EP1409728A2 - Analyse non enzymatique a liaison liposomique avec electrodes en reseau etroitement espacees (nel-ela) pour detecter et quantifier des acides nucleiques - Google Patents

Analyse non enzymatique a liaison liposomique avec electrodes en reseau etroitement espacees (nel-ela) pour detecter et quantifier des acides nucleiques

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Publication number
EP1409728A2
EP1409728A2 EP02735236A EP02735236A EP1409728A2 EP 1409728 A2 EP1409728 A2 EP 1409728A2 EP 02735236 A EP02735236 A EP 02735236A EP 02735236 A EP02735236 A EP 02735236A EP 1409728 A2 EP1409728 A2 EP 1409728A2
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Prior art keywords
affinity
liposomes
oligonucleotides
molecules
analyte
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EP02735236A
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German (de)
English (en)
Inventor
Reinhard Bredehorst
Rainer Hintsche
Anton Heuberger
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Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to non-enzymatic electrochemical assay procedures for the detection and quantification of nucleic acids and amplicons thereof using novel affinity liposomes containing electrochemically active reporter molecules.
  • Reporter systems most frequently utilized in assays for detecting and quantifying nucleic acids in fields such as biotechnology, environmental protection, and public health include detection by fluorescence, chemiluminescence, and colorimetry. These labels have been linked or conjugated directly to nucleic acid reactants and products, or generated via nucleic acid-enzyme conjugates similar to ELISA techniques.
  • the vast majority of assays for detecting and quantifying nucleic acids is based on enzyme-linked methods in combination with optical detection systems. As a consequence, these assays depend on clean and unsoiled measurement chambers, optical clarity and compatible viscosity of sample and support media, and a neutral optical background.
  • An electrochemical amplification step has been realized by redox recycling of electrochemically redox active reporter molecules such as p-aminophenol between the adjacent microband electrodes of an interdigitated electrode array, with each electrode within the diffusion layer of the other. Redox recycling occurs when both the oxidation and the reduction potential of the reversible redox species are applied to pairs of interdigitated electrodes.
  • array microelectrodes for the selective detection of redox species in the presence of interfering molecules has been described by several investigators (e.g. Sanderson, D.G., and Anderson, L.B. Anal. Chem. 57, 2388, 1985; Niwa, O., et al., W.R. Anal. Chem. 65, 1559, 1993). For these analyses, however, interdigitated arrays with relatively large electrodes spaced apart from each other by several micrometer were used.
  • enzymatic amplification systems for electrochemical detection procedures poses additional problems in that the reporter molecules have to be chemically modified to generate substrates which are electrochemically inactive prior to enzymatic action.
  • p-aminophenol has been adjusted for the alkaline phosphatase amplification system by converting the reporter molecule to the electrochemically inactive p-aminophenylphosphate.
  • reporter molecules such as osmium- or ruthenium-containing redox mediators, however, a similar adjustment has not been reported and is likely to pose substantial synthetic hurdles.
  • osmium- as well as ruthenium-containing redox mediators would be very useful for electrochemical detection systems since they provide a combination of important advantages for redox recycling including a reversible redox behaviour at electrodes, low oxidation/reduction potentials, slow oxidation by oxygen, and an excellent solubility in aqueous media.
  • reporter molecules may be immobilized onto solid support matrices via enzymatically cleavable spacer compounds.
  • This approach allows immobilized redox mediators to remain electrochemically active since access to the electrodes requires enzymatic cleavage of the spacer compound.
  • the technology requires additional solid support systems and is likely to provide only moderate amplification due to the limited amount of redox mediators that can be immobilized onto solid supports and the unfavorable enzyme kinetics at solid-liquid interfaces.
  • N-Ferrocenoyl-6-amino- 2,4-dimethylphenyl phosphate disodium salt is used as substrate for the alkaline phosphatase amplification system and the corresponding phenol product is entrapped selectively and irreversibly in a Nafion film electrode as a ferricinium salt by applying an anodic potential.
  • this method has the limitation that the Nafion film electrode can be used only once since the product is strongly retained within the polymer and phenyl derivative radicals are electropolymerized during the course of the anodic preconcentration.
  • the sodium salt of ferrocene ethyl phosphate ester (FcEtOPOs) 2- has been synthesized as a new alkaline phosphatase substrate (Limoges, B., and Degrand, C. Anal. Chem. 68, 4141 , 1996).
  • the enzyme product ferrocene ethanol
  • N- ferrocenoyl-6-amino-2,4-dimethylphenol is less hydrophobic than N- ferrocenoyl-6-amino-2,4-dimethylphenol and can be released from the Nafion film in its neutral form by cathodic stripping.
  • the limited efficiency of the cathodic stripping procedure restricts the reuse of the same electrode to a small number of analyses which is not in accordance with the requirements of cost effective assay procedures.
  • the assay principle is based on a competitive binding reaction of triazine pesticides and hapten-tagged liposomes to the immobilized monoclonal antibodies, followed by immunomigration of nonbound liposomes to a zone containing detergent at a concentration sufficient to release the liposome-entrapped ascobic acid.
  • the method is capable of detecting triazine pesticides in tap water at a concentration of approximately 1 ⁇ g/liter.
  • the technique described in this reference is neither applicable for detecting and quantifying nucleic acids nor does it teach how to design such an assay procedure.
  • Liposomes as carriers of entrapped reporter molecules have also been employed in many other non-enzymatic assay procedures.
  • the most frequently performed non-enzymatic liposome-based assays utilize liposomes that contain encapsulated fluorophores and surface-attached antigens or antibodies for the determination of proteinaceous antigens or antibodies in homogeneous or heterogeneous immunoassays (Diamandis, E.P., and Christopoulos, T.K. (eds.) Immunoassay, Academic Press, San Diego, p. 322 (1996).
  • liposome immunosorbent assays are also not suitable to detect and quantify nucleic acids via non-enzymatic electrochemical liposome- based techniques.
  • a reporter system for detecting an analyte, selected from nucleic acids and their amplicons, in a solution comprising the following components: (i) a solid support containing immobilized thereon capture oligonucleotides capable of binding to said analyte, preferably by forming specific helical complexes therewith, (ii) affinity liposomes containing encapsulated electrochemically detectable reporter molecules, preferably redox mediators, and surface-attached affinity components capable of specifically binding to said analyte and/or said capture oligonucleotide in a condition where analyte and capture oligonucleotides are bound to each other, but not to free capture oligonucleotides, and (iii) an electrochemical sensor for voltammetric, e.g. amperometric detection of electrochemical detectable reporter molecules.
  • a method for detecting and optionally quantifying an analyte, selected from nucleic acids and their amplicons, in a liquid sample to be analyzed comprising the steps of (a) providing (i) a solid support containing immobilized thereon capture oligonucleotides capable of binding said analyte, preferably by forming specific helical complexes therewith, and (ii) an electrochemical sensor, (b) contacting the sample to be analyzed with the immobilized capture oligonucleotides, (c) adding affinity liposomes or systems comprising affinity liposomes which contain encapsulated electrochemically detectable reporter molecules, preferably redox mediators, the affinity liposomes or systems comprising further surface-attached affinity components capable of specifically binding to the analyte to be detected and/or the said capture oligonucleotides in a condition where analyte and capture oligonucleotides are bound to each other
  • the electrochemical sensor is a closely spaced array (micrometer or submicrometer scale) of electrodes for voltammetric, e.g. and preferably amperometric detection of electrochemically detectable reporter molecules, the electrodes consisting e.g. of thin film nobel metal or carbon.
  • the components of this invention allow the design of various electrochemical assay configurations.
  • Preferred assay procedures include binding of affinity liposomes to captured nucleic acids and amplicons thereof, release of redox mediators from specifically bound affinity liposomes, and voltammetric quantification of released redox mediators via redox recycling.
  • the release of encapsulated redox mediators from specifically bound liposomes may be effected e.g. by detergent-mediated lysis or by a moderate increase of the ambient temperature.
  • the quantity of redox mediators is a proportional measure of the quantity of target nucleic acid or amplicons thereof in the specimen.
  • polymeric carrier molecules capable of binding multiple affinity liposomes and/or preformed complexes of affinity liposomes are utilized for amplified assay procedures.
  • Figure 1 shows the general assay procedure using affinity liposomes having surface-attached intercalating agents which serve as affinity components capable of specifically binding to the analyte in a condition where analyte and capture oligonucleotides are bound to each other as defined above.
  • Figure legend solid support (1 ); surface bound capture oligonucleotide (2); target DNA (3); region of DNA double helix (4); affinity liposome (5); intercalating residues (In) (6); flexible spacer molecules (7); redox mediators (8).
  • Figure 2 shows the general assay procedure using affinity liposomes having surface-attached oligonucleotides which serve as affinity components capable of specifically binding to the analyte in a condition where the said analyte is bound to the capture oligonucleotides as defined above .
  • Figure legend solid support (1); surface bound capture oligonucleotide (2); target DNA (3); region of DNA double helix (4); affinity liposome (5); flexible spacer molecules (7); redox mediators (8); single-stranded oligonucleotide (9).
  • Figure 3 shows an assay procedure comparable to that of figure 1 , but using additional polymeric carrier moecules for signal amplification.
  • Figure legend solid support (1); surface bound capture oligonucleotide (2); target DNA (3); region of DNA double helix (4); affinity liposome (5); intercalating residues (In) (6); flexible spacer molecules (7); redox mediators (8); polymeric carrier molecule (10); hapten (11 ); anti-hapten antibody (12).
  • Figure 4 shows an assay procedure comparable to that of fugure 1 , but using preformed complexes of affinity liposomes for signal amplification.
  • solid support (1) optionally comprising a sensor surface; surface bound capture oligonucleotide (2); target DNA (3); region of DNA double helix (4); affinity liposome (5); intercalating residues (In)
  • the novel electrochemical reporter technology of this invention is capable of detecting and quantifying target nucleic acids or their amplicons by molecular biological and immunochemical procedures for analytical and clinical applications.
  • Analyses of specific nucleic acids or nucleic acid sequences are commonly, but not exclusively used to examine body fluids or tissues for the presence of infectious microorganisms, malignancies, inherited genetic defects, pharmacogenomics, forensic medical evidence, and paternity / maternity identification.
  • the present invention employs a solid-support with immobilized capture oligonucleotides, affinity liposomes, and an electrochemical sensor.
  • Capture oligonucleotides are selected from a group including single-chain nucleic acids (ribo and deoxyribo nucleic acids), single-chain oligonucleotides (ribo and deoxyribo oligonucleotides), and 'preorganized' oligonucleotide structures including peptide nucleic acid (PNA) analogues.
  • PNA peptide nucleic acid
  • the affinity liposomes of the present invention contain encapsulated electrochemically detectable reporter molecules and surface-attached affinity components capable of specifically binding to the analyte to be detected and/or to the said oligonucleotide capture oligonucleotides in a condition where analyte and capture oligonucleotides are bound to each other, but not not free captureoligonucleotides.
  • Respective affinity components include but are not limited to single-chain nucleic acids (ribo and deoxyribo nucleic acids), single- chain oligonucleotides (ribo and deoxyribo oligonucleotides), 'preorganized' oligonucleotide structures including peptide nucleic acid (PNA) analogues, intercalating agents, intercalating agents conjugated to oligonucleotides or nucleic acids, immunoglobulins or fragments of immunoglobulins with specificity for double- and/or triple-stranded nucleic acids), and nucleic acid binding proteins (e.g., the prokaryotic lac repressor or the eukaryotic cyclic AMP responsive element binding protein (CREB)).
  • PNA peptide nucleic acid
  • intercalating agents intercalating agents conjugated to oligonucleotides or nucleic acids
  • the nucleic acids and oligonucleotides are designed for hybridization to single-stranded segments of captured target nucleic acids or amplicons thereof, while binding to the capture oligonucleotides should be substantially not possible.
  • Suitable intercalating agents are selected from a group exhibiting a preference for double-stranded and/or triple-stranded nucleic acids including actinomycin D derivatives, anthracycline derivatives, acridine derivatives, cyanine dye derivatives, hydroxystilbamidine derivatives, imidazole derivatives, indole derivatives, phenanthridine derivatives, and psoralen derivatives.
  • encapsulated electrochemically detectable reporter molecules are those which can undergo oxidation or reduction e.g. hydrochinones, naphthols, or organimetals like complexes of Os, Ru or Co or the like.
  • the reperter molecules are capable to undergo redox recycling.
  • Such molecules may be aromatic redox mediators or organic and anorganic metal complexes containing osmium, ruthenium, iron, copper, and chromium.
  • aromatic redox mediators include p-aminophenol and derivatives thereof, catechol and derivatives thereof, dopamine and derivatives thereof, methoxytyraminin and derivatives thereof, aromatic compounds with more than one aromatic ring structure such as anthracene derivatives, and heterocyclic aromatic compounds such as serotonin, hydroxyindol acetic acid, and derivatives thereof.
  • metal complexes contain the metal complexed by various aromatic and heterocyclic aromatic compounds, the side chains of which are derivatized with residues such as carboxyl groups, halogens, aminoethyl groups, and pyridine derivatives for adjustment of the redox potential according to the specific assay requirements.
  • the release of electrochemically detectable reporter molecules encapsulated in specifically bound affinity liposomes is effected e.g. by an increase of the ambient temperature (Method A) or the addition of liposome-lysing solvents such as organic solvents or detergents (Method B).
  • Detection and quantification with an electrochemical sensor preferably comprises a closely spaced array (micrometer and submicrometer scale), the electrodes for example consisting of thin film nobel metal.
  • the electrodes for example consisting of thin film nobel metal.
  • Preferred are interdigitated arrays where anodes and cathodes have a width between 10 and about 5000 nm and the electrodes are spaced apart from each other with a distance between 5 and 5000 nm. More preferred are interdigitated arrays where the electrodes are spaced apart from each other with a distance between 100 and about 800 nm.
  • closely spaced array microelectrodes are utilized where the electrodes are spaced apart from each other with a distance of 10 to 1000 nm, more preferred of 100 to 800 nm. Since the mass transfer of the redox reporter molecules between anodes and cathodes is accomplished by diffusion only, a narrow interelectrode gap reduces the diffusion length and, thereby, increases the efficiency of recycling processes, minimizes intramolecular cyclization of redox reporter molecules and potential electron transfer processes as described for oxidized catecholamines and L- ascorbic acid (Niwa o., Morita, M., and Tabei, H. Electroanalysis 3, 163, 1991).
  • An electrochemical sensor comprised of an interdigitated array of microelectrodes of the kind useful in the pactice of the present invention is published in International Patent Application (Hintsche, R., et al., PCT Publication No. WO 94/29708).
  • the publication discloses an array consisting of four pairs of comb- shaped interdigitated anodes and cathodes parallel arranged on a planar silicon chip. Conductors connecting the electrodes to electrical contact surfaces are covered by an insulating layer.
  • the basic assay procedure includes binding of the analyte to immobilized capture oligonucleotides by molecular biological interactions, detection of captured analyte by analyte-specific binding of affinity liposomes containing encapsulated electrochemically detectable reporter molecules, removal of non-bound affinity liposomes, release of electrochemically detectable reporter molecules from the said specifically bound liposomes, and detection and optionally quantification of such reporter molecules by means of voltammetry or amperometry, preferably using interdigitated array electrodes ( Figures 1 and 2).
  • the quantity of released electrochemically detectable reporter molecules is a proportional measure of the amount of target nucleic acids (analyte) in the specimen.
  • affinity liposomes does not only mean binding of affinity liposomes to analyte bound to capture oligonucleotides , but also includes binding of affinity liposomes to structures of the capture oligonucleotides which are affected by the binding event with the analyte or to structures of the capture oligonucleotide-analyte combination ("complex") thus formed (e.g.
  • double stranded nucleic acid formed upon adding single stranded nucleic acid analyte to a capture oligonucleotide comprising single stranded nucleic acid) and binding to polymeric carriers which in turn are bound to affected capture oligonucleotide and/or bound analyte.
  • Reporter systems and assay procedures including additional or modified steps are specific embodiments of the basic concept illustrated above.
  • additional amplification molecules are included in the assay procedure for signal amplification.
  • captured analyte target nucleic acids or amplicons thereof
  • polymeric carrier molecules containing two different affinity components one for specific binding to captured nucleic acids and/or to capture oligonucleotides in a condition where analyte and capture oligonucleotides are bound to each other, but not to free capture oligonucleotide (e.g., intercalating agents, oligonucleotides, or antibodies with specificity for double- and/or triple-stranded nucleic acids) and the other for binding of affinity liposomes.
  • capture oligonucleotide e.g., intercalating agents, oligonucleotides, or antibodies with specificity for double- and/or triple-stranded nucleic acids
  • Preferred affinity components for binding of affinity liposomes to polymeric carrier molecules are hapten / anti-hapten antibody systems, enzyme inhibitor / enzyme systems, and the biotin / (strept)avidin system. Since polymers allow covalent attachment of multiple affinity components, each captured nucleic acid molecule binds a multitude of affinity liposomes leading to an effective signal amplification.
  • Preferred examples of synthetic and natural polymer derivatives include, but are not limited to derivatives of polysaccharides, polyamino acids, polyvinyl alcohols, polyvinylpyrrolidinones, polyacrylic acids, various polyurethanes, polyphosphazenes, and copolymers of such polymers. In a more preferred embodiment, derivatives of dextran are employed as polymeric carrier molecules.
  • captured analyte i.e., captured target nucleic acids or amplicons thereof
  • dextran polymers containing two types of low molecular weight affinity components One of the covalently linked affinity components is capable of specifically binding to the captured analyte in a structure restricted manner (e.g. intercalating agents or oligonucleotides).
  • the other covalently linked affinity component e.g., hapten molecules, enzyme inhibitors, or biotin residues
  • more than one of the other such component is linked to the polymeric carrier molecule.
  • affinity liposomes containing surface-attached proteinaceous affinity components (e.g., anti-hapten antibodies, enzyme molecules, or (strept)avidin) capable of binding to the affinity components on the dextran polymers (Fig. 3). Quantitation of electrochemically detectable reporter molecules encapsulated in specifically bound affinity liposomes is performed as described.
  • surface-attached proteinaceous affinity components e.g., anti-hapten antibodies, enzyme molecules, or (strept)avidin
  • analyte (captured target nucleic acids or amplicons thereof) is detected by dextran polymers containing two types of proteinaceous affinity components.
  • One of the covalently linked affinity components is capable of specifically binding to a specific captured target nucleic acid or its amplicons in a structure restricted manner (e.g., antibodies with specificity for double- and/or triple-stranded nucleic acids).
  • the other covalently linked affinity components e.g., anti-hapten antibodies, enzyme molecules, or (strept)avidin
  • bound dextran polymers are detected by affinity liposomes containing surface-attached low molecular weight affinity components (e.g., hapten molecules, enzyme inhibitors, or biotin residues) capable of binding to the proteinaceous affinity components on the dextran polymers (Fig. 3).
  • affinity liposomes containing surface-attached low molecular weight affinity components e.g., hapten molecules, enzyme inhibitors, or biotin residues
  • Quantitation of electrochemically detectable reporter molecules encapsulated in specifically bound affinity liposomes is performed as described.
  • captured target nucleic acids or amplicons thereof are detected by dextran polymers containing proteinaceous affinity components derivatized with low molecular weight affinity components.
  • the proteinaceous affinity component is capable of specifically binding to a specific captured target nucleic acid or its amplicons in a structure restricted manner (e.g., antibodies with specificity for double- and/or triple-stranded nucleic acids)
  • the low molecular weight affinity components e.g., hapten molecules, enzyme inhibitors, or biotin residues
  • the low molecular weight affinity components are capable of specifically binding affinity liposomes containing surface-attached proteinaceous affinity components (e.g., anti-hapten antibodies, enzyme molecules, or (strept)avidin) capable of binding to the low molecular weight affinity components on the dextran polymers.
  • the proteinaceous affinity components e.g., anti- hapten antibodies, enzyme molecules, or (strept)avidin
  • the proteinaceous affinity components are capable of specifically binding affinity liposomes containing surface-attached low molecular weight affinity components (e.g., hapten molecules, enzyme inhibitors, or biotin residues) capable of binding to the proteinaceous affinity components on the dextran polymers. Quantitation of electrochemically detectable reporter molecules encapsulated in specifically bound affinity liposomes is performed as described.
  • signal amplification is achieved by using preformed complexes of affinity liposomes.
  • One preferred method for the preparation of complexed affinity liposomes utilizes affinity liposomes containing two types of surface-attached affinity components, one for specific binding to captured nucleic acids or amplicons thereof in a structure restricted manner (e.g., intercalating agents or oligonucleotides) and the other for complexation of affinity liposomes via bridging molecules.
  • Suitable bridging molecules are selected from a group including a) bi- or oligovalent anti-hapten antibodies or fragments thereof, as well as conjugates or fusion constructs thereof; b) enzymes, enzyme conjugates, and fusion constructs of enzymes providing more than one inhibitor-binding site; and c) avidin and streptavidin. Any bridging molecule that provides more than one binding site is useful for this type of amplification methodology.
  • a first group of preformed complexes of affinity liposomes useful for this invention include complexes generated by reaction of i) (strept)avidin with affinity liposomes containing surface-attached biotin residues and surface-attached nucleic acid- reactive components (e.g., intercalating agents or oligonucleotides), ii) anti-hapten antibody molecules providing more than one hapten-binding site with affinity liposomes containing surface-attached hapten molecules and surface-attached nucleic acid-reactive components (e.g., intercalating agents or oligonucleotides), and iii) enzyme conjugates providing more than one inhibitor-binding site with affinity liposomes containing surface-attached inhibitor molecules and surface- attached nucleic acid-reactive components (e.g., intercalating agents or oligonucleotides).
  • strept)avidin with affinity liposomes containing surface-attached biotin residues
  • polymeric carrier molecules containing covalently coupled affinity components are employed as bridging molecules for the preparation of preformed complexes of affinity liposomes.
  • the application of polymeric carrier molecules for complexation of affinity liposomes is useful when affinity components are utilized as bridging molecules which contain only a single binding site for the correponding affinity partner (e.g., anti-hapten single-chain antibodies (scFv) and enzymes containing only a single inhibitor binding site).
  • affinity components are utilized as bridging molecules which contain only a single binding site for the correponding affinity partner (e.g., anti-hapten single-chain antibodies (scFv) and enzymes containing only a single inhibitor binding site).
  • scFv anti-hapten single-chain antibodies
  • complexed affinity liposomes are employed which are prepared utilizing two types (type I and type II) of affinity liposomes containing different affinity components.
  • the affinity components on type I affinity liposomes are capable of specifically binding to specific captured nucleic acids or amplicons thereof (e.g., single-stranded oligonucleotides complementary to single-stranded segments of captured target nucleic acid or amplicons thereof) and/or to capture oligonucleotides in a condition where analyte and capture oligonucleotides are bound to each other, but not to free capture oligonucleotide.
  • the affinity components on type II affinity liposomes are capable of specifically binding to the affinity components on type I affinity liposomes (e.g., single-stranded oligonucleotides complementary to type I single-stranded oligonucleotides).
  • the analyte to be detected is bound to immobilized capture oligonucleotides by molecular biological interactions and detected by specific binding of said preformed complexes of affinity liposomes containing surface- attached nucleic acid-reactive affinity components (Fig. 4).
  • signal amplification is achieved by a combination of polymeric carrier systems and preformed complexes of affinity liposomes.
  • One example for such a combination uses dextran polymers containing two types of covalently linked affinity components, one being capable of specifically binding to the captured target nucleic acid or its amplicons to be detected in a structure restricted manner, and the other being capable of specifically binding preformed complexes of affinity liposomes.
  • the present invention provides a detection system that eliminates i) the necessity for optical clarity of the sample solution and other ambient optical density requirements, and ii) the necessity of enzymatic amplification technology.
  • One small affinity liposome provides up to 10 5 molecules of redox mediators to allow excellent detectability of a binding event.
  • Using a silion microchip-formatted array of closely spaced (micrometer and submicrometer scale) thin fim nobel metal electrodes for quantitation of liposome-encapsulated redox mediators a volume of only a few microliters is required for voltammetric or amperometric detection.
  • the liposome-encapsulated redox mediators can be released into a very small volume, thereby generating a relatively high concentration of released redox mediators.
  • the release of 10 5 molecules of redox mediator from a single affinity liposome into a detection volume of 2 ⁇ l would create a redox mediator concentration of approximately 0.1 pM.
  • the signal provided by a single analyte-bound affinity liposome can be easily amplified by one to two orders of magnitude if polymeric carrier systems and/or preformed complexes of affinity liposomes are used for detection.
  • the liposomal encapsulation technique allows the use of a large variety of redox mediators including those which provide highly efficient redox recycling, but are difficult to chemically adjust for enzyme amplification systems. There is no need for chemical derivatization of redox mediators using the non-enzymatic liposome- linked interdigital array electrodes assay. As a result, the present invention reduces costs and analysis time without compromising detection sensitivity.
  • the reporter system as described above may be provided in the form of a kit, the components of which are (i) a solid support containing the said immobilized capture oligonucleotides, (ii) the affinity liposomes containing at least one surface- attached affinity component, which comprise the said electrochemically detectable reporter molecules, and (iii) the said electrochemical sensor ("transducer").
  • a specific arrangement is not required because measurement of the reporter molecules is independent of the immediate presence of the capture oligonucleotides. It is one of the advantages of the present invention that contacting the sample to be measured for the presence of analyte may be performed independent of performing the other steps of the inventive method, as time and/or area of performing the single method steps are concerned, provided that the components are sufficiently stable.
  • measurement of the reporter molecules may be performed in/on the same or in another vessel or plate (e.g. microtiter plate) in which the analyte had been bound to the capture oligonucleotides, and also possibly within a time limit which is suitably selected.
  • the electrochemical sensor is on the same support which carries the immobilized capture oligonucleotides, but this is not a necessary requirement.
  • the capture oligonucletides may be immobilized in the space between single electrodes.
  • the support carrying the immobilized capture oligonucleotides is provided on a flat substrate which may be inserted into the container in which the electrochemical detection is intended to be made.
  • the solid support containing immobilized capture oligonucleotides is at least a part or structure of the container or vessel In which the detection or a part thereof shall be performed.
  • the solid support is part of a variety of wells in a plate (e.g. microtiter plate), or is part of the bottom or the walls of the container or microtiter plate.
  • the support carrying the immobilized capture oligonucleotides is provided having the form of beads or a stripe or bar which is added to the sample which possibly contains the analyte.
  • the support carrying the immobilized capture oligonucleotides may be added before or after the addition of the affinity liposomes to the sample in which the analyte is to be detected. Care should be taken that the affinity liposomes bound to capture oligonucleotides remain wet or submersed in the liquid als long as they shall be kept intact. In this way, release of reporter molecules therefrom may be deferred.
  • the method of the present invention results in the release of reporter molecules caused by the presence of analyte to be detected, while detection of the said reporter molecules may be performed separately (in regard to place and time). Alternatively, all steps to be performed in the present method may be performed in only one vessel and/or in an immediate sequence. Subsequently, the components of the present invention are described in more detail.
  • the invention comprises a solid support with immobilized capture oligonucleotides for specific binding of target nucleic acids.
  • Capture oligonucleotides may be selected e.g. from single-chain nucleic acids (ribo and deoxyribo nucleic acids) and single-chain oligonucleotides (ribo and deoxyribo oligonucleotides).
  • the oligonucleotides are designed to form specific helical complexes with target nucleic acids.
  • ribo and deoxyribo nucleic acids ribo and deoxyribo oligonucleotides
  • 'preorganized' oligonucleotide structures are used as capture oligonucleotides (for a review, see Kool, E.T., Chem. Rev. 97, 1473, 1997).
  • the rational for using 'preorganized' oligonucleotide structures is based on the observation that the affinity of oligonucleotides for the target nucleic acid can be increased by modifications rigidifying the oligonucleotide prior to binding so that it more resembles the bound conformation.
  • An oligonucleotide rigidly held in the binding position prior to complexation shows less free internal bond rotations that need to be 'frozen' during complexation.
  • the molecule is organized into a shape which is more complementary to the desired target than to undesired ones. This increases selectivity because mismatched targets will cause unfavorable responses such as non-optimum bond angles or streric clashes.
  • Strategies for the construction of 'preorganized' oligonucleotide structures include i) enhancement of base stacking, ii) limitation of bond rotations, and iii) linking of binding domains.
  • Double-, triple-, and quadruple-stranded nucleic acid helices are stabilized by base stacking and hydrogen bonding interactions. Since the majority of the base-stacking interaction in nucleic acids is between bases within a strand, the strengthening of stacking will have the tendency to cause single- stranded oligonucleotides to become preorganized into a more regular helical conformation. This will therefore favor complexation by lowering the entropic cost.
  • Examples for strategies for increasing stacking are the addition of simple substituents to DNA bases (e.g., methylation of pyrimidines at C-5), an increase of the surface area of DNA bases (e.g., by addition of aromatic heterocyclic groups to the C-5 position of pyrimidines), and the use of nonpolar DNA base analogues.
  • Examples for strategies for limiting bond rotations prior to complexation employ covalent bonds and include the synthesis of i) backbones with restricted freedom, ii) bicyclo-DNA, iii) hexose-DNA, and iiii) circular DNA.
  • Prefered examples of nucleic acid derivatives with rigid backbones include peptide nucleic acid (PNA) analogues containing amide bonds. Since an amide has restricted rotation about the carbonyl-nitrogen bond, PNAs are capable of forming strong duplexes with DNA at lowered ionic strength and very strong triplexes even at normal ionic strength.
  • PNA peptide nucleic acid
  • the normally flexible furanose ring is rigidified by addition of an ethylene bridge from C-3' to C-5', thereby forming a second five-membered ring to the natural structure. Since five-membered rings are considerably more flexible than six-membered rings, DNA analogues have been synthesized in which the furanose ring is expanded to a six-membered ring. In some cases, such oligonucleotide analogues hybridized more strongly to DNA than the natural furanose-based structures. Another preferred way to significantly limit the conformational freedom of a flexible oligonucleotide is to cyclize the chain. A circular oligonucleotide can bind a single-stranded target RNA or DNA by forming standard Watson-Crick bonds. However, such binding is limited because of the helical twist of DNA.
  • a linker should be both rigid and orient the binding domains in the productive geometry.
  • Noncovalent links between binding domains have the advantage of simplifying the synthesis, but have the disadvantage of being relatively weak, thereby limiting effective preorganization.
  • covalent links between binding domains For example, thiols may be placed into opposite strands of a duplex-forming sequence and used for disulfide cross-linking upon oxidation. Such duplexes become stabilized thermodynamically, presumably beause of the entropic benefit.
  • oligonucleotide structures can be prepared that lead to triplex formation on single-stranded targets.
  • Triple- helical nucleic acid structures are known since 1957.
  • a purine DNA base can form hydrogen-bonded contacts on two sides, one termed the Watson-Crick face and the other Hoogsteen face.
  • Watson-Crick face and the other Hoogsteen face.
  • a purine stretch presents sites in the major groove for Hoogsteen complexation by a third strand.
  • Single- stranded DNA can also serve as a target for triple helix formation, since a purine stretch can be bound on two sides by a molecule carrying both a Watson-Crick complementary domain and a Hoogsteen complementary domain.
  • a second motif for triplex formation is the so-called purine motif, in which purine-purine-pyrimidine base triads are formed.
  • a purine strand is in the middle, with two other strands forming hydrogen-bonded contacts.
  • the Watson-Crick complementary pyrimidine strand represents the target and the other two strands the binding domains which are preorganized by linking.
  • a simple way to link two such triplex-forming domains is to connect them with extra non-pairing nucleotides or by a non-nucleotide linker such as hexaethylene glycol (clamp or fold-back oligonucleotides).
  • a preferred strategy is to link two triplex-forming domains by two loops at both ends using nucleotide loops or non- nucleotide linkers (circular and looped oligonucleotides). Clamp-like oligonucleotides have been shown to bind target sequences with an 11°C advantage in T , whereas closure of the clamp into a full circle gave a 19°C advantage.
  • the clamp and circular oligonucleotide approaches are strategies in which two DNA-binding domains are linked at their end or ends. Another preferred approach is to link them across the center, thereby generating molecules with an 'H'-form. Examination of the base triads involved in triple helix shows that a bridge can easily link two C-5 positions on pyrimidines in opposite strands. Experimental results have shown that such a molecule cross-linked by a disulfide bridge (via thiopropyne-substituted thymidine nucleosides) binds a target strand more strongly than a clamp-like oligonucleotide (Chaudhuri, N.C., and Kool, E.T. J. Am.
  • tethered DNA For maximum cooperativity, a rigid linking domain is preferred.
  • flexible linkers may not maximize affinity and selectivity, they may provide utility in some cases.
  • flexible tethers may be used to link two DNA-binding sequences for hybridization to separate sites such as purine stretches separated by non-homopurine segments.
  • the solid support serving for immobilization of capture oligonucleotides may be the same support which carries the electrochemical sensor or may be a different one.
  • the support may have e.g. a flat structure, or it may have the form of beads or the like, e.g. glassy, polymeric, or magnetic beads.
  • the support may be part of microtiter plates used for the detection of the analyte, or be a porous, impervious, fibrous, or metallic matrix or membrane or the like, as is well-known in the art.
  • Such matrices may be utilized as derivatized or non-derivatized solid supports for immobilization of capture oligonucleotides.
  • Immobilization of capture oligonucleotides or derivatives thereof may be accomplished non-covalently or covalently by any of the well-known chemical coupling methods. For most applications, covalent immobilization techniques are preferred. Electrically mediated coupling procedures are equally applicable for use with the current invention. Also applicable for this invention are non-covalent, non-adsorptive immobilization techniques. For example, capture oligonucleotides derivatized at their termini with biotin residues may be immobilized onto solid supports functionalized by adsorption or covalent binding of streptavidin beforehand. The methods by which capture oligonucleotides may be derivatized with reactive residues for immobilization onto solid supports are numerous. Preferred methods include but are not limited to those which allow selective derivatization of the termini to guarantee efficient hybridization with target nucleic acids.
  • capture oligonucleotides containing a 5'-phosphate group are used which are derivatized with amine or sulfhydryl terminal spacer molecules for immobilization onto amine-reactive or sulfhydryl-reactive solid supports (Hermanson, G.T. Bioconjugate techniques, Academic Press, San Diego, 1996).
  • the 5'-phosphate groups of capture oligonucleotides may be reacted with carbodiimide in the presence of imidazole to form active phosphorimidazolide intermediates.
  • These derivatives are highly reactive with diamines or bis- hydrazide compounds, forming amine terminal spacer molecules via phosphoramidate linkages.
  • Derivatization of the 5'-phosphate groups with cystamine creates an amine terminal spacer containing a disulfide group. Reduction of the cystamine-labeled oligonucleotide using a disulfide reducing agent releases 2-mercaptoethylamine and generates a terminal thiol group.
  • sulfhydryl groups are introduced at the 5'- termini of oligonucleotides by automated solid-phase synthesis using S- triphenylmethyl O-methoxymorpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan (1) ol, or 6-mercaptohexan (1) ol (Connolly, B.A., and Rider, P., Nucleic Acids Res. 13, 4485, 1985). After clevage from the resin and removal of the phosphate and base protecting groups, oligonucleotides are obtained which contain an S-triphenylmethyl group attached to the 5'-phosphate group via a two, three, or six carbon chain.
  • the triphenylmethyl group can readily be removed with silver nitrate to give the free thiol.
  • primary amino groups can be introduced at the 5'-termini of oligonucleotides by automated solid-phase synthesis using N-monomethoxytrityl-O-methoxydiisopropylaminophosphinyl 3- aminopropan (1) ol (Connolly, B.A. Nucleic Acids Res. 15, 3131 , 1987). After cleavage from the resin and removal of the phosphate and base protecting groups, a monomethoxytrityl-NH(CH2)3P ⁇ 4 - oligomer is obtained. The monomethoxytrityl group can be removed with acetic acid to give the amine- containing oligonucleotide.
  • nucleotide derivatives suitable for immobilization onto solid supports are incorporated into capture oligonucleotides during automated chemical oligonucleotide synthesis.
  • oligonucleotides may be derivatized at their 5'-terminus by coupling of a uridine moiety via a 5'-5' linkage using 2',3'-di-O-acetyluridine 5'-(2-cyanoethyl N,N-diisopropylphosphoramidite).
  • the derivatized oligonucleotide can be immobilized onto amine- containing solid supports via reductive amination.
  • Other nucleotide derivatives such as N-6 or C-8 derivatives of dATP carrying protected amine or protected sulfhydryl groups are equally applicable for this invention.
  • nucleotide derivatives containing reactive residues are incorporated into capture oligonucleotides by enzymatic means.
  • Preferred examples of purine nucleotides include but are not limited to dATP derivatized with a reactive residue at its N-6 position or C-8 position via long linker arms.
  • 8-aminohexyl-dATP is a preferred derivative for coupling to the 3' terminal of DNA oligonucleotides by terminal transferase (Hermanson, G.T. (ed.) Bioconjugate techniques, Academic Press, San Diego, 1996).
  • Preferred examples of pyrimidine nucleotides include but are not limited to dUTP and dCTP modified with a reactive residue at their C-5 position via long linker arms.
  • Preorganized fold-back or looped capture oligonucleotides may be modified in the non-pairing nucleotide region with reactive groups by incorporation of nucleotide derivatives such as N-6 or C-8 derivatives of dATP carrying protected amine or protected sulfhydryl groups via long spacer arms. After deprotection, such derivatized preorganized fold-back or looped capture oligonucleotides can be immobilized onto amine- or sufhydryl-recative solid supports or further derivatized with heterobifu notional reagents.
  • preorganized fold-back or looped capture oligonucleotides may be formed by non-nucleotide linker molecules providing a functional group for immobilization onto a solid support in addition to the other two functional groups required for the formation of a preorganized oligonucleotide structure by cross-linking of the termini.
  • a convenient molecule from which to build trifunctional linker molecules is the amino acid L-lysine. Its three functional groups, ⁇ -carboxy, ⁇ -amino, and ⁇ -amino, can be derivatized independently to contain three arms carrying different reactiv ⁇ groups.
  • spacer molecules may be incorporated between the solid support and the oligonucleotide termini.
  • Preferred spacer molecules are sufficiently long and flexible to allow efficient hybridization of the immobilized oligonucleotides with captured target nucleic acids.
  • spacer molecules are derivatives of 6- aminohexanoic acid (6-[6-((amino)hexanoyl) amino] hexanoate), providing a spacer arm with 14 atoms, oligomeric derivatives of 6-aminohexanoic acid (6-[6- ((amino)hexanoyl)amino]-hexanoate), and oligoethylene glycol derivatives (Levenson, C, and Chang, C. Nonisotopically labelled probes and primers.
  • PCR Protocols A Guide to Methods and Applications (M.A. Innis et al., eds.) pp. 99-112, Academic Press, San Diego, 1990).
  • oligoethylene glycol provides water solubility and a great degree of freedom for the linked oligonucleotides.
  • oligoethylene glycol containing a t-Boc-protected amine on one end and an unprotected amine or an amine-reactive residue on the other end. After reaction of the unprotected functional residue, the t-Boc protecting group can be easily removed by treatment with trifluoroacetic acid.
  • a wide range of derivatives of oligoethylene glycol may be used as spacer molecules between the solid support and the oligonucleotide termini. Conjugation of capture oligonucleotides or derivatives thereof to spacer molecules may be accomplished by any of the well-known chemical coupling methods.
  • primary amino groups are introduced at the 5'-termini of oligonucleotides by automated solid-phase synthesis using N-monomethoxytrityl- O-methoxydiisopropylaminophosphinyl-3-aminopropan(1 )ol as mentioned above.
  • the terminal amino group may be derivatized with an amine-reactive heterobifunctional reagent containing a long spacer arm such as succinimidyl 6-[3- (2-pyridyldithio) propionamidojhexanoate (LC-SPDP), succinimidyl 6-[6- ⁇ ((iodoacetyl) amino) hexanoyl ⁇ amino]hexanoate (SIAXX), or oligoethylene glycol derivatives containing a succinimidyl ester on one end and a vinylsulfone residue on the other end.
  • the sulfhydryl-reactive residue of such reagents may be used for coupling of the oligonucleotide-spacer conjugates to thiol-containing solid supports.
  • nucleotide derivatives containing reactive residues for covalent attachment of spacer molecules are incorporated into oligonucleotides by enzymatic means.
  • 8-aminohexyl-dATP may be utilized for coupling to the 3' terminal of DNA oligonucleotides by terminal transferase (Hermanson, G.T. (ed.) Bioconjugate techniques, Academic Press, San Diego, 1996).
  • the terminal amino group of the incorporated nucleotide may be further derivatized with heterobifunctional cross-linking reagents such as LC-SPDP, SIAXX, or oligoethylene glycol derivatives containing a succinimidyl ester on one end and a vinylsulfone residue on the other end.
  • heterobifunctional cross-linking reagents such as LC-SPDP, SIAXX, or oligoethylene glycol derivatives containing a succinimidyl ester on one end and a vinylsulfone residue on the other end.
  • oligonucleotides can be derivatized at their 3'-terminus with a variety of spacer arms including 6-[6- ((amino)hexanoyl)amino]-hexanoate and oligoethylene glycol derivatives of various length.
  • terminal sulfhydryl-reactive groups of such spacer molecules can be used for subsequent coupling of the oligonucleotide-spacer conjugates to sulfhydryl-containing solid supports.
  • the pyridyl disulfide residues of LC-SPDP-derivatized oligonucleotides can be reduced to generate free sulfhydryl groups for subsequent coupling of the oligonucleotide-spacer conjugates to sulfhydryl-reactive solid supports.
  • oligonucleotides may be derivatized at their 5'-terminus by coupling of a uridine moiety via a 5'-5' linkage using 2',3'-di-O-acetyluridine-5'- (2-cyanoethyl-N,N-diisopropylphosphoramidite) (Kuijpers, W.H.A., et al, Bioconjugate Chem. 4, 94, 1993). After oxidation of the 2',3" cis-diol of the terminal uridine residue at the 5'-terminus by treatment with periodate, 6-[6-
  • ((amino)hexanoyl)amino]hexanoate or oligoethylene glycol derivatives containing an amine residue on one end and a carbyoxyl residue on the other end may be coupled to the aldehyde groups of the oligonucleotide via reductive amination. Thereafter, the terminal carboxyl residues of the spacer molecules may be activated for subsequent coupling of the oligonucleotide derivative to amine- containing solid supports.
  • preorganized fold-back or looped oligonucleotides are modified in the non-pairing nucleotide region with reactive groups by incorporation of nucleotide derivatives such as N-6 or C-8 derivatives of dATP carrying protected amine groups via long spacer arms (e.g., a protected derivative of 8- aminohexyl-dATP).
  • nucleotide derivatives such as N-6 or C-8 derivatives of dATP carrying protected amine groups via long spacer arms (e.g., a protected derivative of 8- aminohexyl-dATP).
  • derivatized preorganized fold-back or looped oligonucleotides may be further derivatized with heterobifunctional cross- linking reagents such as LC-SPDP, SIAXX, or oligoethylene glycol derivatives containing a succinimidyl ester on one end and a vinylsulfone residue on the other end.
  • preorganized fold-back or looped oligonucleotides are formed by non-nucleotide linker molecules such as L-lysine providing a functional group for covalent attachment of spacer molecules in addition to the other two functional groups required for formation of the preorganized oligonucleotide structure.
  • trifunctional linker molecules are L-lysine residues in which the ⁇ -amino and ⁇ -amino are derivatized with hydroxyl terminal spacer molecules and the ⁇ -carboxyl group with 6-[6-((amino)hexanoyl)- aminojhexanoate or oligoethylene glycol derivatives containing an amine residue on one end and a carboxyl residue on the other end.
  • the liposomes to be used in the present invention contain encapsulated reporter molecules and are derivatized on the surface with affinity components capable of binding to captured target nucleic acids or derivatives thereof.
  • Preferred affinity components include but are not limited to single-chain nucleic acids (ribo and deoxyribo nucleic acids), single-chain oligonucleotides (ribo and deoxyribo oligonucleotides), 'preorganized' oligonucleotide structures including peptide nucleic acid (PNA) analogues, intercalating agents, intercalating agents conjugated to oligonucleotides or nucleic acids, immunoglobulins or fragments of immunoglobulins with specificity for double- and/or triple-stranded nucleic acids), and nucleic acid binding proteins (e.g., the prokaryotic lac repressor or the eukaryotic cyclic AMP responsive element binding protein (CREB)).
  • PNA peptide nucleic acid
  • Intercalating agents are molecules which are characterized by planar aromatic ring structures of appropriate size and geometry that can be inserted between baise pairs in double-stranded (ds) DNA. During intercalation neighboring base pairs in DNA are separated to allow for the insertion of the intercalating ring system, causing an elongation of the double helix by stretching.
  • Examples include but are not limited to phenanthridines and acridines (e.g., ethidium bromide; propidium iodide; hexidium iodide; acridine orange; 9-amino-6-chloro-2-methoxyacridine (ACMA)), indoles and imidazoles (e.g., the bisbenzimide dyes Hoechst 33258 and Hoechst 33342; 4',6-diamidino-2- phenylindole (DAPI); 4 ⁇ 6-(diimidazolin-2-yl)-2-phenylindole (DIPl)), anthracyclines (e.g., doxorubicin; daunorubicin), cyanine dyes (e.g., benzoxazolium-4-pyridinium dyes, benzothiazolium-4-pyridinium dyes; benzoxazolium-4-quinolinium dyes
  • DNA intercalating agents used as affinity components possess preference for double-stranded and/or triple-stranded DNA.
  • DNA intercalating agents may be actinomycin D, anthracyclines, e.g. doxorubicin and daunorubicin, DAPI, and the bisbenzimide intercalators Hoechst 33258 and Hoechst 33342.
  • DAPI associates in the minor groove of double-stranded DNA, preferentially binding to AT clusters, although there is evidence that DAPI also intercalates in those DNA sequences containing as few as two AT base pairs.
  • Binding of DAPI to double-stranded DNA occurs with an approximately 20-fold fluorescent enhancement, apparently due to the displacement of water molecules from both DAPI and the minor groove. Fluorescence enhancement does not occur with single-stranded DNA.
  • the relatively non-toxic and water-soluble (2%) bisbenzimide intercalators Hoechst 33258 and Hoechst 33342 are also minor groove-binding DNA intercalators with a preference to contiguous AT base pairs.
  • homodimers and heterodimers of intercalating agents may be used as affinity components. It has been shown that appropriately designed dimers of intercalating agents have DNA binding affinities several orders of magnitude greater than those of their parent compounds. For example, the intrinsic DNA binding affinity of ethidium bromide and ethidium homodimer are 1.5 x 10 5 M _1 and 2 x 10 8 M _1 , respectively. Recently, several sets of dimers of intercalating agents have been developed (Haugland, R.P. Handbook of fluorescent probes and research chemicals, M-T.Z. Spence, ed., Molecular Probes, Inc., Eugene, OR, USA, 1996).
  • TOTO-1 dimeric cyanine dye
  • TO-PRO-1 Molecular Probes, Inc., Eugene, OR, USA
  • TOTO-1 exhibits a higher affinity for double-stranded DNA than even the ethidium homodimers.
  • NMR studies of TOTO-1 interactions with a double-stranded 8-mer indicated that it is a bis-intercalator with interactions in the minor groove and that it distorts the helix by unwinding it where the dye is bound.
  • TOTO-1 exhibits strong sequence selectivity for the site CTAG
  • intercalating agents covalently linked to oligonucleotides are used as affinity components.
  • the oligonucleotides provide binding specificity by hybridizing to complementary single-stranded sequences of the target nucleic acids.
  • capture oligonucleotides with a limited length are used. Thereby, only a part of the target nucleic acid is hybridized to the immobilized capture oligonucleotide, while the rest of the target nucleic acid remains in a single-stranded configuration.
  • the intercalating agent should interact with the hydrid duplex structure formed by the nucleic acid target and the oligonucleotide-intercalating agent conjugate, provided the length of the linker is sufficient to allow for appropriate folding.
  • This approach has been demonstrated with an acridine derivative (2-methoxy-6-chloro-9-aminoacridine) covalently linked to the 3'-phosphate of a series of oligodeoxythymidylat.es via a polymethylene linker [(Tp) n (CH2) m ACr] (Helene, C, et al., Biochem. Soc. Transact. 14, 201 , 1986).
  • oligonucleotide-intercalator conjugates include conjugates in which intercalating agents with a preference for double-stranded and/or triple-stranded DNA are covalently linked to oligonucleotides via flexible oligomethylene linkers.
  • oligonucleotides containing a 3'-phosphate group and a uridine residue at the 5'-terminus via a 5'-5'-linkage are used.
  • the 2',3' cis-diol of the terminal uridine residue at the 5'-terminus is oxidized by treatment with periodate and the resulting aldehyde functions are used to attach long spacer molecules via reductive amination for covalent coupling of the oligonucleotide derivatives to the surface of affinity liposomes.
  • the 3'-phosphate group is derivatized with an intercalating agent (with a preference for double- stranded and/or triple-stranded DNA) using a flexible oligomethylene linker.
  • Actinomycin D has a phenoxazin-2-amino-3-one chromophore and two cyclic pentapeptide lactones attached to the 1 ,9-positions of the chromophore. It binds to dG-dC base pairs in double-standed DNA by intercalation of its chromophore and by hydrogen bonding and hydrophobic interactions of its peptide lactones. The unsubstituted 3-oxo, and 4- and 6-methyl groups of the chromophore as well as the integrity of the cyclic pentapeptide lactones are necessary for the biochemical properties of actinomycin D.
  • actinomycin D and the actinomycin D analogue 7-[(3,4-dichlorobenzyl)amino]-actinomycin D intercalate into calf thymus DNA with apparent binding constants of 2.3 x 10 7 M" 1 and 2.6 x 10 7 M -1 , respectively.
  • the flexible NH-CH2 linkage of the actinomycin D analogue allows for the formation and aids the stabilization of the intercalated complex with DNA.
  • Preferred examples of actinomycin D derivatives for the preparation of affinity liposomes include but are not limited to actinomycin D analogues containing a reactive residue attached to the N 2 - or C-7-position via rotationally flexible linker molecules.
  • derivatives of the anthracyclines doxorubicin and daunorubicin may be used as intercalating agents for the preparation of affinity liposomes.
  • Doxorubicin is a glycoside antibiotic that differs from daunorubicin by a single hydroxyl group on C-14. Both molecules contain an aminosugar, daunosamine, linked through a glycosidic bond to the naphthacenequinone nucleus. They intercalate into the DNA double helix in such a fashion that the aglycone moiety is between the adjacent base pairs and parallel to them.
  • the daunosamine moiety lies in the major groove of the double helix and the protonated amine of the aminosugar binds electrostatically to the negatively charged phosphate groups of the DNA.
  • Both antracyclines intercalate into the DNA double helix with a binding affinity constant of approximately 10 6 M _1 , which is the same order of magnitude as those found for actinomycin and the acridines.
  • doxorubicin as well as daunorubicin derivatives containing a daunosamine moiety with a modified or substituted amino group are still able to form complexes with DNA.
  • the N-acetyl derivative of daunorubicin binds to DNA, athough less strongly than non-derivatized daunorubicin.
  • the 3'- deamino-3'-(3-cyanomorpholinyl)-derivative of doxorubicin represents another example.
  • the complex of this compound with DNA exhibits a typical irreversible binding (Menozzi, M. et al., Stud. Biophys. 104, 113, 1984).
  • antracyclines cinerubin A and B containing pyrromycinone as aglycone and a trisaccharide instead of a monosaccharide are also capable of intercalating into DNA.
  • the stiffening and elongating effects of cinerubins on DNA resemble the changes produced by acridine dyes.
  • examples of doxorubicin and daunorubicin derivatives for the preparation of affinity liposomes are derivatives containing a flexible spacer attached to the daunosamine moiety.
  • the amine group of the daunosamine moiety is derivatized with 2-iminothiolane (Jue, R.
  • oligonucleotides and oligonucleotide derivatives are employed as affinity components.
  • the oligonucleotides are designed to form specific helical complexes with those regions of captured target nucleic acids that are still in a single-stranded configuration.
  • 'preorganized' oligonucleotide structures are used as affinity components. The rational for using such structures has been illustrated in detail above (in connection with the description of capture oligonucleotides).
  • spacer molecules will usually be incorporated between the liposomal surface and the oligonucleotide termini.
  • Preferred spacer molecules are sufficiently long and flexible to allow efficient hybridization of the tethered oligonucleotides with captured target nucleic acids or amplicons thereof.
  • Preferred examples of spacer molecules include those which have been described above in connection with the introduction of spacer molecules between solid support and capture oligonucleotides.
  • Derivatization of the oligonucleotides or derivatives thereof with spacer molecules may be accomplished by any of the well-known chemical coupling methods. Preferred methods may be e.g. those which allow selective derivatization of the termini to guarantee efficient hybridization with target nucleic acids.
  • oligonucleotides containing a 5'-phosphate group are derivatized in a carbodiimide-mediated reaction with derivatives of oligoethylene glycol containing a t-Boc-protected amine on one end and an unprotected amine on the other end.
  • the 5'-phosphate groups are reacted with carbodiimide in the presence of imidazole to form active phosphorimidazolide intermediates.
  • amine nucleophiles such as amine derivatized oligoethylene glycol spacer molecules.
  • terminal t-Boc protecting group is removed by treatment with trifluoroacetic acid for subsequent coupling of the amine terminal oligonucleotide-spacer conjugates to amine-reactive lipid derivatives in the liposomal bilayer.
  • primary amino groups are introduced at the 5'- termini of oligonucleotides by automated solid-phase synthesis using N- monomethoxytrityl-O-methoxydiisopropylaminophosphinyl 3-aminopropan (1 ) ol (Connolly, B.A. Nucleic Acids Res. 15, 3131 , 1987). Thereafter, the terminal amino group may be derivatized with an amine-reactive heterobifunctional reagent containing a long spacer arm (LC-SPDP), (SIAXX), or oligoethylene glycol derivatives containing a succinimidyl ester on one end and a vinylsulfone residue on the other end.
  • LC-SPDP long spacer arm
  • SIAXX oligoethylene glycol derivatives containing a succinimidyl ester on one end and a vinylsulfone residue on the other end.
  • the sulfhydryl-reactive residue of such reagents may be used for coupling of the oligonucleotide-spacer conjugates to thiol-containing lipid molecules (e.g., 2-iminothiolane-derivatized phosphatidyl ethanolamine) in the liposomal bilayer. .
  • thiol-containing lipid molecules e.g., 2-iminothiolane-derivatized phosphatidyl ethanolamine
  • nucleotide derivatives containing reactive residues for covalent attachment of spacer molecules are incorporated into oligonucleotides by enzymatic means.
  • modified nucleotides include those derivatives in which the reactive residue is incorporated in a way that does not affect enzyme recognition and activity.
  • purine nucleotides have been detailed above under "Enzymatic attachment of nucleotide derivatives to the termini of capture oligonucleotides”.
  • Preferred examples of the pyrimidine nucleotides include but are not limited to dUTP and dCTP modified with a reactive residue at their C-5 position via long linker arms.
  • 8-aminohexyl-dATP is utilized for coupling to the 3' terminal of DNA oligonucleotides by terminal transferase (Hermanson, G.T. (ed.) Bioconjugate techniques, Academic Press, San Diego, 1996).
  • the terminal amino group may further be derivatized with LC-SPDP.
  • the oligonucleotides are derivatized at their 3'-terminus with spacer arms which are similar in length as 6-[6-((amino)hexanoyl)amino]-hexanoate.
  • the terminal pyridyl disulfide groups can be used for subsequent coupling of the oligonucleotide derivative to sulfhydryl-containing lipid molecules in the liposomal bilayer.
  • the pyridyl disulfide residues can be reduced to generate free sulfhydryl groups for subsequent coupling of the oligonucleotide derivative to sulfhydryl-reactive lipid molecules in the liposomal bilayer (e.g., pyridyl disulfide-, maleimide- or iodoacetyl-containing derivatives of phosphatidyl ethanolamine).
  • oligonucleotides are derivatized at their 5'- terminus during automated chemical oligonucleotide synthesis by coupling of a uridine moiety via a 5'-5' linkage using 2',3'-di-O-acetyluridine 5'-(2-cyanoethyl N,N-diisopropylphosphoramidite) as detailed above for chemical attachment of nucleotide derivatives to the termini of capture oligonucleotides.
  • 6-[6-((amino)hexanoyl) aminojhexanoate is coupled to the aldehyde groups of the oligonucleotide via reductive amination.
  • the terminal carboxyl residues of the spacer molecules are activated with N-hydroxysuccinimide in a carbodimide-mediated reaction for subsequent coupling of the oligonucleotide derivative to amine- containing lipid molecules (e.g., phosphatidyl ethanolamine) in the liposomal bilayer.
  • amine- containing lipid molecules e.g., phosphatidyl ethanolamine
  • preorganized fold-back or looped oligonucleotides are modified in the non-pairing nucleotide region with reactive groups during automated chemical oligonucleotide synthesis by incorporation of nucleotide derivatives such as N-6 or C-8 derivatives of dATP carrying protected amine groups via long spacer arms (e.g., a protected derivative of 8-aminohexyl- dATP).
  • nucleotide derivatives such as N-6 or C-8 derivatives of dATP carrying protected amine groups via long spacer arms (e.g., a protected derivative of 8-aminohexyl- dATP).
  • derivatized preorganized fold-back or looped oligonucleotides are further derivatized with the heterobifunctional cross-linking reagent LC-SPDP.
  • the terminal pyridyl disulfide groups can be used for subsequent coupling of the oligonucleotide derivative to sulfhydryl-containing lipid molecules in the liposomal bilayer.
  • the pyridyl disulfide residues can also be reduced to generate free sulfhydryl groups for subsequent coupling of the oligonucleotide derivative to sulfhydryl-reactive lipid molecules in the liposomal bilayer (e.g., pyridyl disulfide-, maleimide- or iodoacetyl-containing derivatives of phosphatidyl ethanolamine).
  • preorganized fold-back or looped oligonucleotides are formed by non-nucleotide linker molecules providing a functional group for covalent attachment of spacer molecules in addition to the other two functional groups required for formation of the preorganized oligonucleotide structure.
  • a preferred molecule for the construction of such trifunctional linker molecules is the amino acid L-lysine. Its three functional groups, -carboxy, ⁇ -amino, and ⁇ -amino, can be derivatized independently to contain three spacer arms wth different terminal reactive groups.
  • trifunctional linker molecules include L- lysine residues inwhich the ⁇ -amino and ⁇ -amino are derivatized with hydroxyl terminal spacer molecules and the ⁇ -carboxyl group with 6-[6-((amino)hexanoyl)- aminojhexanoate in a carbodiimide-mediated reaction.
  • antibodies with specificity for double and/or triple helical structures of DNA are employed as liposome-attached affinity components.
  • the term "antibody” refers to immunoglobulins of any isotype or subclass as well as any fragment (e.g., Fab' of Fv fragments) of the aforementioned.
  • Antibodies of any source are applicable including polyclonal materials obtained from any animal species, monoclonal antibodies from any hybridoma source, and all immunoglobulins (or fragments) generated with the aid of viral, prokaryotic or eukaryotic expression systems.
  • Biologic recognition molecules with specificity for double and/or triple helical structures of DNA other than antibodies are equally applicable for use with the current invention.
  • reporter molecules comprise those which may undergo oxidation or reduction and preferably are capable to act as redox mediators.
  • V.4.1 Reporter molecules capable of being oxidized or reduced
  • Reporter molecules which may be electrochemically oxidized or reduced comprise hydrochinones, napthtols or organometals, e.g. organometallic complexes of osmium, ruthenium, cobalt and others.
  • redox mediators are used as reporter molecules which are susceptible to redox recycling in closely spaced arrays of thin film nobel metal electrodes. Redox mediators are more sensitive than reporter molecules which may only be oxidized or reduced Preferred redox mediators of this invention are characterized by several important properties. To allow for efficient encapsulation in affinity liposomes, preferred redox mediators possess high solubility in aqueous media. The redox potentials should exhibit reversible peaks in cyclic voltammograms and in aqueous solutions the potentials should be within the limits of - 600 mV (generation of oxygen) and + 800 mV (generation of hydrogen).
  • Potentials close to 0 mV since they guarantee minimal background current as well as minmal instrumental background noise of the electrode, thereby providing optimal signal-to-noise ratios.
  • Potentials close to 0 mV provide an additional advantage in that they minimize or avoid interference by electroactive compounds present in samples to be analyzed such as ascorbic acid in blood or catecholamines in urine.
  • One preferred class of redox mediators contains one or more aromatic ring structures and various organic substituents and side chains that allow switching from a regular aromatic ring structure to a quinone structure and vice versa.
  • the benzoquinone/hydoquinone couple and the p-aminophenol/quinoneimine couple are well known redox couples of this class.
  • redox mediators include but are not limited to catechol and catechol derivatives (e.g., adrenalin, dihydroxyphenylalanine, epinine, adrenalone, norhomoepinephrine, and protocatechnic acid), dopamine, methoxytyraminin, aromatic compounds with more than one aromatic ring structure such as anthracene derivatives, as well as heterocyclic aromatic compounds such as serotonin and hydroxyindolacetic acid.
  • Aromatic redox mediators may be further derivatized with appropriate substituents to generate optimal redox potentials.
  • redox mediators are organic and anorganic metal complexes that can be reversibly oxidized and reduced.
  • the metal complexes contain osmium, ruthenium, iron, copper, or chromium.
  • Some of these metal complexes such as the ferricinium ion/ferrocene couple and bipyridyl derivatives of the Os(ll)/Os(lll) couple have been employed in numerous amperometric and voltammetric assay procedures.
  • Metal complexes provide several important advantages as redox mediators. Most important, metals can be complexed by various aromatic and heterocyclic aromatic compounds, the side chains of which may be used to adjust the redox potential according to the specific assay requirements.
  • the redox potential of ferrocene has been changed to both more anodic and more cathodic redox species by derivatization with residues such as carboxyl groups, halogens, aminoethyl groups, or pyridine derivatives.
  • residues such as carboxyl groups, halogens, aminoethyl groups, or pyridine derivatives.
  • Preferred are electron positive side chains since they tend to change the potential of redox mediators towards O mV.
  • Metal complexes provide a second important advantage in that they offer the possibility to introduce water-solubility by derivatization of the complexes with polar residues or groups that can be protonated.
  • covalent derivatization of the phenyl residues of Os-bipyridyl-complexes with carboxyl groups improve the water-solubility and, thereby, the access of theses complexes to the electrodes by a factor of 10 as compared to Os-bipyridyl-complexes formed with non- derivatized, hydrophobic phenyl residues.
  • carboxyl-derivatized Os- bipyridyl-complexes provide both, high water-solubility and highly efficient redox reycling.
  • captured analytes are detected by affinity liposomes containing encapsulated reporter molecules, preferably redox mediators.
  • Affinity components attached to the liposomal surface are utilized to mediate analyte- specific binding of affinity liposomes.
  • the release of redox mediators encapsulated in specifically bound affinity liposomes is dependent of the kind of liposomes selected; often it may be effected by an increase of the ambient temperature or the addition of liposome lysing solvents (e.g., detergents).
  • Liposomes are artificial structures primarily composed of phospholipid bilayers exhibiting amphiphilic properties. Other molecules, such as cholesterol, fatty acids, or lipid derivatives also may be included in the bilayer construction.
  • the morphology of liposomes can be classified according to compartmentalization of aqueous regions between bilayer shells. If the aqueous regions are segregated by only one bilayer each, the liposomes are called unilamellar vesicles (ULV). If there is more than one bilayer surrounding each aqueous compartment, the liposomes are termed multilarnellar vesicles (MLV). ULV are further classified according to their relative size.
  • the diameter of small unilamellar vesicles is less than 100 nm with a minimum of about 25 nm, whereas the diameter of large unilamellar vesicles (LUV) is more than 100 nm with a maximum of about 2500 nm.
  • MLVs typically form large complex honeycomb structures. As a consequence of the almost infinite number of ways each bilayer can be associated and interconnected with other bilayers, MLVs are difficult to categorize or exactly to reproduce. MLVs are the simplest to prepare, the most stable, and the easiest to scale up to large production levels. Although the many kinds of liposomes may be used in the present invention, the most useful form thereof consists of small, spherical ULVs containing hydrophilic electrochemically active reporter molecules that are protected from the outer environment by the lipid shell.
  • the outside surface of the liposomes of the present invention is derivatized to contain covalently attached molecules designed to target the liposomes for specific interactions with nucleic acids or derivatives thereof.
  • liposomes are termed affinity liposomes in this invention.
  • lipid components of affinity liposomes Phospholipids are the most important constituents of affinity liposomes. Two main forms of lipid derivatives exist biologically, molecules containing a glycerol backbone and those containing a sphingosine backbone. Naturally occurring phospholipids can be isolated from a variety of sources including egg yolk. The composition of egg phospholipids, however, can vary considerably depending on age of the eggs, the diet of the chickens, and the method of processing.
  • egg lecithin for example is not a single compound, but consists of a mixture of phosphatidyl cholines containing about 31 % saturated fatty acid having a chain length of 16 carbons, 16% saturated fatty acid with 18 carbons, about 48% also with 18 carbons but having at least 1-2 points of unsaturation, and the rest a variety of other fatty acid constituents.
  • Preferred for this invention are synthetic phospholipids of known chemical purity.
  • fatty acid derivatives of synthetic phospholipids are used primarily in affinity liposome preparation: (1 ) myristic acid (n-tetradecanoic acid; containing 14 carbons), (2) palmitic acid (n-hexadecanoic acid; containing 16 carbons), and (3) stearic acid (n-octadecanoic acid, containing 18 carbons).
  • affinity liposome preparations may be cholesterol.
  • cholesterol modulates the permeability and fluidity of the associated membrane, increasing both parameters at temperatures below the phase transition point and decreasing both above the phase transition temperature.
  • lipid compositions for the preparation of stable affinity liposomes of the present invention contains phosphatidyl ethanolamine (PE) derivatives.
  • PE phosphatidyl ethanolamine
  • examples include compositions with molar ratios of phosphatidyl choline (PC): cholesterol: negatively charged phospholipid (e.g., phosphatidyl glycerol, PG): derivatized PE of 8: 10: 1 : 1.
  • Another preferred composition using a maleimide derivative of PE without PG is PC: cholesterol: maleimide-PE of 85: 50: 15 (Friede, M. et al., F. Anal. Biochem. 211 , 117, 1993).
  • the PE derivatives do not exceed a ratio of 1-10 mol PE per 100 mol of total lipid to maintain membrane stability.
  • liposomes may be used in the present invention which allow the release of encapsulated reporter molecules, e.g. redox mediators, by a moderate increase of the ambient temperature.
  • encapsulated reporter molecules e.g. redox mediators
  • Liposomes are known to release encapsulated water-soluble contents more quickly near their liquid crystalline phase-transition temperature (T m ) than at other temperatures (Blok, M.C. et al., J. Biochim. Biophys. Acta 433, 1 , 1976).
  • T m liquid crystalline phase-transition temperature
  • Such a temperature-sensitive release can be engineered by the selection of pure lipids that undergo sharp transition temperatures or by using mutually miscible mixtures of pure lipids to adjust the transition temperature to the desired point.
  • DPPC dipalmitoyl phosphatidylcholine
  • DPPG dipalmitoyl phosphatidyglycerol
  • DSPC distearoyl phosphatidylcholine
  • Typical lipid compositions of temperature-sensitive SUV, LUV, and MLV liposomes are described by various authors (for example, see, Magin, R.L., and Weinstein, J.N. In: Liposome Technology. (G. Gregoriadis, ed.) vol. III., pp. 137-155, CRC Press, Boca Raton, FI., 1984).
  • affinity liposomes which are useful for this invention.
  • One group of them comprises the following steps: (i) dissolving the lipid mixture in organic solvent, (ii) dispersion in an aqueous phase containing redox mediators, and (iii) fractionation to isolate the correct liposomal population.
  • Organic solvents are preferably maintained under a nitrogen or argon atmosphere to prevent introduction of oxygen.
  • Water and buffers are preferably degassed using a vacuum and bubbled with inert gas before lipid components are introduced.
  • the lipid components are dissolved in organic solvent (e.g., chloroform: methanol, by volume 2:1).
  • organic solvent e.g., chloroform: methanol, by volume 2:1.
  • This mixture will include any phospholipid derivatized with reactive groups or affinity components soluble in organic solvents as well as other lipids used to form the liposomal structure.
  • organic solvent e.g., chloroform: methanol, by volume 2:1.
  • organic solvent e.g., chloroform: methanol, by volume 2:1.
  • This mixture will include any phospholipid derivatized with reactive groups or affinity components soluble in organic solvents as well as other lipids used to form the liposomal structure.
  • Preferred methods include (i) mechanical dispersion, (ii) detergent-assisted solubilization, and (iii) solvent- mediated dispersion.
  • mechanical dispersion methods for the preparation of affinity liposomes, an aqueous solution containing the redox mediators is added to the dried, homogenous lipid mixture and manipulated to effect dispersion.
  • mechanical dispersion methods include but are not limited to simple shaking, non-shaken aqueous contact, high-pressure emulsification, sonication, extrusion through small-pore membranes, and various freeze-thaw techniques. Most of these methods result in a population of vesicles ranging from SUVs of only 25 nm diameter to very large MLVs.
  • detergent-assisted dispersion methods for the preparation of affinity liposomes, the amphipathic nature of detergent molecules is utilized to bring more effectively the lipid components into the aqueous phase for dispersion.
  • the detergent molecules bind and mask the hydrophobic tails of lipids from the surrounding water molecules of the aqueous phase containing the redox mediators.
  • Detergent treatment may be performed using a dried lipid mixture or small vesicles.
  • non-ionic detergents such as the Triton X family, alkyl glycosides, or bile salts such as sodium deoxycholate are employed for this procedure.
  • the lipid micelles aggregate to form larger liposome structures. Liposomes of up to 100 nm containing a single bilayer may be created using detergent-asssisted methods.
  • the lipid mixture is first dissolved in a organic solvent to create a homogeneous solution and thereafter introduced into a aqueous phase containing the redox mediators.
  • the solvent may or may not be soluble in the aqueous phase to effect this process.
  • a solvent-mediated dispersion method is described by Batzri, S., and Korn, E.D. (Biochim. Biophys. Acta 298, 1015, 1973). Phospholipids and other lipids to be part of the liposomal membrane are first dissolved in ethanol.
  • This ethanolic solution is then rapidly injected into a redox mediator-containing aqueous solution of 0.16 M KCI using a Hamilton syringe, resulting in a maximum concentration of no more than 7.5% ethanol.
  • a redox mediator-containing aqueous solution of 0.16 M KCI using a Hamilton syringe, resulting in a maximum concentration of no more than 7.5% ethanol.
  • single bilayer liposomes of about 25 nm diameter can be formed.
  • Other preferred solvent-mediated dispersion methods utilize solvents that are insoluble in the aqueous phase
  • the production of liposomes by this procedure involves the formation of a 'water-in-oil' emulsion.
  • a small quantity of aqueous phase containing the redox mediators is introduced into a large quantity of organic phase containing the dissolved liposomes.
  • the result is a milky dispersion.
  • the emulsification process involves the use of mechanical means (shaking, stirring, or sonication) to effect the formation of small droplets of aqueous solution uniformly dispersed in the lipid- organic phase. Excess of organic solvent is then removed by rotary evaporation (reverse-phase-evaporation method) until the mixture becomes a viscous gel. To facilitate liquification of the gel, a small volume of aqueous phase is added. Finally, residual organic solvent and untrapped redox mediators are removed by dialysis.
  • the encapsulation efficiency of reporter molecules, preferably water-soluble redox-mediators, within affinity-liposomes depends on the liposome type.
  • a comparative analysis of the encapsulation effiency in SUVs, LUVs, and MLVs of the water-soluble marker compounds 5(6)-carboxyfluorescein (CF) and tritiated cytosine-1- ⁇ -D-arabinofuranoside ([ 3 H]-Ara-C) has been performed by Magin, R.L., and Weinstein, J.N. (In: Liposome Technology (G. Gregoriadis, ed.) vol. III., pp. 137-155, CRC Press, Boca Raton, FI., 1984). Typical values of this study are given in Table I. For a fixed quantity of lipid, LUVs have the highest encapsulation efficiency.
  • a suitable mixture of lipids is provided to obtain a stable liposomal layer which is preferably solved in an organic solvent.
  • One method to prepare the said liposomes is to dry the lipid mixture, e.g. under reduced pressure at or close to ist transition temperature (the highest transition temperature of any one lipid in the mixture is taken into consideration) and to hydrate the lipid mixture with the reporter molecules dissolved in a suitable aqueous buffer. Vortexing or sonification assists the forming of liposomes.
  • Another method to prepare the said liposomes is to provide the lipid mixture in a suitable organic solvent, to add the reporter molecules in an aqueous buffer, to sonicate the mixture until it appears homogeneous and to evaporate the organic phase, e.g. under reduced pressure (reverse-phase evaporation technique).
  • the size of the liposomes thus obtained may be modified, e.g. SUV ' s filled with enzyme activator may be converted into LUVs.
  • the liposomes may be separated from excess enzyme activator in their environment by a variety of methods, for example using gel chromatography or washing steps. Alternatively or in addition, they may be centrifuged or dialyzed against buffer. Further, the liposomes may be filtered, e.g. under pressure or with suction through a 0.3 to 1.0 ⁇ m pores containing filter. Via filtering, not only a separation from excess enzyme activator is possible, but also controlling their size and/or size distribution.
  • the release of encapsulated reporter molecules may be mediated by the addition of detergent (i.e., Triton X-100 or sodium deoxycholate) or organic solvent. Further, the release of encapsulated reporter molecules may be mediated by an increase of the ambient temperature (phase-transition release).
  • detergent i.e., Triton X-100 or sodium deoxycholate
  • organic solvent i.e., Triton X-100 or sodium deoxycholate
  • phase-transition release A comparative analysis of phase-transition-mediated release of water-soluble marker compounds from different types of temperature-sensitive liposomes has been performed by Magin, R.L., and Weinstein, J.N. (The design and characterization of temperature- sensitive liposomes. In: Liposome Technology (G. Gregoriadis, ed.), vol. III., pp. 137-155, CRC Press, Boca Raton, FI., 1984).
  • Typical values obtained with the marker compounds 5(6)-carboxyfluorescein (CF) and tritiated cytosine-1- ⁇ -D- arabinofuranoside ([ 3 H]-Ara-C) are summarized in Table II below.
  • Preferred heating rates at passage through T m are 10 to 15 °C/min.
  • detergent-mediated release of encapsulated redox mediators may be the preferred mechanism when ultimate detection sensitivity is required.
  • the addition of detergent requires to provide an additional container, whereas heating to approximately 45 °C is relatively easy to accomplish. Therefore, phase-transition-mediated release may be the preferred choice of release mechanism for less sensitive but for more cost-efficient assay procedures.
  • Table I Encapsulation and release characteristics of different types of temperature-sensitive liposomes
  • DPPC dipalmitoyl phosphatidylcholine
  • T m 41 °C
  • DPPG dipalmitoyl phospha- tidylglycerol
  • a purified lipid component e.g., phosphatidyl ethanolamine or cholesterol
  • a purified lipid component may be derivatized with an affinity component prior to incorporation into the lipid bilayer construction.
  • a purified lipid component may be activated prior to incorporation into the lipid bilayer construction and further derivatized with an affinity component after formation of the intact liposome.
  • the activation process and subsequent coupling step of an affinity component may be performed after formation of the intact liposome.
  • lipid functional groups Numerous methods are available for derivatization of lipid functional groups. However, three main strategies are preferably used to couple affinity components to purified lipid components or lipid components incorporated into the lipid bilayer of liposomes: (i) heterobifunctional cross-linker-mediated conjugation reactions, (ii) carbodiimide-mediated reactions for coupling of amine residues to carboxyl groups or amine residues to phosphate groups, and (iii) reductive amination reactions for coupling of amine residues to aldehydes. V.6.1. Activation of lipid components with heterobifunctional reagents
  • heterobifunctional reagents for the activation of lipid components includes amine- and sulfhydryl-reactive cross-linking reagents containing an N-hydroxy-succinimide (NHS) ester on one end and a maleimide, iodoacetyl, or pyridyl disulfide group on the other end.
  • NHS N-hydroxy-succinimide
  • reagents used for activation of lipid components include but are not limited to succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), N-succinimidyl-4-(p-maleimido-phenyl)butyrate (SMPB), N-succinimidyl-(4-iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl-6- [(iodoacetyl)amino]hexanoate (SIAX), and N-succinimidyl-3-(2-pyridyl- dithio)propionate (SPDP).
  • SMCC succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate
  • MCS m-maleimidobenzoyl
  • cross-linking reagents which contain long spacer molecules between the two functionalities.
  • the length of the spacer is important in providing sufficient distance from the liposomal surface to ensure binding of the liposome-attached affinity component with another affinity partner.
  • heterobifunctional cross-linking reagents with long spacer molecules include but are not limited to the long-chain versions of regular cross-linking reagents such as (LC-SPDP) and (SIAXX) (formulas see V.2.5).
  • phosphatidyl ethanolamine (PE) used as a component of the liposomes is derivatized with heterobifunctional cross-linking reagents, preferably with those which derivatize the amino group of PE with a pyridyl disulfide group (e.g., by reaction with LC-SPDP), a maleimide residue (e.g., by reaction with SMPB, an iodoacetyl group (e.g., by reaction with SIAXX), a thioester moiety (e.g., by reaction with SATP, succinimidyl acetyl-thiopropionate), or an aldehyde function (e.g., by reaction with SFPA, succinimidyl-p- formylphenoxyacetate).
  • a pyridyl disulfide group e.g., by reaction with LC-SPDP
  • a maleimide residue e.g., by reaction with SMPB
  • Purified PE phospholipid may be modified in organic solvent prior to incorporation into liposomes, or intact liposomes containing PE may be activated while suspended in an aqueous solution. It is more preferred that the PE derivative is prepared before the liposome is constructed. In this way, a stable stock preparation of modified PE can be made and used in a number of different liposome recipes.
  • the PE employed is of a synthetic variety having fatty acid constitutents of dimyristoyl (DMPE), dipalmitoyl (DPPE), or distearoyl (DSPE) forms.
  • heterobifunctional reagents are preferred which are not modified with a sulfo residue at the N- hydroxysuccinimide ester moiety, since activation of PE is performed under non- aqueous conditions.
  • reporter molecules e.g. redox mediators to be entrapped within the liposome are reactive with the PE derivatives, PE molecules are activated after formation of the liposomal structures to ensure derivatization of only the outer half of the lipid bilayer.
  • heterobifunctional reagents are preferred which are modified with a sulfo residue at the N-hydroxysuccinimide ester moiety, since sulfo-derivatized cross-linking reagents cannot penetrate lipid bilayers.
  • Examples of useful derivatives of affinity components for covalent attachment to activated lipid components include but are not limited to daunorubicin derivatized at its daunosamine moiety with (LC-SPDP), (SIAXX), or 2-iminothiolane (2-IT). N 2 -(3'aminopropyl) actinomycin D derivatized at its terminal amino group with LC-SPDP, SIAXX, or 2-IT, and 5'-amine terminal oligonucleotides derivatized with LC-SPDP or SIAXX.
  • affinity components containing nucleophilic moieties such as a primary amine or a thiol may be reacted with lipid components that have been derivatized with an electrophilic moiety.
  • electrophilic moieties include but are not limited to alkyl halides, alkyl sulfonates, active esters such as N-hydroxysuccimide esters, aldehydes, ketones, isothiocyano, maleimido, pyridyl disulfide, and carboxylic acid chloride residues.
  • lipid components or lipid derivatives containing a nucleophilic moiety can be reacted with an electrophilic moiety on the affinity component.
  • any of a wide range of functional groups may be utilized for conjugation provided these groups are complementary.
  • lipid components after formation of intact liposomes is the preferred method of this invention, if proteinaceous affinity components such as antibodies with specificity for double- and/or triple-stranded nucleic acids are covalently attached to the liposomal surface or reporter molecules to be entrapped within the liposome are reactive with activated lipid components.
  • lipid components such as PE molecules are activated after formation of the liposomal structures to ensure derivatization of only the outer half of the lipid bilayer.
  • affinity components may be covalently coupled to intact liposomes are similar to those employed for derivatization of lipid components and well known in the art.
  • sulfo-N-succinimidyl ester variety of cross-linking reagents is preferred for activation of intact liposomes in aqueous suspension since they are incapable of penetrating membranes. Thus, only the outer surface of the liposomes will be modified.
  • affinity proteins proteinaceous affinity components
  • binding reagents such as antibodies
  • carbodiimides by reductive amination, or by N-hydroxysuccinimide (NHS) ester activation of carboxylates.
  • affinity proteins are coupled via their primary amine groups to N-hydroxysuccinimide ester-derivatized palmitic acid followed by incorporation of the protein-palmitate conjugates into the bilayer construction of intact liposomes by detergent dialysis as described by Huang, A., Huang, L., and Kennel, S.J. (J. Biol. Chem. 255, 8015, 1980).
  • affinity proteins are coupled via their primary amine groups to DMS (dimethyl suberimidate)-derivatized PE molecules incorporated into the bilayer construction.
  • DMS dimethyl suberimidate
  • DMS is a homobifunctional cross-linking agent containing amine-reactive imidoesters on both ends. The resulting amidine linkages are positively charged at neutral pH, thus maintaining the positive charge contribution of the original amine.
  • affinity proteins are first derivatized with amine-reactive heterobifunctional cross-linking reagents to introduce pyridyl disulfide groups (e.g., by reaction with with LC-SPDP), maleimide residues (e.g., by reaction with succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB)), or iodoacetyl groups (e.g., by reaction with SIAXX).
  • pyridyl disulfide groups e.g., by reaction with LC-SPDP
  • maleimide residues e.g., by reaction with succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB)
  • iodoacetyl groups e.g., by reaction with SIAXX.
  • Such derivatized affinity proteins are coupled to sulfhydryl-derivatized PE molecules incorporated into the bilayer construction.
  • affinity proteins containing a free sulfhydryl group are coupled to incorporated PE molecules derivatized with sulfhydryl-reactive residues such as pyridyl disulfide, maleimide, or iodoacetyl groups.
  • Affinity proteins without a free sulfhydryl group are first derivatized with amine-reactive heterobifunctional cross-linking reagents to introduce free or protected sulfhydryl residues such as thioester moieties (e.g., by reaction with SATP).
  • the thioester moieties can be deprotected by treatment with an excess of neutral hydroxylamine.
  • affinity proteins are used which are attached to the liposomal surface through non-covalent, high affinity interactions such as the biotin-(strept)avidin interaction.
  • biotinylated liposomes may be derivatized with biotinylated proteinaceous affinity components using (strept)avidin as bridging molecule or may be derivatized with a proteinaceous affinity component covalently conjugated with (strept)avidin. It is preferred to employ a long-chain spacer in constructing the biotin-PE derivative to allow sufficient spatial separation from the bilayer surface to accomodate (strept)avidin docking.
  • the basic assay procedure of the present invention includes binding of analyte (target nucleic acids or amplicons thereof) to immobilized capture oligonucleotides by molecular biological interactions, detection of captured nucleic acids or amplicons thereof by specific binding of affinity liposomes containing encapsulated reporter molecules, e.g. redox mediators, removal of non-bound affinity liposomes, release of reporter molecules encapsulated in specifically bound liposomes, and detection and preferably quantitation of released reporter molecules by means of amperometry or voltammetry using interdigitated array electrodes.
  • analyte target nucleic acids or amplicons thereof
  • immobilized capture oligonucleotides by molecular biological interactions
  • detection of captured nucleic acids or amplicons thereof by specific binding of affinity liposomes containing encapsulated reporter molecules, e.g. redox mediators, removal of non-bound affinity liposomes, release of reporter molecules encapsulated in specifically bound liposome
  • affinity liposomes allow excellent detectability of a binding event. Binding of one small affinity liposome to a captured target nucleic acid provides up to 10 5 encapsulated molecules of redox mediators. However, for applications that require higher detection sensitivity, an amplification step may be required.
  • Preferred amplication techniques of this invention are non-enzymatic amplication methods.
  • captured target nucleic acids or amplicons thereof are detected by hydrophilic polymers containing two different affinity components, one for specific binding to captured nucleic acids and the other for binding of affinity liposomes.
  • hydrophilic polymers containing two different affinity components one for specific binding to captured nucleic acids and the other for binding of affinity liposomes.
  • each captured nucleic acid molecule binds a multidude of affinity liposomes since polymers allow covalent attachment of multiple affinity components.
  • the signal may be amplified by one to two orders of magnitude.
  • Preferred examples of synthetic and natural polymer derivatives are selected from the o coprising derivatives of polysaccharides, poly(amino acids), po!y(vinyl alcohols), poly(vinyl pyrrolidinones), poly(acrylic acids), various polyurethanes, polyphosphazenes, and copolymers of these polymers.
  • derivatives of dextran are employed as polymeric carrier molecules.
  • Preferred amplified assay procedures utilizing polymeric carrier systems are those in which captured target nucleic acids or amplicons thereof are detected by dextran polymers containing two types of affinity components.
  • One of the covalently linked affinity components is capable of specifically binding to a specific captured target nucleic acid or its amplicons in a structure restricted manner (e.g., intercalating agents or oligonucleotides).
  • the other covalently linked affinity components are capable of specifically binding multiple affinity liposomes.
  • bound dextran polymers are detected by affinity liposomes containing surface-attached affinity components capable of binding to the affinity components on the dextran polymers.
  • the release of redox mediators encapsulated in specifically bound affinity-liposomes is effected by an increase of the ambient temperature or the addition of liposome-lysing solvents such as organic solvents or detergents.
  • the quantity of released redox mediators is a proportional measure of the amount of target nucleic acids in the specimen.
  • affinity liposomes include but are not limited to hapten / anti-hapten antibody affinity systems, enzyme inhibitor / enzyme affinity systems, and the biotin / (strept)avidin affinity system.
  • affinity liposomes containing surface-attached proteinaceous affinity components e.g., anti-hapten antibodies, enzyme molecules, or (strept)avidin
  • the corresponding low molecular weight affinity partner e.g., hapten molecules, enzyme inhibitors, or biotin residues.
  • enzyme inhibitors and corresponding enzymes are used for additional affinity binding steps.
  • Several considerations are important for the choice of enzyme inhibitors suitable for use in the present invention. High affinity binding of the inhibitor to the corresponding enzyme is the most important requirement.
  • the overall binding constant (K 0 ff/K 0 n) should be in the low nanomolar to picomolar range to guarantee tight binding.
  • Methotrexate represents one example of such an inhibitor. Methotrexate binds to dihydrofolate reductase (DHFR) with an overall binding constant of 2.1 x 10" 10 M.
  • DHFR dihydrofolate reductase
  • multisubstrate adduct inhibitors are constructed of multisubstrate adduct inhibitors (Broom, A.D. J. Med. Chem. 32, 2, 1989).
  • such inhibitors can be designed for any enzyme that binds two or more substrates simultaneously (cofactors are considered to be substrates in this context).
  • multisubstrate adduct inhibitors for enzymes catalyzing bimolecular reactions can be synthesized by covalent conjugation of both substrates.
  • the binding affinity of potent multisubstrate adduct inhibitors is 10 3 -10 6 times the binding affinity of either substrate.
  • Another preferred approach of increasing the affinity of inhibitor-enzyme interactions is to combine several covalently linked inhibitor molecules with an enzyme complex consisting of two or more copies of the enzyme. Provided the binding sites are in sufficiently close position, simultaneous binding of covalently linked inhibitor molecules is likely to occur. This will increase the apparent affinity of the inhibitor- enzyme interaction significantly.
  • Suitable enzyme inhibitors include i) low molecular weight, ii) solubility in aqueous solutions, and iii) the ability of chemical conjugation of the inhibitor to polymers without impairment of the binding affinity.
  • Suitable for use in the invention are water-soluble, small molecular weight inhibitors.
  • methotrexate L-4-amino-N 10 - methylpteroyl-glutamic acid
  • DHFR dihydrofolate reductase
  • RPI placental ribonucleae inhibitor
  • RPI is a 50kD protein that forms tight complexes with ribonucleases.
  • RPI inhibits RNase A with an extremely low Kj value of 4 x 10 _14 M, approaching the affinity of avidin for biotin.
  • RPI binds to angiogenin, a blood vessel-inducing protein with 35% sequence homology to pancreatic RNase, with an even lower Ki value of 4 x 10 -16 M.
  • DHFR represents one example of such enzymes. DHFR is found in microorganisms as well as in vertebrates and is easy to purify by affinty chromatography techniques using methotrexate- derivatized matrices. The molecular weight of DHFR of different sources varies between 18 kD and 24 kD. Mammalian DHFR are in general slightly larger
  • DHFR has been derivatized with heterobifunctional cross-linking reagents such as LC-SPDP and conjugated to other proteins with good retention of methotrexate binding affinity.
  • haptens and corresponding anti-hapten antibodies are used for additional affinity binding steps. Due to the high affinity of some anti-hapten antibodies, hapten-anti-hapten antibody systems provide sensitive detection reagents. For example, human chromosomes have been probed by hapten-modified cosmid probes with a sensitivity equal to that achieved with biotinylated cosmid probes.
  • antibody refers to immunoglobulins of any isotype or subclass as well as any fragment (e.g., Fab' of Fv fragments) of the aforementioned.
  • Antibodies of any source are applicable including polyclonal materials obtained from any animal species, monoclonal antibodies from any hybridoma source, and all immunoglobulins (or fragments) generated with the aid of viral, prokaryotic or eukaryotic expression systems.
  • Non-biologic binding molecules such as "molecular imprints" (synthetic polymers with pre-determined specificity for binding or complex formation) are also applicable to the invention.
  • Preferred examples of haptens include but are not limited to digoxin, digoxigenin, dinitrophenol (DNP), trinitrophenol (TNP), biotin, fluorescein, tetramethyl- rhodamin, Texas Red, dansyl residues, lucifer yellow, and Cascade Blue fluorophores.
  • biotin and (strept)avidin are used for additional affinity binding steps.
  • Avidin is a glycoprotein found in egg whites that contains four identical subunits of 16,400 daltons each, giving an intact molecular weight of approximately 66 kD. Each subunit contains one binding site for biotin.
  • the biotin interaction with avidin is among the strongest non-covalent affinities known, exhibiting an affinity constant of about 1.3 x 10 -15 M.
  • the tetrameric native structure of avidin is resistant to denaturation under extreme chaotropic conditions.
  • One disadvantage of the use of avidin is its tendency to bind nonspecifically to components other than biotin due to its carbohydrate content and its high pi of about 10.
  • the strong positive charge on the protein causes ionic interactions with more negatively charged molecules such as nucleic acids.
  • Streptavidin is another biotin binding protein from Streptomyces avidinii that can overcome some of the nonspecificities of avidin. Similar to avidin, streptavidin contains four subunits, each with a single biotin binding site. The intact tetrameric protein has a molecular mass of about 60 kD, slightly less than that of avidin. Like avidin, streptavidin is an extremely robust protein that can tolerate a wide range of buffer conditions, pH values, and chemical modification processes without loss of biotin binding activity. The primary structure of streptavidin, however, differs considerably from that of avidin despite the fact that they both bind biotin with similar avidity. This variation in the amino acid sequence results in a much lower pi of 5-6.
  • affinity components may be derivatized and covalently coupled to polymers.
  • affinity components containing nucleophilic moieties such as a primary amine, a thiol, or a hydroxyl group may be reacted with residues on polymeric carrier molecules that contain electrophilic moieties or have been derivatized with such a moiety.
  • electrophilic moieties include, but are not limited to alkyl halides, alkyl sulfonates, active esters such as N-hydroxysuccimide esters, aldehydes, ketones, isothiocyano, maleimido, and carboxylic acid chloride residues.
  • any of a wide range of functional groups on both the affinity components and the polymeric crrier molecules may be utilized for conjugation provided these groups are complementary.
  • proteinaceous affinity components such as enzymes, antibodies, and streptavidin
  • one strategy involves the use of hetero- or homobifunctional cross-linking reagents.
  • protein components may be derivatized with pyridyl disulfide groups (e.g., by reaction with SPDP) and subsequently coupled to sulfhydryl-containing polymeric carrier molecules via disulfide linkages.
  • SPDP pyridyl disulfide groups
  • the introduced sulfhydryl residues may be reduced with disulfide reducing agents to create terminal sulfhydryl groups for coupling to sulfhydryl-reactive polymeric carrier molecules.
  • SPDP should affect the binding activity of one of these proteins, there are a number of additional cross-linking reagents for coupling via disulfide bonds such as 2-iminothiolane (2-IT) and N-succinimidyl S-acetylthioacetate (SATA).
  • 2-IT reacts with primary amines, instantly incorporating an unprotected sulfhydryl group.
  • SATA also reacts with primary amines, but incorporates a protected sulfhydryl group, which is subsequently deacetylated using neutral hydroxylamine to produce a free sulfhydryl group.
  • Other cross-linkers are available that can be used in different strategies for covalent coupling of proteinaceous affinity components to polymeric carrier molecules.
  • TPCH S-(2-Thiopyridyl)-L-cysteine hydrazide
  • TPMPH S-(2-thiopyridyl)mercaptopropiono hydrazide
  • cross-linking reagents may be used.
  • N-( ⁇ -maleimidobutyryloxy)- succinimide (GMBS) and SMCC (see above) react with primary amines, thereby introducing a maleimide group for coupling to sulfhydryl-containing polymeric carrier molecules via stable thioether linkages.
  • cross-linking reagents may be used which introduce long spacer arms if steric hindrance problems interfere with the binding activity of the covalently coupled affinity component.
  • suitable cross-linking reagents which could be used. Suitable reactions would be well known to one skilled in the art based on the nature of the reactive groups that are available or have been introduced to the molecules and information about the binding site requirements of the affinity components.
  • low molecular weight affinity components include but are not limited to D-biotinyl-aminocaproic acid-N-hydroxy-succinimide ester and digoxigenin-3-O-methyl-carbonyl- ⁇ -aminocaproic acid-N-hydroxy-succinimide ester for coupling to amine-containing polymeric carrier molecules, and methotrexate- ⁇ -hydrazide (Rosowsky, A., Forsch, R., Uren, J., and Wick, M. J. Med. Chem. 24, 1450, 1981 ) for coupling to aldehyde-containing polymeric carrier molecules.
  • methotrexate- ⁇ -hydrazide Rosowsky, A., Forsch, R., Uren, J., and Wick, M. J. Med. Chem. 24, 1450, 1981
  • Dextran is a naturally occurring, mainly a linear polysaccharide consisting of repeating units of D-glucose linked together in glycosidic bonds wherein the carbon-1 of one monomer is attached to the hydroxyl group at the carbon-6 of the next residue. Occasional branch points also may be present in a dextran polymer, occurring as ⁇ -1 ,2, ⁇ -1 ,3, or ⁇ -1 ,4 glycosidic linkages.
  • the monomers contain at least three hydroxyls that may undergo derivatization reactions.
  • dextran polymers of molecular weight of 10,000 - 50,000 have been used in the past as a modifying agent for proteins and other molecules including the application as a carrier of biotin residues and hapten molecules. Furthermore, dextran polymers have been used as a multifunctional linker to cross-link monoclonal antibodies with low molecular weight compounds.
  • dextran polymers are used which are oxidized with periodate to produce aldehydes. This procedures results in two aldehyde groups formed per glucose monomer, thus producing a highly reactive, multifunctional polymer able to couple with numerous amine-containing molecules (Bernstein, A. et al., J. Natl. Cancer Inst. 60, 379, 1978).
  • Polyaldehyde dextran may be conjugated with amine groups by Schiff base formation followed by reductive amination to create stable secondary (or tertiary amine) linkages. Amine- containing affinity components may be coupled to oxidized dextran polymers under mild conditions using sodium cyanoborohydride as reducing agent.
  • the optimal pH for the reductive amination reaction is an alkaline environment between pH 7 and 10.
  • dextran derivatives containing carboxy or amine-terminal groups are used for coupling of affinity components.
  • Amine- terminal derivatives may be prepared by coupling diamine compounds such as ethylene diamine or diaminodipropylamine (3,3"-iminob/spropylamine) in excess to polyaldehyde dextran.
  • Carboxyl-terminal derivatives may be prepared similarly by coupling molecules such as 6-aminocaproic acid or ⁇ -alanine to polyaldehyde dextran.
  • reactive alkyl halogen compounds containing a terminal carboxylate group on the other end such as chloroacetic acid or 6-bromohexanoic acidmay be used.
  • the carboxylates may then be aminated with ethylene diamine to form an amine-terminal spacer and further reacted with amine-reactive heterobifunctional reagents to prepare dextran derivatives carrying terminal sulfhydryl residues or sulfhydryl-reactive groups such as pyridyl disulfide, maleimide, or iodoacetyl groups.
  • dextran derivatives are used for covalent coupling of affinity components containing free sulfhydryl groups or derivatized with sulfhydryl-reactive residues.
  • complexes of affinity liposomes are utilized for signal amplification.
  • Complexes of affinity liposomes may be prepared from affinity liposomes containing two types of surface-attached affinity components, one (type I) for specific binding to captured nucleic acids or amplicons thereof in a structure restricted manner (e.g., intercalating agents or oligonucleotides) and the other (type II) for complexation of affinity liposomes via bridging molecules providing at least two binding sites for type II affinity components.
  • polymeric carrier molecules e.g., derivatives of dextran polymers
  • bridging molecules for the preparation of preformed complexes of affinity liposomes.
  • the application of polymeric carrier molecules for complexation of affinity liposomes may be useful when affinity components containing only a single binding site for the correponding affinity partner are utilized as bridging molecules.
  • affinity components may be coupled covalently to polymeric carrier molecules are described in detail in previous sections of the preferred embodiment.
  • complexes of affinity liposomes are used which are prepared with two types of affinity liposomes containing different affinity components.
  • the affinity components on type I affinity liposomes are capable of specifically binding to a specific captured target nucleic acid or its amplicons in a structure restricted manner (e.g., single-stranded oligonucleotides complementary to single-stranded segments of captured target nucleic acid).
  • the affinity components on type II affinity liposomes are capable of specifically binding to the affinity components o type I affinity liposomes (e.g., single-stranded oligonucleotides complementary to type I single-stranded oligonucleotides).
  • captured target nucleic acids or amplicons thereof are detected by specific binding of preformed complexes of affinity liposomes containing surface-attached nucleic acid-reactive affinity components. Subsequent detection and preferably quantitation of reporter molecules encapsulated in specifically bound affinity liposome-complexes is performed as described.
  • captured target nucleic acids or amplicons thereof may be detected by dextran polymers containing two types of covalently linked affinity components, one being capable of specifically binding to a specific captured target nucleic acid or its amplicons in a structure restricted manner, and the other being capable of specifically binding preformed complexes of affinity liposomes.
  • dextran polymers containing two types of covalently linked affinity components one being capable of specifically binding to a specific captured target nucleic acid or its amplicons in a structure restricted manner, and the other being capable of specifically binding preformed complexes of affinity liposomes.
  • Subsequent quantitation of redox mediators encapsulated in specifically bound complexes of affinity liposomes is performed as described.
  • Bridging molecules Suitable bridging molecules include but are not limited to a) bi- or oligovalent anti- hapten antibodies or fragments thereof, as well as conjugates or fusion constructs thereof; b) enzymes, enzyme conjugates, and fusion constructs of enzymes providing more than one inhibitor-binding site; and c) avidin and streptavidin. Any bridging molecule that provides more than one binding site is useful for this type of amplification methodology.
  • streptavidin may be employed as bridging molecule for complexation of affinity liposomes carrying surface-attached biotin residues and nucleic acid-reactive affinity components such as intercalating agents or oligonucleotides. Since streptavidin contains four biotin binding sites, it represents a preferred multivalent bridging molecule for the synthesis of preformed complexes of affinity liposomes.
  • Multivalent complexes formed by reaction of biotinylated liposomes with (strept)avidin have been shown to increase the detection sensitivity by one to two orders of magnitude.
  • high affinity anti-hapten antibodies or fragments thereof which provide more than one hapten-binding site as bridging molecules for the synthesis of preformed complexes of affinity liposomes containing surface- attached hapten molecules and surface-attached nucleic acid-reactive components (e.g., intercalating agents or oligonucleotides).
  • a hapten binding site e.g., a surface- attached hapten molecules and surface-attached nucleic acid-reactive components
  • intact IgG molecules provide two hapten binding sites and have been applied successfully for the preparation of soluble enzyme / anti-enzyme complexes such as peroxidase / anti-peroxidase (PAP) and alkaline phosphatase / anti-alkaline phosphatase (APAAP) complexes.
  • PAP peroxidase / anti-peroxidase
  • APAAP alkaline phosphatase / anti-alkaline phosphatase
  • anti-hapten antibodies of the IgM class providing 10 hapten binding sites per antibody molecule may be employed.
  • dimers or oligomers of IgG antibodies or hapten-binding fragments thereof are prepared by chemical conjugation methods using long heterobifunctional cross-linking reagents such as LC-SPDP.
  • two or more antibody binding regions with specificity for the desired hapten are conjugated by genetic engineering technology and expressed as fusion proteins.
  • scFv variable antibody regions
  • VH and VL genes joined with a short linker DNA which codes for example for a flexible (Gly4Ser)3 peptide (Clackson, T. et al., Nature 352, 624, 1991).
  • Gly4Ser flexible (Gly4Ser)3 peptide
  • 'fusion protein' refers to a genetically engineered protein whose coding region is comprised of the coding region residues of a first anti-hapten scFv molecule fused, in frame, to the coding region residues of a second anti-hapten scFv molecule.
  • the dimeric anti-hapten scFv fusion protein construct is extended with coding region residues of additional anti-hapten scFv molecules as described for the dimeric fusion construct.
  • Such constructs may also contain additional flexible oligopeptide linker fused, in frame, to the coding region residues of the scFv molecules.
  • enzyme conjugates providing at least two inhibitor binding sites as bridging molecule for the synthesis of preformed complexes of affinity liposomes containing surface-attached inhibitor molecules and surface- attached nucleic acid-reactive components (e.g., intercalating agents or oligonucleotides).
  • dimers and higher oligomers of DHFR prepared by chemical conjugation methods are used as bridiging molecules.
  • Preferred conjugation procedures use heterobifunctional cross-linking reagents containing long spacer molecules between the two functionalities such as LC-SPDP.
  • two or more DHFR molecules, DHFR mutants, or methotrexate-binding DHFR fragments are conjugated by genetic engineering technology and expressed as fusion proteins.
  • DHFR from various sources has been cloned and expressed in prokaryotic expression systems since the enzyme consists of a single polypeptide chain containing no disulfide linkages or posttranslational modifications. Due to these characteristics, DHFR represents a useful enzyme for the construction of fusion proteins.
  • the term 'fusion protein' refers to a genetically engineered protein whose coding region is comprised of the coding region residues of a first DHFR molecule fused, in frame, to the coding region residues of a second DHFR molecule.
  • the dimeric DHFR fusion protein construct is extended with coding region residues of additional DHFR molecules as described for the dimeric fusion construct.
  • Such constructs may also contain flexible oligopeptide linker fused, in frame, to the coding region residues
  • Affinity liposomes useful for the preparation of preformed complexes contain two types of surface-attached affinity components, one for specific binding to captured nucleic acids (or to polymeric carrier molecules) and the other for complexation of affinity liposomes (e.g. via bridging systems or via polymeric carrier molecules).
  • the synthesis procedures of such affinity liposomes are identical with those described in previous sections of the present specification.
  • two types of lipid derivatives are incorporated into the lipid bilayer construction, the overall molar ratios of derivatized lipid components versus non-derivatized lipid components remain unchanged in preferred lipid compositions.
  • both affinity components are covalently attached to purified lipid components prior to incorporation into the same lipid bilayer construction.
  • purified lipid components derivatized with different reactive residues are incorporated into the same lipid bilayer construction and then reacted with the two affinity components carrying different complementary functionalities.
  • the activation process and subsequent coupling step of the two affinity components is performed after formation of the intact liposome.
  • This invention comprises a system that consists of a solid support containing immobilized capture oligonucleotides, affinity liposomes containing reporter molecules, e.g. redox reporter molecules, and an electrochemical sensor.
  • the electrochemical sensor comprises miniaturized electrodes, this offers effective mass transfer of redox species and the like between the electrodes independent of artificial convection resulting in enhanced signal to noise ratios.
  • detection devices are further miniaturized by utilization of the microelectrodes as the solid support for immobilization of capture oligonucleotides.
  • sulfhydryl-derivatized capture oligonucleotides may be used for immobilization onto gold microelectrodes.
  • the environment e.g. the casing of the microelectrodes may be used as solid phase. Utilization of the casing as solid-phase may be an advantage for capturing trace amounts of target nucleic acids since the casing allows immobilization of large quantities of capture oligonucleotides.
  • OLIGONUCLEOTIDES VI.1.1 Synthesis of 5"-amine derivatives of oligonucleotides
  • Method A Covalent attachment of an amine terminal spacer molecule to the 5'- phosphate of oligonucleotides according to method A is performed via formation of a phosphorimidazolide intermediate in a carbodiimide reaction (based on the method of Ghosh, S.S., Kao, P.M., and Kwoh, D.Y. Anal. Biochem. 178, 43, 1989).
  • the formation of a phosphorimidazolide intermediate provides better reactivity towards amine nucleophiles than the carbodiimide phosphosdiester intermediate if carboddimide is used without added imidazole.
  • the carbodiimide phosphodiester intermediate also is shorter-lived in aqueous conditions due to hydrolysis than the imidazolide.
  • the 5'-phosphate-containing oligonucleotide (7.5 -15 nmol in 7.5 ⁇ l) is added to a microfuge tube containing 1.25 mg of the water-soluble carbodiimide EDC (1- ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride; Piece Chemical
  • diamine spacer molecules e.g., ethylene diamine or 1 ,6-diaminohexane
  • the diamine compound is dissolved at a concentration of 0.25
  • Method B Using method B, the desired oligonucleotide is prepared using automated standard solid-phase phosphoramidite techniques on a scale of about
  • the solid-phase synthesis protocol includes a) removal of dimethoxytrityl groups with 3% dichloroacetic acid in 1 ,2- dichloroethane, b) coupling of the incoming nucleoside 3'- methoxymorpholinophosphite with tetrazole, c) iodine oxidation of the intermediate phosphite to a phosphate, and d) a capping step with acetic anhydride.
  • the purified monomethoxytrityl protected oligonucleotide is dissolved in 2 ml of 80% acetic acid and incubated for two hours. Thereafter, the acetic acid is removed by evaporation and the detritylated amino-containing oligonucleotide is is redissolved in a small volume of water.
  • Method A For the derivatization of 5'-phosphate-containing oligonucleotides with a terminal sulfhydryl group according to method A (based on a procedure of Ghosh, S.S. et al., Bioconjugate Chem. 1 , 71 , 1990), the oligonucleotide (7.5 -15 nmol in 7.5 ⁇ l) is added to a microfuge tube containing 1.25 mg of the water- soluble carbodiimide EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; Piece Chemical Company, Rockford, IL, USA).
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • the sulfhydryl-derivatized oligonucleotide is purified by gel filtration on Sephadex G-25 using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2.
  • Method B the desired oligonucleotide is prepared by automated standard solid-phase phosphoramidite techniques on a scale of about 2.5 ⁇ mol of bound first nucleoside.
  • the solid-phase synthesis protocol includes a) removal of dimethoxytrityl groups with 3% dichloroacetic acid in 1 ,2-dichloroethane, b) coupling of the incoming nucleoside 3'-methoxymorpho!inophosphite with tetrazole, c) iodine oxidation of the intermediate phosphite to a phosphate, and d) a capping step with acetic anhydride.
  • an extra round of synthesis is carried out using 25 ⁇ mol of an S-trityl-O- methoxymorpholinophosphite derivative of 2-mercaptoethanol (dissolved in 0.3 ml of acetonitile and 0.2 ml of 1 ,2-dichloroethane), 3-mercaptopropan (1) ol (dissolved in 0.5 ml of acetonitrile), or 6-mercaptohexan (1) ol (dissolved in 0.5 ml of acetonitrile) and 75 ⁇ mol of tetrazole (dissolved in 0.5 ml of acetonitrile).
  • the S-trityl-O-methoxymorpholinophosphite derivatives are prepared according to Connolly, B.A., and Rider, P. (Nucleic Acids Res. 13, 4485, 1985). Following coupling, the phosphite intermediate is oxidized by treatment with iodine. Thereafter, the phosphate protecting groups are removed with thiophenolate and cleavage from the resin together with deblocking of the base protecting groups is effected with ammonia.
  • Oligonucleotides that have been modified with an amine-terminal spacer molecule can be reacted further with the heterobifunctional cross-linking reagent SPDP (N- succinimidyl 3-(2-pyridyldithio)propionate; Pierce Chemical Company, Rockford, IL, USA).
  • SPDP N- succinimidyl 3-(2-pyridyldithio)propionate
  • Oligonucleotides derivatized with a terminal pyridyl disulfide residue then can be coupled with sulfhydryl-containing molecules, forming a disulfide bond. Reduction of the terminal pyridyl disulfide residue releases the pyridine-2- thione leaving group and generates a terminal sulhydryl group. This procedure allows conjugation of the 5'-thiolated oligonucleotide to sulfhydryl-reactive derivatives.
  • SPDP is dissolved at a concentration of 6.2 mg/ml in DMF (makes a 20 mM stock solution).
  • the amine-derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 50 ⁇ l of the SPDP solution. After reaction for 1 hour at room temperature, excess reagents are removed from the modified oligonucleotide by gel filtration.
  • the thiolated oligonucleotide is purified from ecxess dithiothreitol by dialysis or gel filtration using 50 mM sodium phosphate, 1 mM EDTA, pH 7.2. The thiolated oligonucleotide is used immediately for further coupling reactions to prevent sulfhydryl oxidation.
  • the NHS ester of SATA introduces a thioester moiety.
  • the acetyl protecting group can be removed by treatment with neutral hydroxylamine.
  • the resulting terminal sulfhydryl group can be used for subsequent conjugation to thiol-reactive molecules.
  • SATA over disulfide-containing thiolation reagents such as SPDP is that the introduction of sulfhydryl residues does not include the use of a disulfide reducing agent.
  • the pyridiyl dithiol group resulting from an SPDP thiolation must be reduced with a reducing agent such as dithiothreitol to free the sulfhydryl group.
  • SATA the sulfhydryl is freed by hydroxylamine, thus eliminating the need for removal of sulfhydryl reductants prior to a conjugation reaction.
  • SATA is dissolved at a concentration of 8 mg/ml in DMF.
  • the amine- derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 250 ⁇ l of the SATA solution. After reaction for 3 hours at 37 °C, excess reagents are removed from the modified oligonucleotide by gel filtration.
  • the cross-linking reagent SFB (succinimidyl p-formylbenzoate) can be used to add aldehyde groups to amine-containing oligonucleotides.
  • SFB succinimidyl p-formylbenzoate
  • acetonitrile makes a 50 mM solution.
  • the amine-derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 50 ⁇ l of the SFB solution. After reaction for 3 hours at room temperature, excess reagents are removed from the modified oligonucleotide by gel filtration.
  • spacer molecules are introduced between the solid support and the immobilized capture oligonucleotides.
  • Method A the heterobifunctional reagents LC-SPDP or sulfo-LC- SPDP (both Pierce Chemical Company, Rockford, IL, USA) are utilized as spacer molecules.
  • the sulfo-NHS form of the cross-linker contains a negatively charged sulfonate group that provides water-solubility to the cross-linker.
  • LC-SPDP is dissolved at a concentration of 8.5 mg/ml in DMF (makes a 20 mM stock solution). If the water-soluble sulfo-LC-SPDP is used, a stock solution in water is prepared just prior to addition of an aliquot to the reaction, since an aqueous solution of the cross-linker will degrade by hydrolysis of the sulfo-NHS ester. A 10 mM stock solution of sulfo-LC-SPDP is prepared by dissolving 5.2 mg/ ml water. The amine-derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 50 ⁇ l of the LC-SPDP solution.
  • water-soluble sulfo-LC-SPDP 100 ⁇ l of the sulfo-LC-SPDP solution is added. After reaction for 1 hour at room temperature, excess reagents are removed from the modified oligonucleotide by gel filtration.
  • Method B water-soluble heterobifunctional derivatives of polyethylene glycol (PEG) are utilized as spacer molecules.
  • PEG polyethylene glycol
  • Heterobifunctional PEG derivatives containing an amine-reactive N-hydroxysuccinimidyl (NHS) moiety and a sulfhydryl-reactive vinylsulfone (VS) moiety are especially useful, since the VS moiety is hydrolytically stable in aqueous media.
  • the VS moiety reacts selectively with sulfhydryl groups. Reaction with amino groups proceeds at higher pH, but is still relatively slow.
  • NHS-PEG-VS (MW 3400; Shearwater Polymers Europe, Enschede, Netherlands) or NHS-PEG-VS (MW 2000; Shearwater Polymers Europe, Enschede, Netherlands) is dissolved in DMF at a concentration of 10 mM.
  • the amine- derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH
  • each of the described procedures allows to modify the density of immobilized capture oligonucleotides by co-immobilizing hydrophilic compounds capable of blocking immobilization sites for capture oligodeoxynucleotides (e.g., for amine-reactive solid supports: aminoethanol or monomethoxy-poly(ethylene glycol) containing a terminal amino group).
  • Sepharose 6B (30 g of moist cake) is washed with water, suction-dried, suspended in 24 ml of 1 M NaOH followed by dropwise addition of epichlorohydrin over 15 min at room temperature with stirring (0.75 ml of epichlorohydrin is added for a degree of subsitution of approximately 50 ⁇ mol of SH-groups per gram beads; 4.5 ml of epichlorohydrin is added for a degree of subsitution of approximately 700 ⁇ mol of SH-groups per gram beads). The suspension is then incubated with continuous stirring for 2 hrs at 60 °C. The product is washed with water and with 0.5 M sodium phosphate buffer, pH 6.25.
  • the mercapto-activated cross-linked agarose beads Prior to coupling of 5'-pyridyl disulfide derivatized oligodeoxynucleotides, the mercapto-activated cross-linked agarose beads are filtered and washed quickly with 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.5. Thereafter, the cake is suspended in 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.5, containing the 5'-pyridyl disulfide-derivatized oligodeoxynucleotide to be immobilized.
  • the pyridyl disulfide-activated cross-linked agarose beads Prior to coupling of 5'-sulfhydryl-derivatized oligodeoxynucleotides, the pyridyl disulfide-activated cross-linked agarose beads are filtered and washed with 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.5. Thereafter, the cake is suspended in 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.5, containing the 5'-sulfhydryl-derivatized oligodeoxynucleotide to be immobilized. After 20 hrs at room temperature with gentle agitation, the residue is filtered and washed with 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.5, and with distilled water.
  • the CDI-activated cross-linked agarose beads Prior to coupling of 5'-amine derivatized oligodeoxynucleotides, the CDI-activated cross-linked agarose beads are filtered and washed quickly with 0.1 M sodium bicarbonate, pH 8.5. Thereafter, the moist beads are resuspended in 0.1 M sodium bicarbonate, pH 8.5, containing the 5'-amine-oligodeoxynucleotide derivative to be immobilized. After 20 hrs at 4 °C with gentle agitation, the residue is filtered and washed with 0.1 M NaHCO3 and with distilled water. Excess active groups are removed by reaction for 1 hr at room temperature with either 0.1 M NH4OH or 0.1 M ethanolamine at pH 9.
  • Sepharose 4B or CL-6B (10 g, wet) is washed successively with 100 ml of each of the following: 30: 70 and 70: 30 of acetone: water (v/v), twice with acetone, and three times with dry acetone (dried with a molecular sieve overnight) using 1 liter of acetone per 35 g of Sepharose 4B.
  • the gel is then transferred to a dried beaker containing 3 ml of dry acetone and 150 ⁇ l of dry pyridine (dried with a molecular sieve).
  • tresyl chloride (2,2,2- trifluoroethanesulfonyl chloride) (Fluka AG, Buchs, Switzerland) is added dropwise. After 10 min at room temperature, the gel is washed twice with 100 ml of each of the following: acetone, 30: 70 of 5 mM HCI: acetone, 50: 50 of 5 mM HCI: acetone, 70: 30 of 5 mM HCI: acetone, and 1 mM HCI.
  • the beaker contains 0.6 g of tosyl chloride (p-toluenesulfonyl chloride) dissolved in 3 ml of dry acetone.
  • the tresyl- activated cross-linked agarose beads Prior to coupling of 5'-amine derivatized oligodeoxynucleotides, the tresyl- activated cross-linked agarose beads are washed quickly with cold 0.2 M sodium phosphate, 0.5 M NaCl, pH 8.2. Thereafter, the moist agarose beads (1 g) are resuspended in 1 ml of 0.2 M sodium phosphate, 0.5 M NaCl, pH 8.2, containing the 5'-amine-oligodeoxynucleotide derivative to be immobilized.
  • the gel is treated with 0.2 M Tris-HCI, pH 8.5, for 5 hrs at room temperature, and washed with a) 0.2 M sodium acetate, 0.5 M NaCl, pH 3.5, b) 0.5 M NaCl, and c) distilled water.
  • the tosyl-activated cross-linked agarose beads Prior to coupling of 5'-amine derivatized oligodeoxynucleotides, the tosyl-activated cross-linked agarose beads are washed quickly with cold 0.25 M NaHCO3 at pH 10.5. Sepharose tosyl groups require a pH of 9 to 10.5 for efficient coupling of amine-containing molecules. Thereafter, the moist agarose beads (0.7 g) are resuspended in 1 ml of 0.25 M NaHCO3, pH 10.5, containing the 5'-amine- oligodeoxynucleotide derivative to be immobilized.
  • the gel is treated with 0.8 M mercaptoethanol, pH 10, for 15 hrs at 40 °C, and washed with a) 0.2 M sodium acetate, 0.5 M NaCl, pH 3.5, b) 0.5 M NaCl, and c) distilled water.
  • oxidized Sepharose is then washed with cold and resuspended in three volumes of aqueous polyacrylhydrazide solution (0.1 - 0.5%) prepared from polymethylacrylate and hydrazine hydrate as described (Wilchek, M., and Miron, T. Meth. Enzymol. 34, 72, 1974). After slow stirring for 16 hrs in the dark at room temperature, the beads are washed extensively with 0.1 M NaCl, and then reduced with 0.3 M sodium borohydride in 0.5 M Tris-HCI, pH 8, for 3 hrs at room temperature. The reduced gel is washed with water on a sintered-glass funnel and stored at 4 °C.
  • Sulfhydryl groups are introduced by treating the gel with N-acetylhomocysteine thiolactone.
  • the thiolactone (1 g) is added to a cold suspension of 10 ml of PAHOS beads in 20 ml of 1 M NaCO3. After slow stirring for 16 hrs at 4 °C, the product is washed extensively with water and 0.1 M NaCl.
  • the sulfhydryl-activated PAHOS beads Prior to coupling of 5'-pyridyl disulfide derivatized oligodeoxynucleotides, the sulfhydryl-activated PAHOS beads are washed quickly with 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.5. Thereafter, the beads are resuspended in 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.5, containing the 5'-pyridyl disulfide-derivatized oligodeoxynucleotide to be immobilized.
  • the beads After incubation for 20 hrs at room temperature with gentle agitation, the beads are washed with 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.5, and with distilled water. Excess active groups can be removed by reaction with iodoacetate.
  • Porous glass spheres (10 ⁇ m diameter, 500 A pore diamter) (LiChrosphere Si, Altex Scientific, Inc., Berkeley, CA, USA) are treated with hot chromic acid cleaning solution follwed by rinsing with 100 ml per 1 g porous glass spheres of hot 1 M HNO3 and water. Thereafter, the cleaned porous glass spheres are suspended in a 10% aqueous solution of glycidoxypropyl trimethoxysilane, degassed with ultrasonic vibration for 10 min, and kept for 2 hrs at 90 °C, during which time the pH is maintained at 3.0 with 1 M HCI. the glass sheres are then collected on a medium-porosity glass frit, rinsed with water and dried overnight at 105 °C in vacuo.
  • the spheres are washed twice with 50 ml of each of the following: acetone, 30: 70 of 5 mM HCI: acetone, 50: 50 of 5 mM HCI: acetone, 70: 30 of 5 mM HCI: acetone, and 1 mM HCI.
  • the tresylated porous glass spheres are washed with water, 50: 50 (v/v) water: acetone, and acetone and dried.
  • the tresylated porous glass spheres are resuspended in 1 ml of 0.2 M sodium phosphate, 0.5 M NaCl, pH 8.2, containing the 5'-amine-oligodeoxynucleotide derivative to be immobilized. After 20 hrs at 4 °C with gentle agitation, the spheres are treated with 0.2 M Tris- HCI, pH 8.5, for 5 hrs at room temperature, and washed with a) 0.2 M sodium acetate, 0.5 M NaCl, pH 3.5, b) 0.5 M NaCl, and c) distilled water.
  • polyester film is a polymer of glycerol and terephthalic acid (Gareware Chemical Co., India) which on partial acid hydrolysis and periodate oxidation provides aldehyde groups (Sarkar, M., and Mandal, C. J. Immunol. Meth. 83, 55, 1985).
  • the film is hydrolyzed with 1 M H2SO4 for 6 hrs at
  • the aldehyde- activated polyester film is incubated with the 5'-hydrazide-oligodeoxynucleotide derivative to be immobilized in 50 mM M sodium phosphate, 150 M NaCl, pH 7.5. After 16 hrs at room temperature with gentle agitation, the film is washed with 50 mM M sodium phosphate, 150 M NaCl, pH 7.5. To reduce the hydrazone bonds to more stable linkages, the film may be further incubated for 1 hr in the presence of 15 mM sodium cyanoborohydride.
  • IDA microelectrodes Closely spaced interdigitated array (IDA) microelectrodes are utilized to select suitable redox mediators as described by Wollenberger, U., Paeschke, M., and Hintsche, R. (Analyst 119, 1245, 1994).
  • the IDA electrodes consist of four pairs of microbands arranged on one silicon chip. Each finger electrode is 900 ⁇ m long and 3.2 ⁇ m wide. The gaps between the finger electrodes are 0.8 ⁇ m.
  • the silicon chip is encapsulated by a 400 nm thick plasma-enhanced chemical vapour deposition (PECVD) Si ⁇ 2 layer, excluding the electrode area and the connecting pads.
  • PECVD plasma-enhanced chemical vapour deposition
  • the chip sensor is electrically connected by means of gold wires bonded to the pads. These bonds are covered with silicon rubber resin.
  • Ferrocene dicarboxylic acid 600 0 0.93 50
  • Electrochemical measurements are carried out at 25 °C in 66 mM potassium phosphate / sodium phosphate, 100 mM KCI, pH 7.0, using an Ag-AgCI reference electrode (Bioanalytical Systems, West Lafayette, IN, USA). A custom-made multipotentiostat is used to control the potential of the electrodes independently. The current response of the individual electrodes is separately preamplified after current-to-voltage conversion and sent to a data acquisition board. Cyclic voltammograms are ontained with an Auto-Lab PSTAT 10 electrochemical analyzer (ECO Chemie, Utrecht, The Netherlands). In order to define the optimum potentials for oxidation and reduction of suitable mediators, cyclic and hydrodynamic voltammograms are recorded. Results are summarized in the table shown above.
  • the cyclic imidoester 2-iminothiolane reacts with amines to form a stable, positively charged linkage, while leaving a sulfhydryl group available for further coupling (Jue, R. et al., Biochemistry 17, 5399, 1978).
  • this heterobifunctional reagent the positive charge of the original amine of the daunosamine moiety is preserved and can bind electrostatically to the negatively charged phosphate groups of the DNA.
  • Daunorubicin hydrochloride (Sigma, St. Louis, MO, USA) is dissolved in dry DMF at a concentration of 4.5 mg/ml and treated with an excess of silver carbonate (Pietersz, G.A. et al., C. In: Antibody-mediated delivery systems. (J.D. Rodwell, ed.) pp. 25-53, Marcel Dekker, New York, 1988). After shaking for 15 min, the mixture is centrifuged at 450 x g for 10 min. The supernatant is decanted and a slight molar excess of 2-iminothiolane (Pierce Chemical Company, Rockford, IL, USA) in DMF is added.
  • LC-SPDP long-chain (LC) version of SPDP provides an additional 6-aminohexanoate spacer group as compared to SPDP.
  • LC-SPDP increases the flexibility of daunorubcin when conjugated to liposomal surfaces and reduces thereby potential steric hindrance problems.
  • Daunorubicin hydrochloride (Sigma, St. Louis, MO, USA) is dissolved in dry DMF at a concentration of 4.5 mg/ml and treated with an excess of silver carbonate (Pietersz, G.A. et al., In: Antibody-mediated delivery systems. (J.D. Rodwell, ed.) pp. 25-53, Marcel Dekker, New York, 1988). After shaking for 15 min, the mixture is centrifuged at 450 x g for 10 min. The supernatant is decanted and a slight molar excess of LC-SPDP (Pierce Chemical Company, Rockford, IL, USA) in DMF is added.
  • LC-SPDP ierce Chemical Company, Rockford, IL, USA
  • SIAXX Since SIAXX contains two aminohexanoate spacer groups, conjugates prepared with this reagent are connected by a spacer arm containing 16 atoms. Thus, SIAXX provides high flexibility to surface-attached daunorubicin molecules.
  • Daunorubicin hydrochloride (Sigma, St. Louis, MO, USA) is dissolved in dry DMF at a concentration of 4.5 mg/ml and treated with an excess of silver carbonate (Pietersz, G.A. et al., In: Antibody-mediated delivery systems. (.D. Rodwell, ed.) pp. 25-53, Marcel Dekker, New York, 1988). After shaking for 15 min, the mixture is centrifuged at 450 x g for 10 min. The supernatant is decanted and a slight molar excess of SIAXX (Molecular Probes, Eugene, OR, USA) in DMF is added.
  • SIAXX Molecular Probes, Eugene, OR, USA
  • NHS-PEG-VS NHS-PEG-VS heterobifunctional poly(ethylene glycol) derivatives containing an amine-reactive N-hydroxysuccinimidyl (NHS) moiety and a sulfhydryl-reactive vinylsulfone (VS) moiety, are commercially available from Shearwater Polymers Europe (Enschede, The Netherlands).
  • PEG-spacer molecules provide water- solubility and a high degree of flexibility.
  • Daunorubicin hydrochloride (Sigma, St. Louis, MO, USA) is dissolved in dry DMF at a concentration of 4.5 mg/ml and mixed with a slight molar excess of NHS-PG- VS (MW 2 kD or 3.4 kD) dissolved in DMF at a concentration of 10 mM. After incubation of the reaction mixture for 3 hours at room temperature with stirring, DMF is evaporated and the residue dissolved in dichloromethane. Diethyl ether is added until precipitation is completed. The precipitate is washed several times with diethyl ether/dichloromethane (2:1) and finally dried and stored at 4 °C in the dark.
  • VS-PG-derivatized daunorubicin is analyzed by TLC using silica gel 60 F254 plates (Merck, Germany) and n-butanol: acetic acid: water at a ratio of 4: 1 : 5 as solvent.
  • Derivatization of the N 2 -position of actinomycin D with 1 ,3-propanediamine is performed in three steps including the synthesis of 2-deamino-2- hydroxyactinomycin D, 2-deamino-2-chloroactinomycin D (both according to Moore, S. et al, J. Med. Chem. 18, 1098, 1975), and N 2 -(3'-aminopropyl) actinomycin D (according to Sengupta, S.K. et al., J. Med. Chem. 24, 1052, 1981 ).
  • the long-chain (LC) version of SPDP provides a propionamidohexanoate spacer group which increases the flexibility of actinomycin D when conjugated to liposomal surfaces.
  • N 2 -(3'-Aminopropyl)actinomycin D is dissoved in dry PMF and mixed with a slight molar excess of LC-SPPP (Pierce Chemical Company, Rockford, IL, USA) in PMF. After incubation of the reaction mixture for 3 hours at room temperature with stirring, PMF is evaporated and the residue dissolved in dichloromethane. Diethyl ether is added until precipitation is completed. The precipitate is washed several times with diethyl ether/dichloromethane (2:1) and finally dried and stored at 4 °C in the dark.
  • LC-SPPP ierce Chemical Company, Rockford, IL, USA
  • SlAXX-derivatized daunorubicin is analyzed by TLC using silica gel 60 F254 plates (Merck, Germany) and n-butanol: acetic acid: water at a ratio of 4: 1 : 5 as solvent.
  • N 2 -(3'-Aminopropyl)actinomycin D is dissoved in dry DMF and mixed with a slight molar excess of SIAXX (Molecular Probes, Eugene, OR, USA) in DMF is added. After incubation of the reaction mixture for 3 hours at room temperature with stirring, DMF is evaporated and the residue dissolved in dichloromethane. Diethyl ether is added until precipitation is completed. The precipitate is washed several times with diethyl ether/dichloromethane (2:1) and finally dried and stored at 4 °C in the dark.
  • SIAXX Molecular Probes, Eugene, OR, USA
  • SIAXX-derivatized daunorubicin is analyzed by TLC using silica gel 60 F254 plates (Merck, Germany) and n-butanol: acetic acid: water at a ratio of 4: 1 : 5 as solvent.
  • N 2 -(3'-Aminopropyl)actinomycin D is dissolved in dry DMF and mixed with a slight molar excess of NHS-PG-VS (Shearwater Polymers Europe, Enschede, The Netherlands, MW 2 kD or 3.4 kD) dissolved in DMF at a concentration of 10 mM. After incubation of the reaction mixture for 3 hours at room temperature with stirring, DMF is evaporated and the residue dissolved in dichloromethane. Diethyl ether is added until precipitation is completed. The precipitate is washed several times with diethyl ether/dichloromethane (2:1 ) and finally dried and stored at 4 °C in the dark.
  • NHS-PG-VS Shearwater Polymers Europe, Enschede, The Netherlands, MW 2 kD or 3.4 kD
  • VS-PG-derivatized daunorubicin is analyzed by TLC using silica gel 60 F254 plates (Merck, Germany) and n-butanol: acetic acid: water at a ratio of 4: 1 : 5 as solvent.
  • RESIDUES FOR ATTACHMENT TO AFFINITY LIPOSOMES Vl.4.1 Synthesis of 5'-amine derivatives of oligonucleotides
  • Method A Covalent attachment of an amine terminal spacer molecule to the 5'- phosphate of oligonucleotides according to method A is performed via formation of a phosphorimidazolide intermediate in a carbodiimide reaction (based on the method of Ghosh, S.S., et al., Anal. Biochem. 178, 43, 1989).
  • a phosphorimidazolide intermediate provides better reactivity towards amine nucleophiles than the carbodiimide phosphosdiester intermediate if carboddimide is used without added imidazole.
  • the carbodiimide phosphodiester intermediate also is shorter-lived in aqueous conditions due to hydrolysis than the imidazolide.
  • the 5'-phosphate-containing oligonucleotide (7.5 -15 nmol in 7.5 ⁇ l) is added to a microfuge tube containing 1.25 mg of the water-soluble carbodiimide EDC (1- ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride; Piece Chemical Company, Rockford, IL, USA).
  • EDC water-soluble carbodiimide
  • EDC 1- ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride; Piece Chemical Company, Rockford, IL, USA.
  • 5 ⁇ l of 0.25 M bis-hydrazide compound e.g., carbohydrazide or adipic acid dihydrazide
  • the reaction mixture is vortexed and centrifuged in a microcentrifuge at maximal rpm for 5 min.
  • hydrazide-labeled oligonucleotide is purified by gel filtration on Sephadex G-25 using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2.
  • diamine spacer molecules e.g., ethylene diamine or 1 ,6-diaminohexane
  • the diamine compound is dissolved at a concentration of 0.25
  • Method B Using method B, the desired oligonucleotide is prepared using automated standard solid-phase phosphoramidite techniques on a scale of about
  • the solid-phase synthesis protocol includes a) removal of dimethoxytrityl groups with 3% dichloroacetic acid in 1 ,2- dichloroethane, b) coupling of the incoming nucleoside 3'- methoxymorpholinophosphite with tetrazole, c) iodine oxidation of the intermediate phosphite to a phosphate, and d) a capping step with acetic anhydride.
  • the purified monomethoxytrityl protected oligonucleotide is dissolved in 2 ml of 80% acetic acid and incubated for two hours. Thereafter, the acetic acid is removed by evaporation and the detritylated amino-containing oligonucleotide is is redissolved in a small volume of water.
  • Method A For the derivatization of 5'-phosphate-containing oligonucleotides with a terminal sulfhydryl group according to method A (based on a procedure of Ghosh, S.S. et al., Kao, Bioconjugate Chem. 1 , 71 , 1990), the oligonucleotide
  • the cystamine-derivatized oligonucleotide For reduction of the cystamine-derivatized oligonucleotide, 20 ⁇ l of 1 M dithiothreitol is added. Thereby, 2-mercaptoethylamine is released from the cystamine modification site and a terminal free sulfhydryl group is created at the 5'-position of the oligonucleotide. After 15 min at room temperature, the sulfhydryl-derivatized oligonucleotide is purified by gel filtration on Sephadex G-25 using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2.
  • Method B Using method B, the desired oligonucleotide is prepared by automated standard solid-phase phosphoramidite techniques on a scale of about 2.5 ⁇ mol of bound first nucleoside.
  • the solid-phase synthesis protocol includes a) removal of dimethoxytrityl groups with 3% dichloroacetic acid in 1 ,2-dichloroethane, b) coupling of the incoming nucleoside 3'-methoxymorpholinophosphite with tetrazole, c) iodine oxidation of the intermediate phosphite to a phosphate, and d) a capping step with acetic anhydride.
  • an extra round of synthesis is carried out using 25 ⁇ mol of an S-trityl-O- methoxymorpholinophosphite derivative of 2-mercaptoethanol (dissolved in 0.3 ml of acetonitile and 0.2 ml of 1 ,2-dichloroethane), 3-mercaptopropan (1) ol (dissolved in 0.5 ml of acetonitrile), or 6-mercaptohexan (1) ol (dissolved in 0.5 ml of acetonitrile) and 75 ⁇ mol of tetrazole (dissolved in 0.5 ml of acetonitrile).
  • the S-trityl-O-methoxymorpholinophosphite derivatives are prepared according to Connolly, B.A., and Rider, P. (Nucleic Acids Res. 13, 4485, 1985). Following coupling, the phosphite intermediate is oxidized by treatment with iodine. Thereafter, the phosphate protecting groups are removed with thiophenolate and cleavage from the resin together with deblocking of the base protecting groups is effected with ammonia.
  • Oligonucleotides that have been modified with an amine-terminal spacer molecule can be reacted further with the heterobifunctional cross-linking reagent SPDP (N- succinimidyl 3-(2-pyridyldithio)propionate; Pierce Chemical Company, Rockford, IL, USA).
  • SPDP N- succinimidyl 3-(2-pyridyldithio)propionate
  • Oligonucleotides derivatized with a terminal pyridyl disulfide residue then can be coupled with sulfhydryl-containing molecules, forming a disulfide bond. Reduction of the terminal pyridyl disulfide residue releases the pyridine-2- thione leaving group and generates a terminal sulhydryl group. This procedure allows conjugation of the 5'-thiolated oligonucleotide to sulfhydryl-reactive derivatives.
  • SPDP is dissolved at a concentration of 6.2 mg/ml in PMF (makes a 20 mM stock solution).
  • the amine-derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 50 ⁇ l of the SPOP solution. After reaction for 1 hour at room temperature, excess reagents are removed from the modified oligonucleotide by gel filtration.
  • the thiolated oligonucleotide is purified from ecxess dithiothreitol by dialysis or gel filtration using 50 mM sodium phosphate, 1 mM EPTA, pH 7.2. The thiolated oligonucleotide is used immediately for further coupling reactions to prevent sulfhydryl oxidation.
  • the pyridiyl dithiol group resulting from an SPPP thiolation must be reduced with a reducing agent such as dithiothreitol to free the sulfhydryl group.
  • a reducing agent such as dithiothreitol
  • SATA the sulfhydryl is freed by hydroxylamine, thus eliminating the need for removal of sulfhydryl reductants prior to a conjugation reaction.
  • SATA is dissolved at a concentration of 8 mg/ml in PMF.
  • the amine- derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 250 ⁇ l of the SATA solution. After reaction for 3 hours at 37
  • the cross-linking reagent SFB (succinimidyl p-formylbenzoate) can be used to add aldehyde groups to amine-containing oligonucleotides.
  • SFB succinimidyl p-formylbenzoate
  • acetonitrile makes a 50 mM solution.
  • the amine-derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 50 ⁇ l of the SFB solution. After reaction for 3 hours at room temperature, excess reagents are removed from the modified oligonucleotide by gel filtration.
  • spacer molecules are introduced between the liposomal surface and the oligonucleotides.
  • Method A the heterobifunctional reagents LC-SPDP or sulfo-LC- SPDP (both Pierce Chemical Company, Rockford, IL, USA) are utilized as spacer molecules.
  • the sulfo-NHS form of the cross-linker contains a negatively charged sulfonate group that provides water-solubility to the cross-linker.
  • LC-SPDP is dissolved at a concentration of 8.5 mg/ml in PMF (makes a 20 mM stock solution). If the water-soluble sulfo-LC-SPPP is used, a stock solution in water is prepared just prior to addition of an aliquot to the reaction, since an aqueous solution of the cross-linker will degrade by hydrolysis of the sulfo-NHS ester. A 10 mM stock solution of sulfo-LC-SPPP is prepared by dissolving 5.2 mg/ ml water. The amine-derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 50 ⁇ l of the LC-SPPP solution.
  • water-soluble sulfo-LC-SPDP 100 ⁇ l of the sulfo-LC-SPDP solution is added. After reaction for 1 hour at room temperature, excess reagents are removed from the modified oligonucleotide by gel filtration.
  • Method B water-soluble heterobifunctional derivatives of polyethylene glycol (PEG) are utilized as spacer molecules.
  • PEG polyethylene glycol
  • Heterobifunctional PEG derivatives containing an amine-reactive N-hydroxysuccinimidyl (NHS) moiety and a sulfhydryl-reactive vinylsulfone (VS) moiety are especially useful, since the VS moiety is hydrolytically stable in aqueous media.
  • the VS moiety reacts selectively with sulfhydryl groups. Reaction with amino groups proceeds at higher pH, but is still relatively slow.
  • NHS-PEG-VS (MW 3400) or NHS-PEG-VS (MW 2000) (both Shearwater Polymers Europe, Enschede, Netherlands) is dissolved in PMF at a concentration of 10 mM.
  • the amine-derivatized oligonucleotide is dissolved in 250 ⁇ l of 50 mM sodium phosphate, pH 7.5, and mixed with 100 ⁇ l of the NHS-PEG-VS solution. After reaction for 1 hour at room temperature, excess reagents are removed from the modified oligonucleotide by gel filtration.
  • Phosphatidylethanolamine 15 mg; 20 ⁇ mol
  • PE pyridyl disulfide residue
  • TEA triethylamine
  • the reaction mixture is mixed well and incubated for 2 h at room temperature while maintained under an argon atmosphere.
  • Reaction progress is determined by thin- layer chromatography (TLC) using silica gel plates developed with a mixture of chloroform : methanohwater (by volume 65:25:4).
  • the activated PE derivative (LC-PPP-PE) develops faster on TLC than the unmodified PE.
  • the methanol is removed from the reaction solution by rotary evaporation and the solid is redissolved in 5 ml of chloroform.
  • the water-soluble reaction by-products are extracted twice from the chloroform with an equal volume of 1% NaCl.
  • the LC-PPP-PE derivative is further purified by chromatography on a column of silicic acid as described by Martin, F.J. et al.
  • PE 100 ⁇ mol
  • TMPB N-succinimidyl-4-(p- maleimidiphenyl) butyrate
  • Pierce Chemical Company Rockford, IL, USA
  • Reaction progress is determined by thin-layer chromatography (TLC) using silica gel 6O-F254 plates (Merck, Germany) developed with a 65:25:4 (by volume) mixture of chloroform: methanol: water.
  • TLC thin-layer chromatography
  • the activated PE derivative develops faster on TLC than the unmodified PE.
  • the methanol is removed from the reaction solution by rotary evaporation and the solid is redissolved in 5 ml of chloroform.
  • the water-soluble reaction by-products are extracted twice from the chloroform with an equal volume of 1% NaCl.
  • the MPB-PE derivative is further purified by chromatography on a column of silicic acid as described above under VI.5.1.
  • Reaction progress is determined by thin-layer chromatography (TLC) using silica gel plates developed with a mixture of chloroform : methanol-.water (by volume 65:25:4).
  • the activated PE derivative (IAXX-PE) develops faster on TLC than the unmodified PE.
  • the methanol is removed from the reaction solution by rotary evaporation and the solid is redissolved in 5 ml of chloroform and purified by chromatography on a column of silicic acid as described above under VI.5.1.
  • Fractions of 2 ml are collected and monitored for the presence of purified IAXX-PE by TLC as described above.
  • the chloroform is removed from the IAXX-PE by rotary evaporation and the derivative is stored at -20°C under a nitrogen atmosphere until use.
  • PE 15 mg; 20 ⁇ mol
  • TEA triethylamine
  • SFPA succinimidyl-p- formylphenoxyacetate
  • the activated PE derivative develops faster on TLC than the unmodified PE.
  • the methanol is removed from the reaction solution by rotary evaporation and the solid is redissolved in 5 ml of chloroform and purified by chromatography on a column of silicic acid as described above under VI.5.1. Fractions of 2 ml are collected and monitored for the presence of purified FPA-PE by TLC as described above. Finally, the chloroform is removed from the FPA-PE by rotary evaporation and the derivative is stored at -20°C under a nitrogen atmosphere until use.
  • PE 15 mg; 20 ⁇ mol
  • TEA triethylamine
  • SATP succinimidyl acetylthiopropionate
  • the activated PE derivative develops faster on TLC than the unmodified PE.
  • the methanol is removed from the reaction solution by rotary evaporation and the solid is redissolved in 5 ml of chloroform.
  • the water-soluble reaction by- products are extracted twice from the chloroform with an equal volume of 1 % NaCl.
  • the ATP-PE derivative is further purified by chromatography on a column of silicic acid as described above under VI.5.1. Fractions of 2 ml are collected and monitored for the presence of purified ATP-PE by TLC as described above.
  • the chloroform is removed from the ATP-PE by rotary evaporation and the derivative is stored at -20°C under a nitrogen atmosphere until use.
  • TNBSA (10 mg, 26 ⁇ mol) is dissolved in 15 ml of 10 mM sodium phosphate, pH 8.
  • 2-lminothiolane-derivatized daunorubicin (15 ⁇ mol) (section Vl.3.1) is dissolved in 15 ml of degassed 10 mM sodium phosphate, 1 mM EPTA, pH 7.5, and mixed with 15 ml of chloroform and 15 ml of methanol containing 7 ⁇ mol SPPP- derivatized PE. After vigorous stirring for 3 hrs at room temperature, 15 ml of chloroform and 15 ml of 10 mM sodium phosphate, pH 7.5, are added. Phase separation is performed by centrifugation for 10 min at 1200 x g.
  • the chloroform layer is washed once with 20 ml of 10 mM sodium phosphate, pH 7.5, and two times with 20 ml of distilled water. To the resulting chloroform layer 60 ml of methanol are added and the daunorubicin-PE conjugate (dissolved in a methanol: chloroform mixture of 2:1) is stored under nitrogen at -20 °C.
  • PD-PPPE pyridyl disulfide-derivatized dipalmitoyl phosphatidylethanolamine
  • Other lipid derivatives including those containing a covalently attached affinity component (e.g., biotin-derivatized dipalmitoyl phosphatidyl ethanolamine) may be used as long as they are soluble in suitable organic solvents.
  • the redox mediator p-aminophenol is dissolved at a concentration of 50 mM in 10 mM HEPES (4-(2-hydroxyethyI)-1-piperazineethanesulfonic acid) buffer in distilled water, pH 7.4.
  • MLV liposomes containing encapsulated p-aminophenol are prepared from a lipid mixture consisting, on a molar ratio basis, of dipalmitoyl phosphatidylcholine (PC), cholesterol, dipalmitoyl phosphatidylglycerol (PG), and PP-PPPE of 8:10:1 :1.
  • PC dipalmitoyl phosphatidylcholine
  • PG dipalmitoyl phosphatidylglycerol
  • PP-PPPE 8:10:1 :1.
  • the integrity of the liposomal bilayer will be stable up to a level of organic solvent addition of about 5%. This is important for subsequent coupling of affinity components added to the liposome suspension as a concentrated stock dilution in an organic solvent.
  • the lipid mixture solved in organic solvent (approximatey a total of 60 ⁇ mol/ml preparation) is pipetted into a round-bottom flask and then dried under reduced pressure at or close to its transition temperature (the highest transition temperature of any one lipid in the mixture is taken into consideration). Thereafter, the lipid mixture is hydrated with p-aminophenol dissolved at a concentration of 50 mM in 10 mM HEPES buffer, pH 7.4, by vortexing for at least 10 min in a water bath at or above the transition temperature of the lipid mixture.
  • the lipid mixture is mechanically shaken in the water bath for about 30 min and after another 30 min at room temperature (without skaking), the liposome mixture is filtered through 0.4 ⁇ m nucleopore filter under nitrogen pressure. Sometimes it is necessary, to perform sequential filtering starting from 1.0, 0.6. , and then 0.4 ⁇ m.
  • the MLVs are dialyzed against 10 mM phosphate-buffered saline, pH 7.4 (at least 1 : 100 volume; three or four changes; 1 hr each; then overnight at 4 °C). To obtain a more concentrated liposome suspension, it can be centrifuged at 100,000 x g (80 min at 20 °C).
  • the SUVs are allowed to equilibrate for approximately 30 min at room temperature before uncaptured redox mediator is removed by dialysis against 10 mM phosphate-buffered saline, pH 7.4 (at least 1 : 100 volume; three or four changes; 1 hr each; then overnight at 4 °C). To obtain a more concentrated liposome suspension, it can be centrifuged at 100,000 x g (80 min at 20 °C).
  • the dried lipids are redissolved in isopropyl ether, freshly redistilled from sodium bisulfite, to a concentration of approximately 20 ⁇ mol/ml ether and transferred to a 50 ml screw- cap Erlenmeyer flask. Thereafter, the aqueous phase consisting of 50 mM p- aminophenol in 10 mM HEPES buffer in distilled water, pH 7.4, is added directly to the lipid solution in a ratio of 1 :3 with ether. The flask is sealed with nitrogen, contents are mixed very well, and the mixture is sonicated at room temperature for at least 5 min until the mixture looks homogeneous. The organic phase is then removed by rotary evaporation under reduced pressure initially at about 450 mm Hg for small preparations and 550 mm Hg for larger preparations. When gel forms, vacuum is increased gradually to a maximum of 700 to 750 mm Hg.
  • Foaming during this process can be eliminiated by quick flushing of nitrogen into the flask.
  • the temperature is also increased gradually from room temperature to about 37 °C towards the end of evaporation.
  • the residue is slightly less in volume than the original aqueous phae (at this stage, no odor of isopropyl ether should be detectable).
  • the SUVs are allowed to equilibrate for approximately 30 to 60 min at room temperature, then extruded through 0.4 _m nucleopore filter under nitrogen pressure. Sometimes it is necessary, to perform sequential filtering starting from 1.0, 0.6. , and then 0.4 _m.
  • Uncaptured redox mediator is removed by dialysis against 10 mM phosphate- buffered saline, pH 7.4 (at least 1: 100 volume; three or four changes; 1 hr each; then overnight at 4 °C). To obtain a more concentrated liposome suspension, it can be centrifuged at 100,000 x g (80 min at 20 °C).
  • the lipid formulation consists of PPPC, DPPG, and PSPC at a molar ratio of 6.5: 0.5: 3.0.
  • the PSPC is added to counterbalance the lowering of T m due to the small radius of curvature of SUV.
  • the lipids are dried from benzene onto a glass vial under a stream of argon and lyophilized overnight.
  • lipid formulation consisting of PPPC and PPPG at a molar ratio of 9: 1 is used.
  • solvent is evaporated to dryness on a rotary evaporator.
  • the dried lipids (approximately a total of 125 mg) are redissolved in an organic phase consisting of 4 ml of chloroform and 8 ml of isopropyl ether, freshly washed with 10% sodium bisulfite.
  • the mixture is transferred to a 50 ml screw-cap Erienmeyer flask.
  • the aqueous phase consists of 10 mM HEPES buffer in distilled water, pH 7.4, containing 50 mM p-aminophenol.
  • the organic phase (12 ml) and the aqueous phase (4 ml) are warmed to 50 °C and combined.
  • the screw-cap Erienmeyer flask is then filled with nitrogen gas and sealed with Teflon tape.
  • the organic / aqueous mixture is placed in a cylindrical bath-type sonicator (e.g., Laboratory Supplies Company, Hicksville, N.Y., USA) filled with water at 45 to 50 °C, and sonicated for 5 min to form a milky, white, homogeneous emulsion.
  • the emulsion is then transferred to a 125 ml tear-drop-shaped rotary evaporation flask.
  • the water around the flask is kept at 50 °C.
  • the organic phase is drawn off. During this process the emulsion foams exuberantly and requires careful venting to adjust the pressure.
  • a lipid formulation consisting of DPPC and DPPG at a molar ratio of 9: 1 is used.
  • the solvent is evaporated to dryness on the wall of a 100 ml round-bottom fask.
  • the aqueous phase consists of 10 mM HEPES buffer in distilled water, pH 7.4, containing 50 mM p-aminophenol. After heating of the aqueous phase to 50 °C, 4 ml are transferred to the flask containing the dried lipids, taking care to keep the flask above the T m of the mixture.
  • the flask is filled with nitrogen gas, closed with a glass stopper, and sealed with Teflon tape. Thereafter, the lipids are hydrated by repeated cycles of vortex-mixing for 15 sec followed by 1.5 min of incubation in a 50 °C water bath. The suspension is cycled 20 times in this manner and then allowed to anneal 30 min or longer in the 50 °C water bath. The liposomes are then rapidly cooled to room temperature in an ice bath and dialyzed over 24 hrs against two 1000 ml volumes of HEPES buffer to remove non-encapsulated p- aminophenol.
  • LUV, SUV, and MLV liposomes containing encapsulated redox mediators are prepared from a lipid mixture consisting, on a molar ratio basis, of PC, cholesterol, PG, and PE of 8:10:1 :1 as described in section VI.6.1.
  • Other lipid recipes may be used as long as they contain about this percentage of PE.
  • this level of cholesterol is maintained in the liposome, then the integrity of the bilayer will be stable up to a level of organic solvent addition of about 5%. This factor is important for adding an aliquot of the cross-linker to the liposome suspension as a concentrated stock dilution in an organic solvent. Any method of liposome formation may be used.
  • Derivatization of PE-liposomes with pyridyl disulfide residues can be performed with SPDP, LC-SPDP, or sulfo-LC-SPDP (all of Pierce Chemical Company, Rockford, IL, USA).
  • SPDP is dissolved at a concentration of 6.2 mg/ml in DMF (makes a 20 mM stock solution).
  • LC-SPDP is dissolved at a concentration of 8.5 mg/ml in DMF (also makes a 20 mM stock solution).
  • water-soluble sulfo-LC-SPDP If the water-soluble sulfo-LC-SPDP is used, a stock solution in water is prepared just prior to addition of an aliquot to the reaction, since an aqueous solution of the cross-linker will degrade by hydrolysis of the sulfo-NHS ester.
  • the sulfo-NHS form of the cross-linker contains a negatively charged sulfonate group that prevents the reagent from penetrating lipid bilayers. Thus, only the outer surfaces of the liposomes are activated using sulfo-LC-SPDP.
  • a 10 mM stock solution of sulfo-LC-SPDP is prepared by dissolving 5.2 mg/ml water.
  • the liposome suspension to be modified, 25 - 50 ⁇ l of the stock solution of either SPDP or LC-SPDP in DMF is added. If sulfo-LC-SPDP is used, 50 - 100 ⁇ l of the stock solution in water is added to each milliliter of the liposome suspension. The reaction mixture is vortexed and reacted for 30 min at room temperature. Longer reaction times, even overnight, have no adverse effects. Finally, the modified liposomes are purified from reaction by-products by dialysis or gel filtration using Sephadex G-50. The derivatized liposomes may be used immediately for subsequent coupling reactions or stored in a lyophilized state in the presence of sorbitol as described by Friede, M. et al., Anal. Biochem. 211 , 117, 1993.
  • LUV, SUV, and MLV liposomes containing encapsulated redox mediators are prepared from a lipid mixture consisting, on a molar ratio basis, of PC, cholesterol, PG, and other glycolipids of 8:10:1 :1 as described in section VI.6.1.
  • the other glycolipids that can be incorporated include phosphatidyl inositol, lactosylceramide, galactose cerebroside, or various gangliosides.
  • Other liposome compositions may be used (e.g., recipes without cholesterol), as long as a periodate-oxidizable component containing vicinal hydroxyls is present. Any method of liposome formation may be used.
  • Temperature-sensitive SUV liposomes containing phosphatidylglycerol and encapsulated redox mediators are prepared from a lipid mixture consisting, on a molar ratio basis, of DPPC, DPPG, and PSPC at a molar ratio of 6.5: 0.5: 3.0 as described in section VI.6.2.1.
  • Temperature-sensitive LUV and MLV liposomes containing phosphatidylglycerol and encapsulated redox mediators are prepared from a lipid mixture consisting, on a molar ratio basis, of PPPC and PPPG at a molar ratio of 9: 1 as described in sections Vl.6.2.2 and Vl.6.2.3, respectively.
  • Sodium periodate is dissolved in water to a concentration of 0.6 M (128 mg of sodium periodate/ ml of H2O) and 200 ⁇ l of this stock solution is added with stirring to each milliliter of PG-liposomes (5 mg/ml) suspended in 20 mM sodium phosphate, 0.15 M NaCl, pH 7.4. After incubation at room temperature for 30 min in the dark, the oxidized liposomes are dialyzed against 20 mM sodium borate, o.15 M NaCl, pH 8.4, to remove unreacted periodate. This buffer is optimal for subsequent coupling with amine-containing affinity components such as antibodies. Alternatively, unreacted periodate can be removed by gel filtration using a column of Sephadex G-50.
  • the periodate-oxidized liposomes may be used immediately for subsequent coupling reactions or stored in a lyophilized state in the presence of sorbitol as described by Friede, M. et al., Anal. Biochem. 211 , 117, 1993.
  • the deacetylated liposomes are purified by gel filtration on Sephadex G-50 (Pharmacia) equlibrated with 50 mM sodium phosphate, 150 mM NaCl, 2.5 mM EPTA, pH 7.5, and used immediately to couple vinylsulfone-derivatized daunorubicin.
  • Daunorubicin derivatized at its daunosamine moiety with the heterobifunctional poly (ethylene glycol) (PEG) derivative NHS-PEG-VS (section VI.3.4.) is dissolved in PMF at a concentration of 10 mM. From this solution, 25 to 50 ⁇ l are added to each ml of purified sulfhydryl-containing liposomes. The bilayer of liposomes containing 50% cholesterol will be stable up to a level of organic solvent addition of about 5%.
  • the daunorubicin-derivatized liposomes are purified by gel filtration on Sepharose 4B (Pharmacia) equlibrated with 20 mM sodium phosphate, 150 mM NaCl, pH 7.4.
  • LC-SPPP-derivatized liposomes (sections VI.5.1. and VI.6.) suspended in 0.1 M sodium phosphate, 0.15 M NaCl, 2.5 mM EPTA, pH 7.5 (5mg lipid/ml).
  • the bilayer of liposomes containing 50% cholesterol will be stable up to a level of organic solvent addition of about 5%.
  • the daunorubicin-derivatized liposomes are purified by gel filtration on Sepharose 4B (Pharmacia) equlibrated with 20 mM sodium phosphate, 150 mM NaCl, pH 7.4.
  • Vlll.8.2.1 Coupling of vinylsulfone-derivatized oligonucleotides to intact thioester-derivatized liposomes Acetylthiopropionate-derivatized liposomes (sections VI.5.5. and VI.6.) suspended in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5, at a concentration of 5 mg lipid/ml, are mixed with 0.5 M hydroxylamine hydrochloride in 50 mM sodium phosphate, 25 mM EPTA, pH 7.5 (100 ⁇ l per ml of liposome suspension).
  • the deacetylated liposomes are purified by gel filtration on Sephadex G-50 (Pharmacia) equlibrated with 50 mM sodium phosphate, 150 mM NaCl, 2.5 mM EPTA, pH 7.5, and used immediately to couple vinylsulfone (VS)-derivatized oligonucleotides prepared from 5'-amine-containing oligonucleotides (section Vl.4.1.) by reaction with the heterobifunctional PEG derivative NHS-PEG-VS (compare section Vl.4.6.).
  • VS vinylsulfone
  • the oligonucleotide- derivatized liposomes are purified by gel filtration on Sepharose 4B (Pharmacia) equlibrated with 20 mM sodium phosphate, 150 mM NaCl, pH 7.5.
  • the oligonucleotide- derivatized liposomes are purified by gel filtration on Sepharose 4B (Pharmacia) equlibrated with 20 mM sodium phosphate, 150 mM NaCl, pH 7.5.
  • the oligonucleotide- derivatized liposomes may be treated with a reducing agent such as cyanoborohydride prior to purification by gel filtration.
  • a reducing agent such as cyanoborohydride
  • 125 mg of sodium cyanoborohydride is dissolved in 1 ml water (makes a 2 M solution). This solution is allowed to sit for 30 min to eliminate most of the hydrogen-bubble evolution that could effect the liposome suspension.
  • An aliquot of 10 ⁇ l of the cyanoborohydride solution is added to each ml of oligonucleotide-derivatized liposomes.
  • the oligonucleotide-derivatized liposomes are purified by gel filtration on Sepharose 4B (Pharmacia) equlibrated with 20 mM sodium phosphate, 150 mM NaCl, pH 7.5.
  • Proteinaceous affinity components include antibodies (e.g., with specificity for haptens and double- and/or triple-stranded nucleic acids), enzymes (e.g., dihydrofolate reductase), and streptavidin (or avidin). Coupling of these proteinaceous affinity components to liposomes occasionally may include liposome aggregation. This may be due to the unique properties or concentration of the protein used, or it may be a result of liposome-to-liposome cross-linking during the conjugation process.
  • antibodies e.g., with specificity for haptens and double- and/or triple-stranded nucleic acids
  • enzymes e.g., dihydrofolate reductase
  • streptavidin or avidin
  • Adjusting the amount of proteinaceous affinity component in the reaction mixture as well as the relative amount of reactive residues (e.g., pyridyl disulfide residues) attached to each proteinaceous affinity component may have to be done to solve an aggregation problem.
  • Vl.8.3.1 Coupling of sulfhydryl-derivatized proteinaceous affinity components to intact LC-SPDP-derivatized liposomes a) Coupling of sulfhydryl-derivatized antibodies to intact LC-SPDP-derivatized liposomes.
  • a) Coupling of sulfhydryl-derivatized antibodies to intact LC-SPDP-derivatized liposomes To 1.0 ml of 20 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 10 mg of IgG antibody, 16 ⁇ l of 20 mM LC-SPDP (Pierce Chemical Company, Rockford, IL, USA) in DMF (8.5 mg/ml) is added and mixed. This gives a molar ratio of 5 mol LC-SPDP per mol of IgG.
  • the solution is subjected to gel filtration on Sephadex G-25 (Pharmacia) equilibrated with 0.1 M sodium acetate, 0.15 M NaCl, pH 4.5. At this point, the derivative is stable and may be stored.
  • the degree of substitution can be determined with an aliquot of the purified LC-SPDP-derivatized antibody at a known protein concentration by measurement of the OD343 (resulting from the release of pyridine-2-thione; extinction coefficient of 8,080 M _1 cm -1 ) after the addition of dithiothreitol (DTT) to a final concentration of 50 mM.
  • DTT reduces the pyridyl disulfide bonds, but not the intrinsic aliphatic disulfide bonds of antibodies.
  • the antibodies are separated from DTT by a second column passage on Sephadex G-25 (Pharmacia) equilibrated with 50 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA, pH 7.4.
  • the purified sulfhydryl-derivatized antibodies are used immediately for coupling to LC-SPDP-derivatized liposomes (sections VIII.5.1. and VIII.6.) suspended at a concentration of 5 mg lipid/ml in 50 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA, pH 7.4.
  • LC-SPDP-derivatized liposomes Sections VIII.5.1. and VIII.6.
  • the SATA-derivatized streptavidin is purified by gel filtration on Sephadex G-25 equilibrated with 0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. At this point, the derivative is stable and may be stored.
  • each ml of SATA-derivatized streptavidin is mixed with 100 ⁇ l of 0.5 M hydroxylamine in 0.1 M sodium phosphate, 10 mM EDTA, pH 7.2. After 2 hrs at room temperature, the thiolated streptavidin is purified by gel filtration on Sephadex G-25 equilibrated with 0.05 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.4.
  • LC- SPDP-derivatized liposomes Sections VI.5.1. and VI.6.
  • LC- SPDP-derivatized liposomes Sections VI.5.1. and VI.6.
  • DHFR sulfhydryl-derivatized dihydrofolate reductase
  • the LC-SPDP-derivatized DHFR is purified by gel filtration on Sephadex G-25 equilibrated with 0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. At this point, the derivative is stable and may be stored. The degree of substitution can be determined with an aliquot of the purified LC-SPDP-derivatized DHFR at a known protein concentration by measurement of the OD343 (resulting from the release of pyridine-2-thione; extinction coefficient of 8,080 M _1 cm" 1 ) after the addition of dithiothreitol (DTT) to a final concentration of 50 mM.
  • DTT dithiothreitol
  • DHFR For reduction of the pyridyl disulfide residues, to purified LC-SPDP-derivatized DHFR in 50 mM sodium phosphate, 0.15 M NaCl, pH 7.4, 0.5 M DTT is added to a final concentration of 50 mM. Since DHFR contains no disulfide bonds, the incorporated pyridyl disulfide residues can be deprotected with DTT without detrimental effects to the enzyme.
  • the sulfhydryl-derivatized DHFR is separated from DTT by a second column passage on Sephadex G-25 (Pharmacia) equilibrated with 50 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA, pH 7.4.
  • LC- SPDP-derivatized liposomes Sections VI.5.1. and VI.6.
  • LC- SPDP-derivatized liposomes Sections VI.5.1. and VI.6.
  • sulfhydryl-derivatized DHFR (adjusted to approximately 0.6 mg protein/ml) in 50 mM sodium phosphate, 150 mM NaCl, 10 mM EDTA, pH 7.4, to each ml of the liposome suspension, the reaction mixture is stirred overnight at room temperature in a nitrogen or argon atmosphere to prevent lipid oxidation.
  • Non-coupled DHFR is removed by gel filtration on Sepharose 4B (Pharmacia) equlibrated with 50 mM sodium phosphate, 150 mM NaCl, pH 7.4.
  • Vl.8.3.2 Coupling of sulfhydryl-derivatized proteinaceous affinity components to intact SMPB-derivatized liposomes Derivatization of proteinaceous affinity components with sulfhydryl groups is performed as described in VI.8.3.1. In this example, sulfhydryl-derivatized IgG antibodies are coupled to intact SMPB-derivatized liposomes. _ After purification by gel filtration (Vl.8.3.1.) sulfhydryl-derivatized IgG antibodies are used immediately for coupling to SMPB-derivatized liposomes (sections VI.5.2.
  • non-derivatized IgG antibodies are coupled to periodate-oxidized liposomes (Method A) or to glutaraldehyde-activated liposomes (Method B).
  • Method A To each ml of periodate-oxidized liposomes (section VI.7.2.) suspended in 20 mM sodium borate, 0.15 M NaCl, pH 8.4, at a concentration of 5 mg lipid/ml, 500 ⁇ l of 20 mM sodium borate, 0.15 M NaCl, pH 8.4, containing non- derivatized IgG antibodies at a concentration of 10 mg/ml, are added. After 2 hrs stirring at room temperature, the Schiff base interactions between the aldehydes on the liposomes and the amines on the antibodies are stabilized by reduction with cyanoborohydride. In a fume hood, 125 mg of sodium cyanoborohydride is dissolved in 1 ml water (makes a 2 M solution).
  • Phosphatidyl ethanolamine (PE) liposomes containing encapsulated redox mediators are prepared as described in section VI.7.1. and suspended in 0.1 M sodium phosphate, 0.15 M NaCl, pH 6.8, at a concentration of 5 mg lipid/ml. All buffers are degassed and bubbled with nitrogen or argon prior to use.
  • PE Phosphatidyl ethanolamine
  • the liposome suspension is reacted overnight at room temperature under a nitrogen atmosphere, and then purified from exess glutaraldehyde by gel filtration on Sephadex G-50 (Pharmacia) equilibrated with 0.1 M sodium phosphate, 0.15 M NaCl, pH 6.8.
  • streptavidin is derivatized with adipic acid dihydrazide according to a method of Bayer, E.A. et al. (Anal. Biochem. 161 , 123, 1987).
  • Adipic acid dihydrazide 160 mg dissolved in 5 ml of 0.1 M sodium phosphate, pH 6 (some heating may be required during solubilization of the compound) is mixed with 50 mg streptavidin.
  • 160 mg of water-soluble 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride is added and mixed thoroughly.
  • hydrazide-derivatized streptavidin is purified by gel filtration on Sephadex G-50 (Pharmacia) equilibrated with 0.1 M sodium phosphate, 0.15 M NaCl, pH 6.8. Hydrazide-derivatized streptavidin may be stored as a freeze-dried preparation without loss of ativity.
  • Coupling of hydrazide-derivatized streptavidin to aldehyde-derivatized liposomes is performed as described in section VI.8.3.3. Stabilization by reduction with cyanoborohydride of the hydrazone linkages formed between the aldehydes on the liposomes and the hydrazide residues on streptavidin may be omitted, since hydrazone linkages provide a much higher stability than Schiff base interactions. However, the addition of a reductant during hydrazide/aldehyde reactions increases the efficiency and yield of the reaction. VI.8.3.5.
  • non-derivatized antibodies are coupled to palmitic acid derivatized with an N-hydroxysuccinimide ester, followed by incorporation of the antibody-palmitate conjugate into intact liposomes by detergent dialysis according to Huang, A. et al. (J. Biol. Chem. 255, 8015, 1980).
  • the activated fatty acid is dissolved in a minimum quantity of hot ethanol and immediately filtered through a filter funnel containing a fluted glass fiber filter pad, both of which have been warmed to the same temperature as the ethanol solution.
  • the NHS-palmitate is recrystallized overnight at room temperature, the solvent is removed by filtration, and the solid is dried under vacuum in a desiccator.
  • the purity of NHS-palmitate can be analyzed by TLC on silica plates using a mixture of chloroform: petroleum diethyl ether (bp 40-60 °C) of 8: 2 as solvent.
  • NHS and NHS-palmitate may be detected by staining with 10% hydroxylamine in 0.1 M NaOH followed after 2 min by a 5% solution of FeCl3 in 1.2 M HCI (creates red-coloured spots).
  • palmitate-antibody conjugates for incorporation of palmitate-antibody conjugates into intact liposomes, palmitate-antibody conjugates in 20 mM sodium phosphate, 0.15 M NaCl, 0.15% deoxycholate, pH 7.4, are added in a ratio of 20: 1 (w/w) to intact liposomes in 20 mM sodium phosphate, 0.15 M NaCl, pH 7.4. After the addition of deoxycholate to a final concentration of 0.7%, the suspension is mixed thoroughly using a vortex mixer, and the liposomes are dialyzed against 20 mM sodium phosphate, 0.15 M NaCl, pH 7.4.
  • liposomes may be prepared containing surface- attached proteinaceous affinity components (e.g., antibodies) which are derivatized with low molecular weight affitnity components (e.g., biotin).
  • the proteinaceous affinity component such as an anti-hapten antibody can mediate binding of the liposomes to hapten-derivatized amplification polymers and the biotin residues complexation of the liposomes via streptavidin as bridging molecule.
  • Covalent attachment of proteinaceous affinity components derivatized with low molecular affinity components to intact liposomes is performed as described in section VI.8.3.
  • Fluorescein isothiocyanate (FITC; Pierce Chemical Company, Rockford, IL, USA) is dissolved in DMF at a concentration of 2 mg/ml (from light protected). An aliquot of 50-100 ⁇ l of this solution is added to each ml of 0.1 M sodium carbonate, pH 9.5, containing streptavidin at a concentration of 2-4 mg/ml. After incubation overnight at 4 °C, excess fluorescein is removed by gel filtration on Sephadex G-25 (Pharmacia) equilibrated with 20 mM sodium phosphate, 0.15 M NaCl, pH 7.2. VI.9. RELEASE OF ENCAPSULATED REDOX MEDIATORS FROM AFFINITY LIPOSOMES
  • Method A Utilizing cholesterol-containing affinity liposomes, the release of encapsulated redox mediators is mediated by the addition of detergent (i.e., Triton X-100 or sodium deoxycholate) or organic solvent.
  • detergent i.e., Triton X-100 or sodium deoxycholate
  • organic solvent i.e., Triton X-100 or sodium deoxycholate
  • the percentage of detergent or organic solvent required for lysis of cholesterol-containing affinity liposomes is dependent on the percentage of cholesterol in the liposomal bilayer.
  • affinity liposomes prepared from a mixture containing phosphatidyl choline, cholesterol, phosphatidyl glycerol, and phosphatidyl ethanolamine derivatized with a reactive residue at a molar ratio of 8: 10: 1 : 1 will be stable up to a level of organic solvent addition of about 5%.
  • affinity liposomes prepared from a mixture containing only phosphatidyl choline and phosphatidyl glycerol at a molar ratio of 4: 1 are completely disrupted in the presence of 0.4% Triton X-100.
  • Method B Utilizing temperature-sensitive, cholesterol-free affinity liposomes, the release of encapsulated redox mediators is mediated by an increase of the ambient temperature (phase-transition release) as described by Magin, R.L. et al. (In: Liposome Technology (G. Gregoriadis, ed.), vol. III., pp. 137-155, CRC Press, Boca Raton, FI., 1984). Preferred heating rates at passage through T m are 10 to
  • AMPLIFIED ASSAY PROCEDURES Soluble dextran polymers of molecular weight between 10,000 and 50,000 have been used extensively as carriers of proteins and other molecules including the application as a carrier of biotin residues (Brandt, H.M. et al., J. Neurosci. Methods 45, 35, 1992) and hapten molecules (Shi, L.B. et al., Cancer Res. 51 , 4192, 1991).
  • Dextran of molecular weight between 10,000 and 40,000 is dissolved in a 30 mM aqueous sodium periodate solution (6.42 g Nal ⁇ 4 in 500 ml deionized water) and stirred overnight at room temperature in the dark. Excess reactant is removed by dialyis against water and the purified polyaldehyde dextran is lyophilized for long- term storage.
  • the degree of aldehyde formation may be assessed by aldehyde-mediated reduction of Cu 2+ to Cu + which can be detected using the bicinchoninic acid (BCA) reagent (Pierce Chemical Company, Rockford, IL, USA) as described by Smith, P.K. et al., Anal. Biochem. 150, 76, 1985.
  • BCA bicinchoninic acid
  • the formation of Cu + is in direct proportion to the amount of aldehydes present in the polymer.
  • BCA forms a purple-colored complex with Cu + which can be measured at 562 nm.
  • adipic acid dihydrazide (or another suitable dihydrazide compound) is dissolved in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 30 mg/ml (heating under a hot water tap is necessary to dissolve the dihydrazide compound completely at this concentration) and the pH is adjusted to 7.2 with HCI.
  • the diamine- or dihydrazide-containing solution is used to dissolve polyaldehyde dextran (prepared as described in section VI.10.1.1.) at a concentration of 25 mg/ml and each ml of the polyaldehyde dextran/diamine (or polyaldehyde dextran/dihydrazide) solution is mixed with 0.2 ml of 1 M sodium cyanoborohydride (in a fume hood). After reaction for at least 6 hrs at room temperature, excess diamine (or dihydrazide) and reaction-by-products are removed by dialysis.
  • Epoxide functional groups react efficiently with sulfhydryl groups at pH values ranging between 7.5 and 8.5, and with amine nucleophiles at moderate alkaline pH values (typically needing pH values of at least 9).
  • 1 ,4- butanediol diglycidyl ether is mixed with an equal part of 0.6 M NaOH containing 2 mg/ml sodium borohydride.
  • 5 mg of dextran are added to each ml of the j /s-epoxide solution. After reaction for 12 hrs at 25 °C, excess reactants are removed by extensive dialysis. For long-term storage, the activated dextran is lyophilized.
  • SPDP is dissolved at a concentration of 6.2 mg/ml in DMF (makes a 20 mM stock solution) and 100 ⁇ l of this solution are added to each ml of 50 mM sodium phosphate, pH 7.5, containing the polyamine-derivatized (or polyhydrazide-derivatized) dextran polymer at a concentration of 20 mg/ml.
  • Dextran polymers suitable for amplified assay procedures contain two types of affinity components.
  • One of the covalently linked affinity components is capable of specifically binding to a specific captured target nucleic acid or its amplicons in a structure restricted manner (e.g., intercalating agents or oligonucleotides).
  • the other covalently linked affinity components are capable of specifically binding multiple affinity liposomes.
  • Suitable affinity systems mediating the binding of affinity liposomes to dextran polymers include hapten / anti-hapten antibody affinity systems, enzyme inhibitor / enzyme affinity systems, and the biotin / (strept)avidin affinity system.
  • affinity liposomes containing surface-attached proteinaceous affinity components may be utilized for the detection of dextran polymers containing the corresponding low molecular weight affinity partner (e.g., hapten molecules, enzyme inhibitors, or biotin residues) and nucleic acid-reactive affinity components (e.g., intercalating agents or oligonucleotides).
  • surface-attached proteinaceous affinity components e.g., anti-hapten antibodies, enzyme molecules, or (strept)avidin
  • dextran polymers containing the corresponding low molecular weight affinity partner e.g., hapten molecules, enzyme inhibitors, or biotin residues
  • nucleic acid-reactive affinity components e.g., intercalating agents or oligonucleotides
  • VI.10.2.1 Coupling of oligonucleotides and methotrexate to polyaldehyde- dextran
  • hydrazide-derivatized methotrexate a high affinity inhibitor of the enzyme dihydrofolate reductase
  • hydrazide-derivatized oligonucleotides prepared as described in section VI.1.1.
  • polyaldehyde derivatives of dextran prepared as described in section VI.10.1.1.
  • Methotrexate (MTX)- ⁇ -hydrazide is prepared from 4-amino-4-deoxy-N - methylpteroic acid (APA) obtained by cleavage of MTX with carboxypeptidase Gi
  • the polyaldehyde derivative of dextran is dissolved in 0.1 M sodium bicarbonate, 0.15 M NaCl, pH 8.5, at a concentration of 20 mg/ml and mixed with a two-fold molar excess (over the molar concentration of aldehyde groups) of each MTX ⁇ - hydrazide and 5'-hydrazide-derivatized oligonucleotides.
  • 0.1 M sodium bicarbonate, 0.15 M NaCl, pH 8.5 at a concentration of 20 mg/ml
  • a two-fold molar excess (over the molar concentration of aldehyde groups) of each MTX ⁇ - hydrazide and 5'-hydrazide-derivatized oligonucleotides In a fume hood, to each ml of this mixture 0.2 ml of 1 M sodium cyanoborohydride are added and the reaction mixture is incubated for at least 6 hrs at room temperature.
  • VI.10.2.2 Coupling of oligonucleotides and biotin to polythioester-dextran
  • pyridyl disulfide-derivatized oligonucleotides prepared as described in section VI.1.3.
  • pyridyl disulfide-containing biotin biotin-HPOP; N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide; Pierce Chemical
  • the sulfhydryl-containing dextran polymer is used immediately for coupling of the pyridyl disulfide derivatives of biotin and the oligonucleotides.
  • VI.10.2.3 Coupling of oligonucleotides and hapten residues to polythioester- dextran
  • vinylsulfone PEG-derivatized oligonucleotides prepared as described in section VI.1.6.
  • vinylsulfone PEG-derivatized fluorescein are coupled to deprotected polythioester-derivatives of dextran (prepared as described in section VI.10.1.7.).
  • non-derivatized IgG antibodies with specificity for double- and/or triple-stranded nucleic acids and non-derivatized streptavidin are coupled to polyaldehyde derivatives of dextran.
  • the polyaldehyde derivative of dextran is dissolved in 0.1 M sodium bicarbonate, 0.15 M NaCl, pH 8.5, at a concentration of 20 mg/ml.
  • Single-stranded (ss) oligonucleotides complementary to the 3'-terminus of short fragments from the direct-repeat region of the Mycobacterium tuberculosis PNA are covalently immobilized onto silica beads to serve as capture oligonucleotides.
  • Captured Mycobacterium tuberculosis PNA is detected with ss oligonucleotide- affinity liposomes containing p-aminophenol.
  • the liposome-attached oligonucleotides are complementary to the 5'-terminus of short fragments from the direct-repeat region of the Mycobacterium tuberculosis PNA.
  • Bound liposomes are lysed by the addition of detergent and released p-aminophenol (PAP) is quantified via redox recycling using interdigitated array (I PA) electrodes.
  • the assay is performed as a combination of liquid chromatography with electrochemical detection (LCEC).
  • the analytical system applied for the detection of Mycobacterium tuberculosis PNA includes a micromachined flow-through assembly, a multipotentiostat, pumps, valves, and a computer (PC type).
  • the flow is accomplished using a peristaltic pump combined with a 6-way selector valve.
  • the micromachined flow- through assembly consists of three main parts: a hybridization chamber, a flow chamber designed as a channel structure, and the interdigitated electrode array (IPA).
  • the hybridization chamber (volume: 100 ⁇ l) contains silica beads with covalently immobilized capture oligonucleotides and is connected via a two-way selector valve to a flow channel fabricated by double-sided anisotropic etching in silicon wafers.
  • the lateral dimensions of the flow channel corresponds to the active electrode area (1mm x 3mm) and the channel hight is set to 200 ⁇ m.
  • the inlet and outlet tubes are pasted in an acrylic holder and arranged on top of the electrode.
  • the microelectrode arrays consist of platinum electrodes fabricated on thermal oxidized silicon wafers by photolithography and the lift-off technique.
  • One chip contains four independent interdigitated electrode pairs and has an area of 8 mm x 8 mm.
  • One of the 70 fingers of each electrode is 1.5 Dm wide. Two adjacent fingers are 0.8 ⁇ m spaced.
  • ss Capture oligonucleotide (18-mer): The oligonucleotide is complementary to the 3'-terminus of the short fragment from the direct-repeat region of the Mycobacterium tuberculosis DNA and includes at the 3'-terminus i) a 2-base thiophosphate thymine-Ts tag for immobilization to tresyl-activated silica beads and ii) a single T-base separating the tag from the sensing oligonucleotide sequence.
  • oligonucleotide is complementary to the 5'-terminus of the short fragment from the direct-repeat region of the Mycobacterium tuberculosis DNA and includes at the 5'-terminus a pyridyl disulfide residue for covalent coupling to sulfhydryl-derivatized affinity liposomes.
  • Derivatization of the 18-mer oligonucleotide with a pyridyl disulfide residue at the 5'-terminus is performed in two steps as described in VI.1.1. and VI.1.3.
  • VI.11.3.2 Immobilized capture oligonucleotides.
  • the thiophosphate thymine- containing capture oligonucleotides are immobilized onto tresyl-activated silica beads as described in VI.1.7.6.
  • VI.11.3.3 Oligonucleotide-affinity liposomes containing p-aminophenol.
  • sulfhydryl-derivatized affinity liposomes are prepared by the injection method (Biochim. Biophys.
  • the lipid is resolubilized in 50 ⁇ l of dry isopropanol and injected with a syringe into 1 ml of 10 mM HEPES, pH7.4, containing 50 mM p-aminophenol, which is being mixed by vortex. Liposomes of uniform size are formed spontaneously by this method. After removal of unencapsulated p-aminophenol by gel filtration, liposomes are suspended in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5, at a concentration of 5 mg/ml and mixed with hydroxylamine hydrochloride as described in VI.8.1.1.
  • the deacetylated liposomes are purified by gel filtration and used immediately for coupling of pyridyl disulfide-derivatized oligonucleotides to the surface-attached sulfhydryl groups (compare VI.8.1.2.).
  • the analytical system is washed with several volumes of 20 mM sodium phosphate, 0.1 M NaCl, pH 7.0. Thereafter, the valve connecting the hybridization chamber with the flow chamber is closed and the sample (50 ⁇ l) containing the short fragment (36-mer) from the direct-repeat region of the Mycobacterium tuberculosis DNA is applied to the hybridization chamber.
  • the hybridization chamber After an incubation of 1 hr at room temperature, the hybridization chamber is washed with several volumes of 20 mM sodium phosphate, 0.1 M NaCl, pH 7.0, captured Mycobacterium tuberculosis DNA is detected by the addition of 50 ⁇ l oligonucleotide-affinity liposomes (approximately 500 ng of liposome-attached oligonucleotide ml "1 ). After an incubation for an additional 1 hr at room temperature, the hybridization chamber is washed again with several volumes of 20 mM sodium phosphate, 0.1 M NaCl, pH 7.0.
  • oligonucleotide-affinity liposomes are lysed by the addition of 100 ⁇ l of 10 mM sodium phosphate, pH 7.0, containing 0.01% Triton X-100. Released p- aminophenol (PAP) is detected by redox recycling. PAP is oxidized to quinoneimine at the anode (+250 mV) yielding an oxidation current, and the quinoneimine is reduced to PAP at the cathode (-50 mV). VI.11.5. Detection sensitivity
  • the detection limit is in the range of
  • the described assay configuration allows the detection of low nanogram quantities per ml of the short fragment (36-mer) from the direct-repeat region of the Mycobacterium tuberculosis DNA (1 ng corresponds to 85 fmol).
  • Single-stranded (ss) oligonucleotides complementary to the 3'-terminus of short fragments from the direct-repeat region of the Mycobacterium tuberculosis DNA are covalently immobilized onto silica beads to serve as capture oligonucleotides.
  • Captured Mycobacterium tuberculosis DNA is detected with preformed complexes of biotinylated oligonucleotides, streptavidin, and affinity liposomes containing p- aminophenol.
  • the biotinylated oligonucleotides are complementary to the 5'- terminus of short fragments from the direct-repeat region of the Mycobacterium tuberculosis DNA.
  • Bound complexes of biotinylated oligonucleotides, streptavidin, and affinity liposomes are lysed by the addition of detergent and released p- aminophenol (PAP) is quantified via redox recycling using interdigitated array (IDA) electrodes.
  • PAP p- aminophenol
  • IDA interdigitated array
  • the assay is performed as a combination of liquid chromatography with electrochemical detection (LCEC).
  • ss Capture oligonucleotide (18-mer): The oligonucleotide is complementary to the 3'-terminus of the short fragment from the direct-repeat region of the Mycobacterium tuberculosis DNA and includes at the 3'-terminus i) a 2-base thiophosphate thymine-Ts tag for immobilization to tresyl-activated silica beads and ii) a single T-base separating the tag from the sensing oligonucleotide sequence.
  • oligonucleotide is complementary to the 5'-terminus of the short fragment from the direct-repeat region of the Mycobacterium tuberculosis DNA and includes at the 5'-terminus a biotin residue.
  • 5'-Biotinylated oligonucleotides are synthesized by derivatization of 5'-amine derivativatives (prepared as described in Vl.4.1.) with succinimidyl-6- (biotinamido)hexanoate (NHS-LC-biotin) as described in Bioconjugate Techniques (G.T. Hermanson, ed.) p. 658, Academic Press, San Diego, CA, 1996.
  • VI.12.3.2 Immobilized capture oligonucleotides.
  • the thiophosphate thymine- containing capture oligonucleotides are immobilized onto tresyl-activated silica beads as described in VI.1.7.6.
  • Biotinylated affinity liposomes containing p-aminophenol are prepared by the injection method (Biochim. Biophys. Acta 298, 1015, 1973) from a lipid mixture of dimyristoylphosphatidylcholine, cholesterol, dicetylphosphate at a molar ratio of 5:4:1 , and N-biotinyldipalmitoyl-L-h ⁇ -phosphatidylethanolamine (B-PE; Molecular Probes, Junction City, OR, USA) at a concentration of 0.1mol% of total lipid.
  • B-PE N-biotinyldipalmitoyl-L-h ⁇ -phosphatidylethanolamine
  • liposomes To prepare liposomes, 2 ⁇ mol of stock lipid mixture in chloroform is evaporated under a stream of nitrogen and then placed in a vacuum desiccator overnight. The lipid is resolubilized in 50 ⁇ l of dry isopropanol and injected with a syringe into 1 ml of 10 mM HEPES, pH 7.4, containing 50 mM p-aminophenol, which is being mixed by vortex. Liposomes of uniform size are formed spontaneously by this method.
  • liposomes are continuously stirred in 20 mM Tris, 0.15 M NaCl, 0.01 % (v/v) NaN 3 , pH 7.4, at a concentration of 0.5 nmol total lipid in 2.5 ml buffer. Streptavidin is quantified using an extinction coefficient of 15.4 cm "1 for a 1 % (w/v) solution at 281 nm.
  • B-PE N-biotinyldipalmitoyl-L-i ⁇ - phosphatidylethanolamine
  • the analytical system is washed with several volumes of 20 mM sodium phosphate, 0.1 M NaCl, pH 7.0. Therafter, the valve connecting the hybridization chamber with the flow chamber is closed and the sample (50 ⁇ l) containing the short fragment (36-mer) from the direct-repeat region of the Mycobacterium tuberculosis DNA is applied to the hybridization chamber.
  • the hybridization chamber is washed with several volumes of of 20 mM sodium phosphate, 0.1 M NaCl, pH 7.0, captured Mycobacterium tuberculosis DNA is detected by the addition of 50 ⁇ l preformed complexes of biotinylated oligonucleotides, streptavidin, and affinity liposomes containing p-aminophenol (approximately 500 ng of biotinylated oligonucleotides ml "1 ). After an incubation for an additional 1 hr at room temperature, the hybridization chamber is washed again with several volumes of 20 mM sodium phosphate, 0.1 M NaCl, pH 7.0.

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Abstract

Selon l'invention, des acides nucléiques cibles ou des amplicons de ces acides fixés sur des oligonucléotides de capture immobilisés sous l'effet de réactions biologiques moléculaires sont détectés et quantifiés au moyen de liposomes d'affinité contenant des molécules rapporteuses détectables par voie électrochimique sensibles à un recyclage redox ainsi que des composants d'affinité fixés en surface capables de se fixer spécifiquement sur des acides nucléiques cibles capturés ou des amplicons desdits acides de manière structuralement restrictive. Les liposomes d'affinité à fixation spécifique sont lysés à l'aide de processus induits par la température ou par un détergent, les molécules rapporteuses libérées étant quantifiées par recyclage redox au moyen d'une voltamétrie conjointement avec un réseau d'électrodes de métal noble à couche mince étroitement espacées. La quantité de molécules rapporteuses libérées est une mesure proportionnelle de la quantité d'acides nucléiques cibles dans l'échantillon. Pour les opérations d'essais amplifiés, on utilise des molécules de support polymères permettant de fixer plusieurs liposomes d'affinité ou complexes préformés de liposomes d'affinité.
EP02735236A 2001-04-09 2002-04-08 Analyse non enzymatique a liaison liposomique avec electrodes en reseau etroitement espacees (nel-ela) pour detecter et quantifier des acides nucleiques Withdrawn EP1409728A2 (fr)

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JP5771366B2 (ja) 2009-09-02 2015-08-26 株式会社バイオメッドコア リポソーム製造装置及び方法
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