WO1998039655A1 - Detection of analyte species - Google Patents
Detection of analyte species Download PDFInfo
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- WO1998039655A1 WO1998039655A1 PCT/GB1998/000688 GB9800688W WO9839655A1 WO 1998039655 A1 WO1998039655 A1 WO 1998039655A1 GB 9800688 W GB9800688 W GB 9800688W WO 9839655 A1 WO9839655 A1 WO 9839655A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
Definitions
- This invention relates to the detection of analyte species, in particular to such detection by a method in which characteristic gaseous or volatile species are detected.
- the well known ELISA (Enzyme Linked Immuno Sorbent Assay) technique is suitable for the detection of a wide variety of antigens.
- the technique involves the use of a substrate to which is bound a first antibody.
- the substrate is first washed with a solution which contains an antigen specific to the antibody, and then treated with a second antibody, also specific to the antigen.
- the second antibody is adapted to comprise a reporter molecule.
- Detection of the presence of the antigen is accomplished by detecting a change occurring due to the reporter molecule.
- the reporter molecule might be an enzyme, in which case some feature of the reaction established by the enzyme is detected. Typically, this feature is a colour change resulting from the enzyme catalysed reaction.
- the reporter molecule might comprise a radioactive isotope.
- the method is applicable to the detection of a very wide range of analyte species, which need not be antigens.
- Microorganisms, DNA, RNA, biomolecules and other molecular species can be detected using the present invention.
- the receptor species being adapted so that the complex formed by the receptor species and the analyte species bound thereto can catalyse a reaction producing a gaseous or volatile species;
- the catalytic production of the gaseous or volatile species results in amplification of the detected signal.
- the gaseous or volatile species produced can be detected with excellent efficiency and sensitivity by judicious selection of the gas sensor employed.
- the analyte species may be bound with a receptor species which possess an enzyme moiety, wherein the enzyme moiety catalyses the reaction producing a gaseous or volatile species.
- the analyte species may be bound by at least two receptor species, wherein the complex holds the receptor species in sufficiently close proximity that the receptor species cooperate to catalyse the reaction producing a gaseous or volatile species.
- the receptor species must be brought together, or at least in close proximity, by the formation of the complex in order to effect a response (the production of the gaseous or volatile species). In this way, detection of gaseous or volatile species can be correlated with the presence of the complex and thus the presence of the analyte species.
- the receptor species may possess enzyme sub-unit moieties which cooperate to catalyse the reaction when held in the complex. Alternatively, or additionally, at least one receptor species may possess a co-factor moiety.
- the complex may be separated from receptor species which have not bound an analyte species before the step of causing the reaction to occur is performed.
- the analyte species may be bound by at least two receptor species, and one of the receptor species may be attached to a substrate.
- the complex may be separated from receptor species which have not bound an analyte species by washing the substrate.
- the detection of the gaseous or volatile species may be performed with an array of sensors of which an electrical property varies according to the exposure to gases or volatile species.
- the detection of the gaseous or volatile species may be performed with at least one semiconducting organic polymer sensor.
- the detection of the gaseous or volatile species may be performed by a mass spectrometry or by GC-MS.
- the enzyme reaction may comprise the cleavage of at least one chemical bond of a substrate in order to produce the gaseous or volatile species.
- a urease enzyme may cause said cleavage.
- the analyte species may be a microorganism, which may be a bacterium, a virus, a microfungus, a yeast, an alga, a mammalian cell or a plant cell.
- the at least one receptor species may be an antibody, a DNA strand or a RNA strand. It is also possible to use other binding proteins, other bio-molecules, macromolecules, supramolecular species or simpler molecular species.
- the invention also provides a method for detecting a plurality of analyte species in which each analyte species is bound by different receptor species so that a plurality of complexes are formed by the receptor species and the analyte species, the receptor species being adapted so that complexes comprising different analyte species can catalyse reactions producing different gaseous or volatile species, and wherein the detection of one gaseous or volatile species is correlated with the presence of the corresponding analyte species.
- Figure 1 shows a modified ELISA scheme for detecting a single anti genie species
- Figure 2 shows a modified ELISA scheme for detecting a plurality of anti genie species
- Figure 3 shows the detection of an analyte using DNA receptors
- Figure 4 shows the detection of an analyte using a locally generated functional enzyme.
- the invention provides a method for detecting an analyte species comprising the steps of:
- the analyte species 14 with at least one receptor species 12, 16, the receptor species 12, 16 being adapted so that the complex formed by the receptor species 12, 16 and the analyte species 14 bound thereto can catalyse a reaction (A ⁇ B + C) producing a gaseous or volatile species (B);
- a generalised ELISA system is shown in Figure 1.
- a substrate 10 such as a membrane or a bead has antibodies of a first type 12 attached thereto.
- Treatment with a solution containing an anti genie species 14 leads to binding of the anti genie species 14 with the antibodies of the first type 12.
- Subsequent treatment with antibodies of a second type 16 leads to binding thereof with the anti genie species 14.
- the antibodies of the second type 16 comprise an enzyme moiety 18, which catalyses some diagnostic reaction.
- the enzyme moiety 18 catalyses the production of a gaseous or volatile species (B) which is subsequently detected.
- B gaseous or volatile species
- the antigenic species may be a bacterium, virus, microfungus, yeast, alga, mammalian or plant cell, protein or steroid, depending on the specific system employed.
- Figure 3 shows an DNA assay employing the method of the present invention in which a DNA analyte 50 is attached to a substrate 52 and a receptor species 54 comprises a DNA strand having an enzyme moiety 56.
- the presence of the analyte species 50 is detected by detecting a generalised gaseous or volatile species B. It is also possible to utilise RNA strands, one example being 16S RNA which is useful for bacteria recognition.
- receptor species such as suitably modified binding proteins, other biomolecules, macromolecules or simpler molecular species.
- So-called "supramolecular” species may be useful as receptor species.
- Supramolecular species such as calixarenes and crown ethers , can selectively bind a wide range of molecules, from simple organic chemicals to complex bio-molecules, usually by providing binding or chelating sites in cavities of dimensions which correspond to the molecular size of the analyte species. The strength of the binding, and the specificity of the binding towards the particular analyte species in question can be selected depending on the specific application envisaged.
- analyte species detectable is very broad, including microorganisms, DNA, RNA, biomolecules and other molecular species.
- One use is to detect chemicals which are not themselves readily detectable by gas sensors. This is done by binding the chemical with a receptor species which can catalyse a reaction producing a gaseous or volatile species which is readily detectable by gas sensing.
- the detection of the gaseous or volatile species may be accomplished by any suitable gas detection technique suitable for the particular species to be detected. Ionisation techniques, mass spectrometry and GC-MS are examples of such techniques.
- the detection of the gaseous or volatile species is performed with an array of sensors of which an electrical property varies according to exposure to gases or vapours.
- the array comprises semiconducting organic polymers, which exhibit a number of advantageous properties such as rapid response times, good sensitivity, discrimination and robustness (see for example, J V Hatfield, P Neaves, P J Hicks, K Persaud and P Travers, Sensors & Actuators B, 18-19 (1994) 221-228).
- arrays of other devices, such as MOS sensors are within the scope of the invention.
- a single semiconducting organic polymer sensor may be employed, in conjunction with ac interrogation.
- the enzyme catalyses the cleavage of at least one chemical bond of a substrate in order to produce the gaseous or volatile species.
- the reaction is a unimolecular elimination reaction such as shown in the Figure, where the enzyme catalyses cleavage of the substrate A to produce product C and gaseous or volatile product B.
- the enzyme is a urease enzyme, producing ammonia as an easily identifiable gaseous species, detectable with excellent sensitivity.
- Figure 4 shows an alternative approach to the catalytic production of a gaseous or volatile species, in which an analyte species 60 is bound by a first receptor species 62 and a second receptor species 64, and wherein the complex (defined by the bound analyte species 60 and the two receptor species 62, 64) holds the receptor species 62, 64 in sufficiently close proximity that the receptor species cooperate to catalyse the reaction producing a gaseous or volatile species (B).
- “cooperate” it is meant that the receptor species 62, 64 interact in some way to catalyse the reaction, neither the first receptor species 62 nor the second receptor species 64 being able to catalyse the reaction in its own.
- receptor species 62, 64 possess enzyme sub-unit moieties 66, 68 which cooperate to catalyse the reaction when held in the complex, by forming a "functional enzyme".
- one receptor species might possess a co-factor which enables an enzyme or a functional enzyme to catalyse the reaction.
- an organic phase and an aqueous phase with the analyte species in one phase and one or more receptor species in the other phase, in which the complex is preferentially partitioned into the analyte containing phase.
- a single receptor species in order to detect an analyte species.
- the receptor species do not individually catalyse the gas producing reaction, as described above in relation to Figure 4, then it is not necessary to separate the complex from unreacted, free receptor species. It may still be desirable to attach one receptor species to a substrate.
- the invention also provides a method for detecting a plurality of analyte species in a single test, using the same principles as described above, in which each analyte species is bound by different receptor species so that a plurality of complexes are formed by the receptor species and the analyte species, the receptor species being adapted so that complexes comprising different analyte species catalyse reactions producing different gaseous or volatile species, and wherein the detection of each gaseous or volatile species is correlated with the presence of the corresponding analyte species.
- Figure 2 shows a modified ELISA scheme which enables a plurality of antigenic species to be detected, comprising the steps of:
- three different antibody types 22, 24, 26 are attached to the surface 20, permitting simultaneous and selective binding of three different types of antigenic species 28, 30, 32. Detection of all these antigenic species 28, 30, 32 is accomplished with the second plurality of different antibody types, which in this example comprise three antibody types 40, 42, 44.
- Each antibody type 40, 42, 44 has a different enzyme 34, 36, 38 which catalyses a different reaction viz, A ⁇ B+C; D ⁇ E+F; G ⁇ H+I.
- the different gaseous volatile species produced are B, E and H, and therefore simultaneous but selective detection of the species B, E, and H provides simultaneous and selective detection of the three antigenic species antigenic species 28, 30, 32.
- One way of detecting the different gaseous or volatile species simultaneously is to provide a plurality of gas sensors, each gas sensor being specific to a particular gaseous or volatile species.
- Another way is to employ a single device which is capable of detecting the individual components of gaseous mixtures. The latter approach is more powerful, since a single device can be used to detect a variety of gaseous species or combinations thereof.
- Arrays of semiconducting organic polymer sensors using dc interrogation are suitable in this regard. Most preferably, however, is the use of one or more semiconducting organic polymer sensors together with ac interrogation. This technique is capable of quantitively identifying the individual components in quaternary gaseous mixtures. It will be appreciated that other experimental aspects, discussed above in relation to the detection of a single antigenic species, are also applicable in the detection of a plurality of antigenic species.
- the catalytic activity of the enzyme or enzymes results in the production of detectable quantities of a gas or gases.
- the gas or gases may be continuously sampled by the detector, or a headspace may be allowed to develop which is subsequently sampled.
- the reaction medium may be aqueous, another solvent, or an oil.
- Water vapour in particular, can provide interfering background signals during gas detection. Such interfering signals may be reduced or even eliminated if a gas permeable but water rejecting membrane is employed as a barrier between the reaction medium and the gas detector.
- a possible variant is to bind the antibody of the first type onto the surface of this membrane itself.
- the use of a gas permeable membrane is in itself advantageous since it permits positioning of the sensor very close to the source of the gas, thereby increasing sensitivity.
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Abstract
There is disclosed a method for detecting an analyte species comprising the steps of: binding the analyte species with at least one receptor species, the receptor species being adapted so that the complex formed by the receptor species and the analyte species bound thereto can catalyse a reaction producing a gaseous or volatile species; causing said reaction to occur, thereby producing said gaseous or volatile species; detecting said gaseous or volatile species; and correlating detection of said gaseous or volatile species with the presence of the analyte species.
Description
DETECTION OF ANALYTE SPECIES
This invention relates to the detection of analyte species, in particular to such detection by a method in which characteristic gaseous or volatile species are detected.
The well known ELISA (Enzyme Linked Immuno Sorbent Assay) technique is suitable for the detection of a wide variety of antigens. The technique involves the use of a substrate to which is bound a first antibody. The substrate is first washed with a solution which contains an antigen specific to the antibody, and then treated with a second antibody, also specific to the antigen. The second antibody is adapted to comprise a reporter molecule. Detection of the presence of the antigen is accomplished by detecting a change occurring due to the reporter molecule. The reporter molecule might be an enzyme, in which case some feature of the reaction established by the enzyme is detected. Typically, this feature is a colour change resulting from the enzyme catalysed reaction. Alternatively, the reporter molecule might comprise a radioactive isotope.
International Publication WO 95/33848 describes a method for identifying bacteria on the basis of characteristic gases and vapours emitted as by-products of bacterial metabolism. The gases and vapours are detected by an array of gas sensing devices, in particular arrays based on semiconducting organic polymers.
International Publication WO 94/04705 describes the detection of Escherichia coli in which the bacteria is incubated with a glucuronide conjugate which is cleaved by the action of β-glucuronidase (present in Escherichia coli). The cleavage produces a gaseous or volatile compound, such as o-nitrophenol, which is detected by a gas detection system such as an ionising ultraviolet radiation based detector or a gas chromatograph.
The present invention concerns inter alia a modification of the ELISA methodology in which the enzymatic reaction produces a volatile or gaseous species which is detected in the gas phase. However, it should be noted that the method is applicable to the detection of a very wide range of analyte species, which need not be antigens. Microorganisms, DNA, RNA, biomolecules and other molecular species can be detected using the present invention.
According to the invention there is provided a method for detecting an analyte species comprising the steps of:
binding the analyte species with at least one receptor species, the receptor species being adapted so that the complex formed by the receptor species and the analyte species bound thereto can catalyse a reaction producing a gaseous or volatile species;
causing said reaction to occur, thereby producing said gaseous or volatile species;
detecting said gaseous or volatile species; and
correlating detection of said gaseous or volatile species with the presence of the analyte species.
An important advantage is that the catalytic production of the gaseous or volatile species results in amplification of the detected signal. The gaseous or volatile species produced can be detected with excellent efficiency and sensitivity by judicious selection of the gas sensor employed.
The analyte species may be bound with a receptor species which possess an enzyme moiety, wherein the enzyme moiety catalyses the reaction producing a gaseous or volatile species.
Alternatively, the analyte species may be bound by at least two receptor species, wherein the complex holds the receptor species in sufficiently close proximity that the receptor species cooperate to catalyse the reaction producing a gaseous or volatile species. The receptor species must be brought together, or at least in close proximity, by the formation of the complex in order to effect a response (the production of the gaseous or volatile species). In this way, detection of gaseous or volatile species can be correlated with the presence of the complex and thus the presence of the analyte species. The receptor species may possess enzyme sub-unit moieties which cooperate to catalyse the reaction when held in the complex. Alternatively, or additionally, at least one receptor species may possess a co-factor moiety.
The complex may be separated from receptor species which have not bound an analyte species before the step of causing the reaction to occur is performed.
The analyte species may be bound by at least two receptor species, and one of the receptor species may be attached to a substrate. In this instance, the complex may be separated from receptor species which have not bound an analyte species by washing the substrate.
The detection of the gaseous or volatile species may be performed with an array of sensors of which an electrical property varies according to the exposure to gases or volatile species.
The detection of the gaseous or volatile species may be performed with at least one semiconducting organic polymer sensor.
The detection of the gaseous or volatile species may be performed by a mass spectrometry or by GC-MS.
The enzyme reaction may comprise the cleavage of at least one chemical bond of a substrate in order to produce the gaseous or volatile species. A urease enzyme may cause said cleavage.
The analyte species may be a microorganism, which may be a bacterium, a virus, a microfungus, a yeast, an alga, a mammalian cell or a plant cell.
The at least one receptor species may be an antibody, a DNA strand or a RNA strand. It is also possible to use other binding proteins, other bio-molecules, macromolecules, supramolecular species or simpler molecular species.
The invention also provides a method for detecting a plurality of analyte species in which each analyte species is bound by different receptor species so that a plurality of complexes are formed by the receptor species and the analyte species, the receptor species being adapted so that complexes comprising different analyte species can catalyse reactions producing different gaseous or volatile species, and wherein the detection of one gaseous or volatile species is correlated with the presence of the corresponding analyte species.
Methods in accordance with the present invention will now be described with reference to the accompanying drawings, in which :-
Figure 1 shows a modified ELISA scheme for detecting a single anti genie species;
Figure 2 shows a modified ELISA scheme for detecting a plurality of anti genie species;
Figure 3 shows the detection of an analyte using DNA receptors; and Figure 4 shows the detection of an analyte using a locally generated functional enzyme.
The invention provides a method for detecting an analyte species comprising the steps of:
binding (as shown in Figure 1) the analyte species 14 with at least one receptor species 12, 16, the receptor species 12, 16 being adapted so that the complex formed by the receptor species 12, 16 and the analyte species 14 bound thereto can catalyse a reaction (A → B + C) producing a gaseous or volatile species (B);
causing said reaction to occur, thereby producing said gaseous or volatile species (B);
detecting said gaseous or volatile species (B); and
correlating detection of said gaseous or volatile species (B) with the presence of the analyte species 14.
A generalised ELISA system is shown in Figure 1. A substrate 10 such as a membrane or a bead has antibodies of a first type 12 attached thereto. Treatment with a solution containing an anti genie species 14 leads to binding of the anti genie species 14 with the antibodies of the first type 12. Subsequent treatment with antibodies of a second type 16 leads to binding thereof with the anti genie species 14. The antibodies of the
second type 16 comprise an enzyme moiety 18, which catalyses some diagnostic reaction. In the present invention, the enzyme moiety 18 catalyses the production of a gaseous or volatile species (B) which is subsequently detected. It will be appreciated that there are many commonly known variations on the generalised ELISA system described above. For example, a plurality of antibodies of the second type might bind to a single antigen, providing further amplification to the amplification inherently provided by the use of a catalytic system.
The antigenic species may be a bacterium, virus, microfungus, yeast, alga, mammalian or plant cell, protein or steroid, depending on the specific system employed.
However, it is important to note that the receptor species do not have to be antibodies, and the analyte species do not have to be antigens. Figure 3 shows an DNA assay employing the method of the present invention in which a DNA analyte 50 is attached to a substrate 52 and a receptor species 54 comprises a DNA strand having an enzyme moiety 56. The presence of the analyte species 50 is detected by detecting a generalised gaseous or volatile species B. It is also possible to utilise RNA strands, one example being 16S RNA which is useful for bacteria recognition.
Other kinds of receptor species are possible, such as suitably modified binding proteins, other biomolecules, macromolecules or simpler molecular species. So- called "supramolecular" species may be useful as receptor species. Supramolecular species, such as calixarenes and crown ethers , can selectively bind a wide range of molecules, from simple organic chemicals to complex bio-molecules, usually by providing binding or chelating sites in cavities of dimensions which correspond to the molecular size of the analyte species. The strength of the binding, and the specificity of the binding towards the particular analyte species in question can be selected depending on the specific application envisaged. Similarly, the range of analyte species detectable is very broad, including microorganisms, DNA, RNA, biomolecules and other molecular
species. One use is to detect chemicals which are not themselves readily detectable by gas sensors. This is done by binding the chemical with a receptor species which can catalyse a reaction producing a gaseous or volatile species which is readily detectable by gas sensing.
The detection of the gaseous or volatile species may be accomplished by any suitable gas detection technique suitable for the particular species to be detected. Ionisation techniques, mass spectrometry and GC-MS are examples of such techniques. In a preferred method the detection of the gaseous or volatile species is performed with an array of sensors of which an electrical property varies according to exposure to gases or vapours. Most preferably the array comprises semiconducting organic polymers, which exhibit a number of advantageous properties such as rapid response times, good sensitivity, discrimination and robustness (see for example, J V Hatfield, P Neaves, P J Hicks, K Persaud and P Travers, Sensors & Actuators B, 18-19 (1994) 221-228). However, arrays of other devices, such as MOS sensors, are within the scope of the invention.
Commercially available arrays of semiconducting organic polymer gas sensors detect the presence of gas by monitoring changes in the dc resistance of the polymer. It should be noted that the use of ac based interrogation can provide excellent sensitivity and discrimination and, furthermore, can identify the components of mixtures of gases (see British Patent GB 2 203 553 and M E H Amrani, R M Dowdeswell, P A Payne and K C Persaud, Proceedings of Eurosensors X, Volume 2 (1996) pp 665-668). Use of the ac interrogation technique with semiconducting organic polymer gas sensors is within the scope of the invention.
In fact, a single semiconducting organic polymer sensor may be employed, in conjunction with ac interrogation.
Preferably, the enzyme catalyses the cleavage of at least one chemical bond of a substrate in order to produce the gaseous or volatile species. In other words, the reaction is a unimolecular elimination reaction such as shown in the Figure, where the enzyme catalyses cleavage of the substrate A to produce product C and gaseous or volatile product B. In a preferred, but non-limiting, example the enzyme is a urease enzyme, producing ammonia as an easily identifiable gaseous species, detectable with excellent sensitivity.
Figure 4 shows an alternative approach to the catalytic production of a gaseous or volatile species, in which an analyte species 60 is bound by a first receptor species 62 and a second receptor species 64, and wherein the complex (defined by the bound analyte species 60 and the two receptor species 62, 64) holds the receptor species 62, 64 in sufficiently close proximity that the receptor species cooperate to catalyse the reaction producing a gaseous or volatile species (B). By "cooperate" it is meant that the receptor species 62, 64 interact in some way to catalyse the reaction, neither the first receptor species 62 nor the second receptor species 64 being able to catalyse the reaction in its own. One way in which this can be achieved is if the receptor species 62, 64 possess enzyme sub-unit moieties 66, 68 which cooperate to catalyse the reaction when held in the complex, by forming a "functional enzyme". Alternatively, or possibly additionally, one receptor species might possess a co-factor which enables an enzyme or a functional enzyme to catalyse the reaction.
It will be apparent to the skilled reader that it is necessary to ensure that the detected gaseous or volatile species has been produced by the catalytic activity of the complex. With the methods described in relation to Figures 1 and 3, there exists the possibility that unreacted receptor species 16 and 54 might catalyze the reaction, and thus produce false positive results. When one of the receptor species is attached to a substrate, as is the case in Figures 1 and 3, thus possibility is averted by washing the substrate before causing the reaction to occur. It may be possible to separate by the
complex from free receptor species by arranging that the complex precipitates out of solution, or by employing a membrane which permits preferential transport of the complex across it. Alternatively, it might be possible to employ an organic phase and an aqueous phase, with the analyte species in one phase and one or more receptor species in the other phase, in which the complex is preferentially partitioned into the analyte containing phase. In all of these examples, it may be possible to employ a single receptor species in order to detect an analyte species.
If the receptor species do not individually catalyse the gas producing reaction, as described above in relation to Figure 4, then it is not necessary to separate the complex from unreacted, free receptor species. It may still be desirable to attach one receptor species to a substrate.
The invention also provides a method for detecting a plurality of analyte species in a single test, using the same principles as described above, in which each analyte species is bound by different receptor species so that a plurality of complexes are formed by the receptor species and the analyte species, the receptor species being adapted so that complexes comprising different analyte species catalyse reactions producing different gaseous or volatile species, and wherein the detection of each gaseous or volatile species is correlated with the presence of the corresponding analyte species.
Figure 2 shows a modified ELISA scheme which enables a plurality of antigenic species to be detected, comprising the steps of:
performing an ELISA test using: a first plurality of different antibody types 22, 24, 26 attached to a surface 20, each of the different antibody types 22, 24, 26 selectively binding a different antigenic species 28, 30, 32; and a second plurality of different antibody types 40, 42, 44, each of said second
plurality of different antibody types 40, 42, 44 selectively binding a different antigenic species 28, 30, 32 and having a different enzyme 34, 36, 38 which enzyme 34, 36, 38 catalyses a reaction producing a different gas or volatile species;
detecting the different gas or volatile species; and
correlating the detection of each gas or volatile species with the presence of the corresponding antigenic species 28, 30, 32.
In Figure 2, three different antibody types 22, 24, 26 are attached to the surface 20, permitting simultaneous and selective binding of three different types of antigenic species 28, 30, 32. Detection of all these antigenic species 28, 30, 32 is accomplished with the second plurality of different antibody types, which in this example comprise three antibody types 40, 42, 44. Each antibody type 40, 42, 44 has a different enzyme 34, 36, 38 which catalyses a different reaction viz, A → B+C; D→E+F; G→H+I. The different gaseous volatile species produced are B, E and H, and therefore simultaneous but selective detection of the species B, E, and H provides simultaneous and selective detection of the three antigenic species antigenic species 28, 30, 32.
In conventional ELISA tests, it is not uncommon to employ a highly specific first antibody attached to the substrate and a less specific second antibody in solution. When detecting a plurality of analytes using the present invention, it may be preferable to employ a single relatively non-specific first receptor species attached to a substrate and highly specific receptor species in solution.
One way of detecting the different gaseous or volatile species simultaneously is to provide a plurality of gas sensors, each gas sensor being specific to a particular gaseous or volatile species. Another way is to employ a single device which
is capable of detecting the individual components of gaseous mixtures. The latter approach is more powerful, since a single device can be used to detect a variety of gaseous species or combinations thereof. Arrays of semiconducting organic polymer sensors using dc interrogation are suitable in this regard. Most preferably, however, is the use of one or more semiconducting organic polymer sensors together with ac interrogation. This technique is capable of quantitively identifying the individual components in quaternary gaseous mixtures. It will be appreciated that other experimental aspects, discussed above in relation to the detection of a single antigenic species, are also applicable in the detection of a plurality of antigenic species.
The catalytic activity of the enzyme or enzymes results in the production of detectable quantities of a gas or gases. The gas or gases may be continuously sampled by the detector, or a headspace may be allowed to develop which is subsequently sampled. The reaction medium may be aqueous, another solvent, or an oil. Water vapour, in particular, can provide interfering background signals during gas detection. Such interfering signals may be reduced or even eliminated if a gas permeable but water rejecting membrane is employed as a barrier between the reaction medium and the gas detector. A possible variant is to bind the antibody of the first type onto the surface of this membrane itself. The use of a gas permeable membrane is in itself advantageous since it permits positioning of the sensor very close to the source of the gas, thereby increasing sensitivity.
Claims
1. A method for detecting an analyte species comprising the steps of:
binding the analyte species with at least one receptor species, the receptor species being adapted so that the complex formed by the receptor species and the analyte species bound thereto can catalyse a reaction producing a gaseous or volatile species;
causing said reaction to occur, thereby producing said gaseous or volatile species;
detecting said gaseous or volatile species; and
correlating detection of said gaseous or volatile species with the presence of the analyte species.
2. A method according to claim 1 in which the analyte species is bound with a receptor species which possesses an enzyme moiety, and wherein the enzyme moiety catalyses the reaction producing the gaseous or volatile species.
3. A method according to claim 1 in which the analyte species is bound by at least two receptor species, and wherein the complex holds the receptor species in sufficiently close proximity that the receptor species cooperate to catalyse the reaction producing a gaseous or volatile species.
4. A method according to claim 3 in which the receptor species possess enzyme sub-unit moieties which cooperate to catalyse the reaction when held in the complex.
5. A method according to claim 3 or claim 4 in which at least one receptor species possesses a co-factor moiety.
6. A method according to any of the previous claims in which the complex is separated from receptor species which have not bound an analyte species before the step of causing the reaction to occur is performed.
7. A method according to any of the previous claims in which the analyte species is bound by at least two receptor species, and one of the receptor species is attached to a substrate.
8. A method according to claim 7 when dependent on claim 6 in which the complex is separated from receptor species which have not bound an analyte species by washing the substrate.
9. A method according to any of the previous claims in which the detection of the gaseous or volatile species is performed with an array of sensors of which an electrical property varies according to exposure to gases or vapours.
10. A method according to any of the previous claims in which the detection of the gaseous or volatile species is performed with at least one semiconducting organic polymer sensor.
11. A method according to any of claims 1 to 8 in which the detection of the gaseous or volatile species is performed by mass spectrometry or by GC-MS.
12. A method according to any of the previous claims in which the reaction comprises the cleavage of at least one chemical bond of a substrate in order to produce gaseous or volatile species.
13. A method according to claim 12 in which a urease enzyme causes said cleavage.
14. A method according to any of the previous claims in which the analyte species is a microorganism.
15. A method according to claim 14 in which the microorganism is a bacterium.
16. A method according to claim 14 in which the microorganism is a virus, a microfungus, a yeast, an alga, a mammalian cell or a plant cell.
17. A method according to any of the previous claims in which the at least one receptor species is an antibody.
18. A method according to any of claims 1 to 16 in which the at least one receptor species is a DNA or RNA strand.
19. A method for detecting a plurality of analyte species according to any of claims 1 to 18 in which each analyte species is bound by different receptor species so that a plurality of complexes are formed by the receptor species and the analyte species, the receptor species being adapted so that complexes comprising different analyte species catalyse reactions producing different gaseous or volatile species, and wherein the detection of each gaseous or volatile species is correlated with the presence of the corresponding analyte species.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9704627.0A GB9704627D0 (en) | 1997-03-06 | 1997-03-06 | Detection of antigenic species |
GB9704627.0 | 1997-03-06 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998039655A1 true WO1998039655A1 (en) | 1998-09-11 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1998/000688 WO1998039655A1 (en) | 1997-03-06 | 1998-03-06 | Detection of analyte species |
Country Status (2)
Country | Link |
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GB (1) | GB9704627D0 (en) |
WO (1) | WO1998039655A1 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994004705A1 (en) * | 1992-08-21 | 1994-03-03 | The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Detection of microorganisms using gas sensors |
WO1995033848A1 (en) * | 1994-06-09 | 1995-12-14 | Aromascan Plc | Detecting bacteria |
-
1997
- 1997-03-06 GB GBGB9704627.0A patent/GB9704627D0/en active Pending
-
1998
- 1998-03-06 WO PCT/GB1998/000688 patent/WO1998039655A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994004705A1 (en) * | 1992-08-21 | 1994-03-03 | The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Detection of microorganisms using gas sensors |
WO1995033848A1 (en) * | 1994-06-09 | 1995-12-14 | Aromascan Plc | Detecting bacteria |
Also Published As
Publication number | Publication date |
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GB9704627D0 (en) | 1997-04-23 |
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