on the standard of Hastings and Weber "J. OPT. SOC. AM., Volume 53, pages 1410-1415, (1963)." All aldehyde stocks were prepared in dimethyl formamide and stored at 4C. Luciferase was purified to homogeneity from the bioluminescent bacterium Beneckea harveyi~ The solid line represents the response of E-ll-tetradecenal and the dotted line represents the response of Z-ll-tetradecenal, of the spruce budworm pheromone.
Table I below shows the results of conducting the procedure described above on samples of the three listed aldehydes. Each aldehyde was assayed to give a curve as shown in Fig. l, and the maximum luminescence and the time required for light emission to decay from 80~ to 40% of its maximum of intensity recQrded. The values given in Table I for each aldehyde are the average of between 10 and 14 determinations.
TABLE I
Bioluminescent Response of Luc;ferase to Spruce Bud~Jorm Pheromone Maximum Half-time (sec) for Aldehyde Luminescence (+ s.d.)Luminescent Decay (+ s.d.) Tetradecanal 16 + 3 0.71 + .04 E-ll-Tetradecanal 16 + 2 0.79 + .09 Z~ Tetradecanal 13 + 1 0.99 + .07 .
EXAMPLE II
Two and three day old Eastern spruce budworms, Choristoneura Fumiferana, maintained in continuous light, were excised between 15 and 17 hours, EST, and extracted with microliters of heptane for 10 minutes. The heptane extract was directly transferred to a 20 ml glass vial, evporated under 113~642 low vacuum for 2 minutes to remove the heptane, vortexed for 10 seconds with 10 ml of water, and then assayed 10 minutes later by the assay procedure according to the invention, i.e.
as described in Example 1 but with 0.01 M hydroxylamine present in the assay mixture to lower the endogeneous activity.
The results are given in Figure 2 and Table II, In Figure 2, the maximum luminescence is plotted versus the amount of added E~ tetradecenal. Each point is the average of two assays with the bars representing the range of the experimental data. The abdomen samples in Table II refer to the tissue in immediate proximity to the gland with the mass of material analysed being approximately twice that for the gland. Each budworm sample was analysed at least twice, the average value corrected for background response for reference heptane samples containing no budworm material and then converted into nanograms based on the luminescent response of standards of E-ll-tetradecenal in heptane carried through the identical process.
TABLE II
Pheromone Levels in the Spruce Budworm . Average Amount Number of Per Budworm - Budworms Analyzed nq + s.d. Range (ng) Female Gland 32 2.3 + 1.6 0.2 - 5.7 Female Abdomen 6 0.1 ~ 0.07 0.0 - 0.2 Female Head/Antennae 6 0.2 + 0.07 0.1 - 0.3 I Male Head/Antennae 6 0.1 + 0.06 0.0 - 0.2 The results in ~igure 2 indicate that amounts of pheromone as low as lO0 fentomoles can be measured according to the process of the present in~vention, with the elimination of the background aldehyde as described. Whilst these specific experiments in Table II were conducted on extract from insect budworms, it is clear that essentially similar results are obtained on atmospherically collected samples, of the same order of concentration.
The capability to detect very low amounts of phero-mones using the luminescence assay according to the present invention is of major advantage for studying the syntheses, regulation and release of the pheromone from the female of the species such as the spruce budworm. The assay is sensitive enough quantitatively to measure the pheromone levels in the gland of a single spruce budworm. The results show that the amount of pheromone obtained from a heptane extract of the gland of a single spruce budworm moth is in the range of 1-25 picamoles. In contrast, heptane extracts of the body of the same insect contained little if any pheromone. The measurement of pheromones by the bioluminescent assay process according to the present invention, as applied to pheronome release into the air, has the advantages of rapidity, sensitivity and ease of quantitation. Trapping of the atmospheric pheromones by 1~3~6 ~Z
condensation with water in cold traps can be undertaken, since the presence of water does not interfere wtih the analysis of samples by this method and the pheromones are relatively stable in water.
Whilst the invention has been described in reference to certain specific examples and procedures, it will be appreciated that it is not to be limited in scope to the precise experimental conditions and procedure described.
The scope of the invention is limited only by the scope of the appended claims.
Claims (6)
1. A process of analysing a liquid sample for content of aldehyde of sex pheromonal origin, which comprises:
preparing a solution of reduced monoflavine nucleotide and bacterial luciferase enzyme;
treating said solution with hydroxylamine;
reacting the so-treated solution with said aldehyde-containing liquid sample in the presence of water and oxygen, to cause oxidation of the reduced flavine mononucleotide with emission of light;
and determining the intensity of the light emission, to determine therefrom the content of aldehyde in said liquid sample.
preparing a solution of reduced monoflavine nucleotide and bacterial luciferase enzyme;
treating said solution with hydroxylamine;
reacting the so-treated solution with said aldehyde-containing liquid sample in the presence of water and oxygen, to cause oxidation of the reduced flavine mononucleotide with emission of light;
and determining the intensity of the light emission, to determine therefrom the content of aldehyde in said liquid sample.
2. The process of claim 1 wherein the reduced flavine mononucleotide is prepared by treating the flavine mononucleo-tide with sodium dithionite.
3. The process of claim 2 wherein the reduction of the flavine mononucleotide takes place in situ, in the presence of the luciferase enzyme.
4. The process of claim 3 wherein the treatment of the reaction solution with hydroxylamine takes place during the in situ reduction of the flavine mononucleotide.
5. The process of claim 4 wherein solution containing the luciferase enzyme contains mercaptoethanol as an enzyme stabilizer.
6. The process of claim 5 wherein the solution containing the luciferase enzyme is buffered to pH 6 - 8 by phosphate buffers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000354584A CA1134642A (en) | 1980-06-23 | 1980-06-23 | Pheromone detection system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000354584A CA1134642A (en) | 1980-06-23 | 1980-06-23 | Pheromone detection system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1134642A true CA1134642A (en) | 1982-11-02 |
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ID=4117241
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000354584A Expired CA1134642A (en) | 1980-06-23 | 1980-06-23 | Pheromone detection system |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109557058A (en) * | 2017-09-26 | 2019-04-02 | 北京华泰诺安探测技术有限公司 | A kind of method and apparatus for detecting the whether aqueous molecule of under test gas |
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1980
- 1980-06-23 CA CA000354584A patent/CA1134642A/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109557058A (en) * | 2017-09-26 | 2019-04-02 | 北京华泰诺安探测技术有限公司 | A kind of method and apparatus for detecting the whether aqueous molecule of under test gas |
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