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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
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

• the present invention relates to a biosensor for use in detecting the presence of a selected nucleic acid sequence in a sample and to a method of detecting the presence of a selected nucleic acid (NA) sequence in a sample.
• Assays for the presence of specific DNA or RNA sequences in samples have applications in many fields. For example, infection of patients with a particular microorganism can be assessed by analyzing a biological sample from the patient for the presence of a NA sequence specific for the microorganism. Other applications include the analysis of food or environmental samples to detect contamination. The detection of genetic disease caused by mutations can also be achieved by these techniques. To date, the widespread use of this highly sensitive method has been restricted by the need for significant amplification of the amount of the specific nucleic acid sequence and/or the need to use specialized techniques to analyze the amplified DNA This amplification is generally achieved using polymerase chain or ligase chain reaction and the amplified DNA is then separated on a gel and the gel analyzed for the presence of specific bands of DNA.
• US 4,840,893 which teaches a method of nucleic acid detection.
• the method uses a probe sequence with attached ligand, an enzyme mediator system linked to a second ligand and which is capable of transferring a charge to an electrical surface when the enzyme is catalytically active, and an antiligand, to which the first and second ligands compete for binding.
• the assay is a competition system whereby the binding of the target sequence in a sample affects the availability of the first ligand and alters the rate of charge transfer to the electrode.
• the present invention has the advantage of providing a direct gating mechanism, which does not rely on a signal generated by enzyme catalysis.
• U.S. 4,868,104 describes a method of detecting a nucleotide sequence by the use of two polynucleotide reagents, which bind to different sequences on the target analyte, the first of which can be polymerized and the second has a detection system.
• nucleic acid detection methods most of which involve labelling of the probes. These include: U.S. 4,968,602; U.S. 5,116,733; U.S. 4,868,105; U.S. 4,716,106;
• Figures 1 to 3 show the results obtained in Examples 1 to 3.
• Fig. 4 shows linker gramicidin B
• Fig. 5 shows membrane spanning lipid
• Fig. 6 shows linker lipid A
• Fig. 7 shows biotinylated gramicidin E
• the present inventors propose an apparatus which can be used to detect the presence of a specific NA sequence in a sample.
• the apparatus is best described as a biosensor in that it involves the use of lipid membranes.
• Membranes for the use in biosensors have been disclosed In international
• Patent Application Nos PCT/AU88/00273, PCT/AU89/00352 PCT/AU90/00025 and PCT/AU92/00132 The disclosure of these applications is included herein by reference.
• suitably modified lipid molecules may be caused to assemble into an electrode/ionic reservoir/insulating bilayer combination that is suitable for incorporation of ion channels and ionophores. It is also disclosed that the conductance of these membranes is dependent on the presence or absence of an analyte. In bilayer membranes in which each layer includes ion channel monomers, the conductance of the membrane is dependent on the lining up of the monomers in each layer to form continuous ion channels which span the membrane. As these continuous ion channels are constantly being formed and destroyed, the conductance of the membrane is dependent on the lifetimes of these continuous ion channels.
• the present invention consists in a biosensor for use in detecting the presence of a selected nucleic acid sequence in a sample, the biosensor comprising an electrode and a bilayer membrane having a top and a bottom layer, the bottom layer being proximal to and connected to the electrode in a manner such that a space exists between the membrane and the electrode, the conductance or impedance of the membrane being dependent on the presence or absence of the selected nucleic acid sequence, the membrane comprising a closely packed array of amphiphilic molecules and a plurality of ion channels comprising first half membrane spanning monomers dispersed in the top layer and second half membrane spanning monomers dispersed in the bottom layer, the first half membrane spanning monomers being capable of lateral diffusion within the upper layer and the second half membrane-spanning monomers being prevented from lateral diffusion within the bottom layer, a first ligand specifically reactive with the selected nucleic acid sequence attached to an end of a proportion of the first half membrane-spanning monomers proximal the surface of the membrane and
• the membrane includes membrane spanning amphiphiles which are prevented from lateral diffusion within the membrane.
• the present invention consists in a biosensor for use in detecting the presence of a selected nucleic add sequence in a sample, the biosensor comprising an electrode and a bilayer membrane having a top and a bottom layer, the bottom layer being proximal to and connected to the electrode in a manner such that a space exists between the membrane and the electrode, the conductance or impedance of the membrane being dependent on the presence or absence of the selected nucleic acid sequence, the membrane comprising a closely packed array of amphiphilic molecules, membrane spanning amphiphiles which are prevented from lateral diffusion within the membrane and a plurality of ion channels comprising first half membrane spanning monomers dispersed in the top layer and second half membrane spanning monomers dispersed in the bottom layer, the first half membrane spanning monomers being capable of lateral diffusion within the upper layer and the second half membrane-spanning monomers being prevented from lateral diffusion within the bottom layer, a first ligand specifically reactive with the selected nucleic acid sequence attached to either an end of at least
• the present invention consists in a method of detecting the presence of a selected nucleic acid sequence within a sample, the method comprising adding the sample to the biosensor of the first or second aspect of the present invention and detecting change in the impedance or conductance of the membrane.
• the biosensor and method of the present invention functions by the binding of the selected nucleic acid sequence to the first and second ligands causing a change in the ability of ions to traverse the membrane via the ion channels.
• the specificity of the detection is provided by the specificity of the first ligand for the selected nucleic acid squence.
• This specifity may be provided in a number of ways, however, it is presently preferred that this is achieved by the use of first ligand which comprises a nucleic acid molecule or PNA which includes a sequence complementary to a first sequence within the selected nucleic acid.
• the sequence within the first ligand may be of any length sufficient to provide the required level of sensitivity, typically at least 10 residues.
• the nucleic acid molecule may include modified bases.
• the second ligand may be an antibody directed against DNA, or binding fragment thereof.
• the second ligand may also be an intercalating agent, major or minor groove binder or an agent capable of triple helix formation or an agent capable of covalently linking to nucleic acids upon activation or combinations thereof. It is preferred that the second ligand is not specific for the selected nucleic acid sequence as this results in simpler assembly of the biosensor as only one specific ligand need be produced for each selected nucleic acid sequence that is to be detected.
• the biosensor may also include a variety of second ligands which vary over the biosensor.
• the second ligand attached to one membrane spanning amphiphile may be a different moiety to the second ligand attached to another membrane spanning amphiphile.
• the essential feature is that the second ligand binds to nucleic acid or to a marker attached thereto. Accordingly, it is to be understood that the term "second ligand" embraces a single or multiple species of moieties which bind nucleic acid or to a marker attached thereto.
• nucleic acid sequence to be detected will be labelled with a marker, for example a biotinylated PCR product.
• both the first and second ligands are specific for the selected nucleic acid sequence.
• the first and second ligands are specific for different sequences within the selected nucleic acid sequence.
• the first and second ligands may be attached in any of a number of ways. For example, streptavidin may be attached as described in the applications setout above. This can then be used to bind biotinylated nucleic acid. It is preferred that the first and second ligands are attached via linkers to the ion channels or membrane spanning lipids. Where a linker is used it is preferred that the linker is hydrophilic. It is further preferred that the linker includes a phosphoramidite group. It is also expected that changing the linker length may affect the gating response detected in the biosensor.
• first and second ligands are nucleic acid sequences this may be advantageously achieved by producing a molecule having the two DNA sequences which are to act as the ligands at each end with an RNA sequence in the middle. The two ends of the molecule are then attached to the biosensor surface and the central RNA portion digested with RNaseH to leave the biosensor with the two separated DNA sequences attached.
• the first ligand comprises a nucleic acid molecule which includes a sequence complementary to a first sequence within the selected nucleic acid. It is further preferred that the second ligand comprises a nucleic acid molecule which includes a sequence complementary to a second sequence within the selected nucleic acid. Due to the presence of such complementary sequences the first and second ligands bind to the selected nucleic acid by hydridisation. It is presently preferred that the first and second ligands are oligonucleotide sequences selected to bind to different regions of the selected nucleic acid sequence in the sample.
• Methods to detect or sense specific NA sequences using the impedance bridge described in International Patent Application Nos PCT/AU88/00273, PCT/AU88/00273, PCT/AU89/00352, PCT/AU90/00025 and PCT/AU92/00132 may, for example, be achieved by manufacturing the membrane with both biotinylated membrane spanning lipids (MSL) and biotinylated gramicidin. Streptavidin, which binds strongly to biotin is immobilized by reaction with these biotinylated compounds.
• MSL biotinylated membrane spanning lipids
• Streptavidin which binds strongly to biotin is immobilized by reaction with these biotinylated compounds.
• a mixture of two biotinylated oligonucleotide sequences are then attached to the streptavidin. These sequences are preferably non complementary.
• One sequence would be a suitable probe for a sequence of interest e.g., an infectious organism probe sequence, and the other sequence would be the complement to another part of the target DNA.
• the present invention consists in a method of detecting the presence of a selected nucleic acid within a sample, the method comprising the following steps:
• a biosensor comprising an electrode and a bilayer membrane having a top and a bottom layer, the bottom layer being proximal to and connected to the electrode in a manner such that a space exists between the membrane and the electrode, the conductance or impedance of the membrane being dependent on the presence or absence or the selected nucleic acid sequence, the membrane comprising a closely packed array of amphiphilic molecules and a plurality of ion channels comprising first half membrane spanning monomers dispersed in the top layer and second half membrane spanning monomers dispersed in the bottom layer, the first half membrane spanning monomers being capable of lateral diffusion within the upper layer and the second half membrane- spanning monomers being prevented from lateral diffusion within the bottom layer, a first ligand specifically reactive with a first nucleic acid sequence attached to an end of a proportion of the first half membrane-spanning monomers proximal the surface of the membrane and a second ligand specifically reactive with a second nucleic acid sequence attached to an end of the remainder of the first half membrane-
• the challenge sequence is unable to bind to both the first and second ligands and thus, crosslink.
• the present invention consists in a method of detecting the presence of a selected nucleic acid sequence within a sample, the method comprising the following steps:
• a biosensor comprising an electrode and a bilayer membrane having a top and a bottom layer, the bottom layer being proximal to and connected to the electrode in a manner such that a space exists between the membrane and the electrode, the conductance or impedance of the membrane being dependent on the presence or absence or the selected nucleic acid sequence, the membrane comprising a closely packed array of amphiphilic molecules, membrane spanning amphiphiles which are prevented from lateral diffusion within the membrane and a plurality of ion channels comprising first half membrane spanning monomers dispersed in the top layer and second half membrane spanning monomers dispersed in the bottom layer, the first half membrane spanning monomers being capable of lateral diffusion within the upper layer and the second half membrane-spanning monomers being prevented from lateral diffusion within the bottom layer, a first ligand specifically reactive with the selected nucleic acid sequence attached to either an end of at least a proportion of the first half membrane-spanning monomers proximal the surface of the membrane or to an end of the membrane-spanning amphi
• the present invention consists in a biosensor for use in detecting the presence of a selected nucleic acid sequence in a sample, the biosensor comprising an electrode and a bilayer membrane having a top and a bottom layer, the bottom layer being proximal to and connected to the electrode in a manner such that a space exists between the membrane and the electrode, the conductance or impedance of the membrane being dependent on the presence or absence of the selected nucleic acid sequence, the membrane comprising a closely packed array of amphiphilic molecules and a plurality of ion channels comprising first half membrane spanning monomers dispersed in the top layer and second half membrane spanning monomers dispersed in the bottom layer, the first half membrane spanning monomers being capable of lateral diffusion within the upper layer and the second half membrane-spanning monomers being prevented from lateral diffusion within the bottom layer, a first ligand specifically reactive with the selected nucleic acid sequence attached to an end of a proportion of the first half membrane-spanning monomers proximal the surface of the membrane and
• the present invention consists in a biosensor for use in detecting the presence of a selected nucleic acid sequence in a sample, the biosensor comprising an electrode and a bilayer membrane having a top and a bottom layer, the bottom layer being proximal to and connected to the electrode in a manner such that a space exists between the membrane and the electrode, the conductance or impedance of the membrane being dependent on the presence or absence of the selected nucleic acid sequence, the membrane comprising a closely packed array of amphiphilic molecules, membrane spanning amphiphiles which are prevented from lateral diffusion within the membrane and a plurality of ion channels comprising first half membrane spanning monomers dispersed in the top layer and second half membrane spanning monomers dispersed in the bottom layer, the first half membrane spanning monomers being capable of lateral diffusion within the upper layer and the second half membrane-spanning monomers being prevented from lateral diffusion within the bottom layer, a first ligand specifically reactive with the selected nucleic acid sequence attached to either an end of at least
• the biosensor detects the presence of the selected nucleic acid sequence by competition.
• the selected nucleic acid sequence if present in sample to be tested, competes with the second ligand for binding to the first ligand.
• the present invention consists in a biosensor for use in detecting the presence of a selected nucleic acid sequence in a sample, the biosensor comprising an electrode and a bilayer membrane having a top and a bottom layer, the bottom layer being proximal to and connected to the A 7/00 1
• the membrane comprising a closely packed array of amphiphilic molecules and a plurality of ion channels comprising first half membrane spanning monomers dispersed in the top layer and second half membrane spanning monomers dispersed in the bottom layer, the first half membrane spanning monomers being capable of lateral diffusion within the upper layer and the second half membrane-spanning monomers being prevented from lateral diffusion within the bottom layer and a first ligand specifically reactive with the selected nucleic acid sequence attached to an end of a proportion of the first half membrane-spanning monomers proximal the surface of the membrane.
• the first ligand upon addition of sample containing the selected nucleic acid sequence the first ligand will bind to the selected DNA sequence and change the mobility of the first half membrane monomers or restrict ion flow through the channel. This will result in a membrane with a different conductance.
• the first and second half membrane spanning monomers are gramicidin or one of its derivatives.
• the bilayer membrane is attached to the electrode via linking molecules such that a space exists between the membrane and the electrode.
• Preferred linking molecules are those disclosed in application PCT/AU92/00132.
• the second half membrane spanning monomers are attached to the electrode via linker groups.
• the bilayer membrane includes membrane spanning lipids, similar to those found in archaebacteria.
• Cross linking is characterized by the cross linking sequence bringing the first half membrane spanning monomers out of alignment with the second half membrane spanning monomers. This results in a membrane having a second conductance which is different from the first conductance. This difference in conductance can be measured and will be indicative of the presence of the selected DNA sequence in the sample.
• the first and second ligands may be the same or different. Oligonucleotide ligands which bind to specific NA sequences are well known in the art.
• the ligands are peptide nucleic acids (PNA).
• PNA peptide nucleic acids
• PNA PNA
• advantages gained by the use of PNA include the ability of PNA to recognize specific sequences in double stranded DNA and to bind thereto either by strand displacement or triple helix formation. This is in contrast-to ohgonucleotides which can bind single strand or double strand but not by strand displacement. It should be noted that PNA can also bind to single stranded DNA.
• the target probe sequence could be a PNA molecule for tighter binding.
• a mixture of two biotinylated oligonucleotide sequences are immobilized onto streptavidin.
• the target analyte is captured.
• the target analyte need only have the sequence of interest. No cross linking occurs, therefore gating is not observed and a first measurement of conductance is taken.
• a second oligonucleotide sequence is then used to challenge the system.
• This challenge sequence contains both the target oligonucleotide sequence and the complementary sequence to the cross linking sequence. If binding of both epitopes occurs, cross linking will take place, thus gating off the ion channels and changing the conductance of the membrane. If binding of only epitope two occurs (owing to the binding of the target DNA to its probe sequence) no cross linking takes place, thus no gating is seen.
• the NA probe sequence in principle, can be of any length and will contain the base sequence complementary to the target analyte for detection.
• the target analyte can be a DNA or an RNA sequence.
• a PNA probe sequence may also be of any length and will contain the base sequence complementary to the target analyte for detection.
• the target analyte can be a
• DNA or an RNA sequence as PNA binds to both.
• PNA binds DNA and RNA more tightly than does DNA, therefore the challenge sequence should not be able to displace the already bound target sequence.
• target sequences are listed below. Listeria monocytogenes; a 19 base pair oligonucleotide that is specific for a 36S rRNA sequence; detection in food industry (milks, cheeses etc.) See: R. F. Wang W. W. Cao and M.G. Johnson, App. Environ.
• Detection of target polynucleotide sequences can be illustrated by the following example in which specific DNA gating is observed for a target DNA concentration of 5 nM (1 pmole). The total lack of any response in the presence of a non-complementary probe sequence can be seen in the controls.
• Streptavidin was added to all wells (5 ⁇ L O.Olmg/mL in PBS) and allowed to react with biotinylated Gramicidin E for 10-15 minutes before washing out excess unbound streptavidin with PBS.
• 5 ⁇ L of a 1:1 mixture of DNA probe F (200nM):DNA probe G (200nM in PBS) was added to sensor wells.
• a DNA non-specific binding probe H (5 ⁇ L 400 nM in PBS) was added to control wells. The probes were allowed to react with streptavidin for 10-15 minutes then excess unbound probes were washed out with PBS.
• lOO ⁇ L of DNA target I (lOnM) in PBS was added to each well. The binding of DNA target I to the sensor wells gave a decrease in the admittance at minimum phase, but no significant change in membrane admittance in control wells ( Figure 1).
• DNA probe F The amount and rate of decrease of admittance at minimum phase is related to the amount of DNA present in the test solution and therefore can be used to determine concentration in test solutions.
• DNA probe F The amount and rate of decrease of admittance at minimum phase is related to the amount of DNA present in the test solution and therefore can be used to determine concentration in test solutions.
• This example illustrates the ability to perform a second, confirmatory step whereby an enzyme can be used to cleave the bound polynucleotide, thus confirming that a specific region of interest is present.
• Electrodes were prepared and 2 n layer added as described in Example 1. Subsequent streptavidin, DNA probe and DNA target sequences were added as described in Example 1, except that they were carried out at 30°C.
• DNase I activation buffer consists of 50 nM Tris.HCl, pH 7.6, 50 nM NaCl, 10 nM MgCl 2 , 10 nM MnCl 2 , 0.2 mg/mL BSA.
• DNase I was added (2 ⁇ L lmg/mL in a 50% w/v glycerol solution of 20 mM Tris.HCl, pH 7.6, 1 mM MgCl z ) to sensor and control wells. Addition of DNase I gave an increase in admittance at minimum phase for sensor wells, but no significant increase for control wells (figure 2), showing that cleavage of DNA was being detected.
• Example 3 DNA gating by addition of 2 biotinylated complementary polynucleotide sequences
• This example illustrates the possibility of detecting only one specific sequence on the polynucleotide of interest by having a capture molecule incorporated in the target. It also illustrates the use of peptide nucleic acid (PNA) sequences.
• PNA peptide nucleic acid
• Electrodes were prepared and 2 n layer added as described in Example 1. All steps were carried out at room temperature. 5 ⁇ L of the 2nd layer was added to the working electrode before addition of a 180 ⁇ L volume of phosphate buffered saline (lOmM Na 2 HPO 4 , ImM KH 2 P0 4 , 137mM NaCl,

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• 1997
  • 1997-05-22 AU AU27578/97A patent/AU734086B2/en not_active Ceased
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