Patent Application
20120309106
The present invention concerns a device, a system, a method and a kit for assaying a target (for example a biological entity) in a sample.
Detection of biological moieties, such as biological molecules, bacteria, viruses and cells, in a sample is a routine procedure in fields such as medicine, industry and defense. In medicine, detection is routinely carried out for monitoring clinically and bio-chemically important analytes in a sample obtained from a patient which may be a blood sample, urine sample, etc.
Typically, detection of biological molecules, such as proteins, is carried out by employing well known immunoassay techniques, such as ELISA, radio-immuno assays, etc.
Assays for the presence of specific DNA or RNA sequences in a sample have various applications including the detection of microorganismal infections in patients, analysis of food or environmental samples to detect a contamination, detection of genetic diseases caused by mutations, etc. Simultaneous detection of a large number of different nucleic acid sequences became important in genome projects, i.e. sequencing of the full genomes of various organisms, particularly in the human genome project. Such sequencing typically involves the detection of a large number of short, partially overlapping nucleic acid sequences and based thereon constructing a full genetic map. Such a technique which is termed “sequencing by hybridization” (SBH), involves the digestion of long DNA molecules into smaller fragments and their subsequent hybridization with an array of short probes. Another application involves the simultaneous detection of a large number of different nucleic acid sequences for the purpose of gene expression and diagnostics.
The presence of a specific DNA or RNA sequence in a sample can be detected by a labeled probe capable of specific hybridization with the DNA or RNA sequence. However, direct detection by a probe is limited in present techniques to relatively high concentrations of the target DNA or RNA. In order to overcome this problem, methods for amplifying nucleic acid sequences have been developed, including PCR (polymerase-chain-reaction), LCR (ligase-chain-reaction) and 3SR (self-sustained sequence replication). All amplification methods consume substantial time and labor, require specific conditions for the activity of the amplifying enzymes and intricate laboratory apparatus.
Attempts have been made to develop electrochemical sensors which can directly measure the concentration of an analyte in a sample. The sensors generally detect a change in physical, electrical or optical properties as a result of interaction with the analyte.
U.S. Pat. No. 4,314,821 discloses a system for detection of antibodies in a sample based on a change in resonance frequency of piezoelectric oscillators, as antibodies bind to the oscillator. U.S. Pat. No. 4,822,566 discloses an apparatus for detecting the presence and/or measuring the concentration of an analyte in a fluid medium, relying on the bio-specific binding of the analyte to a biochemical host system thus modifying the dielectric properties of the sensor.
U.S. Pat. No. 5,312,527 discloses a voltammetric sensor for the detection of a target polynucleotide sequence in a sample which binds to a complementary sequence immobilized on a amperometric electrode. A change in the electrode's electric response then indicates the presence of the target sequence in the sample and includes means for detecting, voltammetrically, immobilized heteroduplexes.
WO 9744651 discloses an apparatus for the detection of a specific nucleic acid sequence in a sample, which involves the use of a biosensor comprising an electrode and a bilayer lipid membrane. The membrane is composed of modified lipid molecules which are assembled into an electrode/ionic reservoir/insulating bilayer combination that is suitable for incorporation of ion channels and ionophores. The conductance of such a membrane is dependent on the presence, or absence of an analyte. The presence of the specific nucleic acid sequence changes the impedance of the membrane which is then measured.
U.S. Pat. No. 5,284,748 discloses a method for detecting the occurrence of a binding, or complex-forming reaction between specific substances by utilizing the binding reaction to modify an electric circuit, and then measuring the change in electrical state of the circuit. According to this method, biogenic substances such as antigens are coated onto a non-conductive base, present between a pair of electrodes superimposed on the base. Other biogenes such as antibodies which specifically bind with the antigen are pretreated so that they become bound to conductive particles. The particles having antibody bound thereto, are then added to the antigen layer deposited on the non-conductive base. The conductive particles are thereby bound to the base due to the binding between the antigen and the antibody, and thus form aggregates of electrically conductive particles which modify the circuit. The particles can then be selectively coated with a conductive substance which binds essentially only to said particles, and does not bind to the remainder of the path between the electrodes in order to enhance conductiveness of the electric circuit formed by the two electrodes and the aggregates formed therebetween.
In the following, use will be made with some terms, some of which and their meaning are as follows:
Biological entity—an entity which is derived from a biological source and may be a biological molecule (nucleic acid, protein, lipid, antibody, hormone, etc.) a complex of several biological molecules, a bacterium, a virus, a cell (of a multicellular or unicellular organism) a cell organelle (nucleus, nucleozyme, mitochondria).
Affinity group—a group of at least two entities, at least one of which is a biological entity, capable of specifically binding (see below) to each other. Examples of affinity groups are: two complementary strands of nucleic acid sequences; antibody-antigen, ligand-receptor; enzyme-substrate, glycoprotein-lectin, bacterium and its antibody; DNA-DNA binding protein; etc. An example of an affinity group containing a non-biological entity is an antibody and its specific non-biological hapten.
Binding—non-covalent specific interactions (ionic, van der Waals, hydrogenic, hydrophobic, etc.) between at least two members of an affinity group, for example, the interactions between a nucleic acid sequence and its specific complementary sequence, the interaction between an antibody and its antigen, etc.
Target—an entity, which is to be assayed in a sample. The target, which may be biological or non-biological entity, is a member of an affinity group with the other member being the recognition moiety (see below). The target may be an unmodified analyte as originally present in the sample, or the target may be such an analyte which has been modified to either improve its binding affinity to the recognition moiety and/or modified by binding thereto of a nucleation-center forming entity (see below) or a group to which nucleation-center forming entity (see below) may subsequently bind.
Recognition moiety—An entity which specifically binds the target. The target and the recognition moiety are thus two members of an affinity group. A recognition moiety is used for the detection of the target in the sample. The recognition moiety may be attached (see below) to an electrode (see below) or to a substrate disposed between two electrodes.
Binding moiety—a term referring collectively to either a recognition moiety or a moiety which can bind to the target in a non-specific manner or in a semi-specific manner (semi-specific binding meaning binding to a group of entities which display a common characteristic, e.g. binding to molecules with a specific charge, binding to mRNA in general, binding to a class of antibodies, e.g. to all human IgG antibodies, etc.).
Attachment (attach)—the interaction between the recognition or binding moiety and a substrate (for example the electrode and/or the substrate between electrodes) whereby said moiety becomes immobilized onto said substrate. The interaction may be by covalent or by non-covalent binding.
Conductive bridge—A physical link between at least two electrodes which conducts electric current. A conductive bridge can form between electrodes only if the recognition moiety binds present on at least one of the electrodes or on the substrate between the electrodes binding to the target to form a complex between recognition moieties and target. The complex of recognition-moiety and the target, together with nucleation-center forming entities (see below) bound to the complex, serves as a base for growth of a conductive substance between the electrodes to yield the conductive bridge. Alternatively, the complex may serve as a base for the binding of polymerizable monomers which, upon polymerization, form a conductive polymer. A conductive bridge may at times also be referred to herein as a “wire”.
Nucleation-center forming entities—Entities which allow specific deposition of a conductive substance or serve as catalysts for growth of a conductive substance. The nucleation-center forming entities bind specifically to the target-recognition moiety complex on an assay set, for example by having a moiety allowing specific or non-specific binding to a group previously attached to the target. The nucleation-center forming entities can also be attached to the target before it is complexed with the recognition moiety on the assay set. Examples for nucleation-center forming entities are: gold colloids, other metal colloids or gold or other metal atom containing species, or conjugated polymer forming precursors. Nucleation-center forming entitles can, for example, be attached to streptavidin that can bind to a target entity having a biotin group, or alternatively to an anti-double stranded DNA antibody.
Electrode—A conducting substrate, which may be made of metal or of any other conducting material or coated by metal or other conducting material, which serves for connection of the recognition or binding moiety to external electronic or electric components or circuitry, thus serving as an input/output (I/O) interface with an external component or circuitry.
External circuitry, external component at times referred to as “electric or electronic module”—An electronic or electric circuitry or an electronic or electric component, situated electrically external to the electrodes and typically comprises prior art electric or electronic components, including standard solid-state microelectronic components.
Linker—An agent (molecule, complex of molecules, supramolecular structure, macromolecule, aggregate, colloid particle, molecular clusters, etc.) that acts in providing a physical link between the recognition moiety and the electrode or a substrate, thus serving to attach the recognition moiety to the electrode or the substrate. The linkers may have chemical groups for covalent or non covalent anchoring, (e.g. complexation or sorption, etc.) to the electrode or substrate, on the one hand, and to the recognition moiety on the other hand. Examples of linkers are: nucleic acid binding proteins; synthetic molecules with a binding ability to a specific nucleic acid sequence; a short, single or multiple stranded nucleic acid sequence (e.g. an oligonucleotide), e.g. having a “sticky end” and being modified at its other end, to allow it binding to the electrode; and non-biological molecules like derivatized alkyl silanes, etc.
Sample—A medium which is to be tested for the presence of the target therein. Typically is a fluid obtained from a biological source, such as blood, urine, milk, food suspension, etc.
Assaying (assay)—a term referring collectively to both qualitative and/or quantitative determination of the target in a sample.
Assay device—a device for use in the assaying of the target.
The present invention is based on a novel concept for detecting a target in a sample. The invention makes use of a system comprising an assay device which comprises one or more sets of electrodes physically separated from one another. Each set of electrodes forms, together with a recognition moiety immobilized either on at least one of the electrodes of the set, or on the substrate between the electrodes or on both electrodes, an assay set (also referred to occasionally hereinafter as “detection site”). The system further comprises external circuitry (electric or electronic module) which can determine the formation of a conductive bridge between at least two electrodes of a set. Formation of the conductive bridge occurs in the presence of reagents for growing a conductive substance between the electrodes, only if a complex between the target at the recognition moiety is formed as will be explained hereinbelow. Measurement of electric current, conductance, or resistance between the electrodes is indicative of the formation of said conductive bridge, which formation is in turn depends on the presence of the target in the sample. In other words, in the presence of the target in the sample, an electrically conducting bridge is created between the electrodes and is then detected by performing electrical measurements utilizing the electric or electronic circuitry. At minimum, the detection assay utilizes the system of the invention gives a qualitative result of the presence of the target in the sample, namely a “yes” or “no” answer; in other cases, the potential/current relationships can serve as a gauge for the concentration of the target in the sample as will be explained in detail hereinbelow.
According to one aspect of the invention, the conductive bridge is formed by growing conductive substance on nucleation-center forming entities which are either bound to or deposited on the complex formed between the recognition moiety and the targets.
In accordance with said first aspect the present invention provides a system for assaying one or more targets in a sample, comprising:
(a) an assay device having one or more assay sets at least one for each target to be assayed; each of the assay sets comprising at least two electrodes and a recognition moiety immobilized either to one or more of the at least two electrodes and/or on a substrate between the at least two electrodes; the recognition moiety being capable of specific binding to one of the targets;
(b) an electric or electronic module for determining electric conductance between the at least two electrodes of each assay set; and
(c) reagents for growing a conducting substance from nucleation-centers forming entities deposited onto or bound to a complex formed between said recognition moiety and said target, which substance forms a conductive bridge between at least two of the electrodes of a set.
Examples of nucleation-center forming entities are colloids of metal particles such as: gold, platinum, palladium rhodium, ruthenium, etc., as well as clusters and complexes of such metals.
The inclusion of the nucleation-center forming entities in the complexes formed between the target and the recognition moiety on the device may be achieved either by allowing the nucleation-center forming entities to bind to the complexes after their formation or, alternatively, by reacting the sample with the nucleation-center forming entities prior to contact with the device to allow their attachment to the target. In accordance with one embodiment, the nucleation-center forming entities bind directly to the complexes or the targets. Alternatively the nucleation-center forming entity is bound to an agent which recognizes a specific moiety on the target or on the complex. For example, the nucleation-center forming entity may be bound to avidin and the target then comprises a biotin entity to allow their binding to one another. Such a biotin entity may be bound to the analyte by a prior step of reacting a sample with appropriate reagents. In the above biotin-avidin example, the nucleation-center may be added either to the target when it is free in the sample or to the complex. Another example, applicable where both the target and the recognition moiety are single-stranded nucleic acid sequences, are nucleation-center forming entities, attached to antibodies against double-stranded nucleic acid sequences. In such a case, the nucleation-center forming entity is attached to the complex formed between the target and recognition moiety.
By another aspect of the present invention the conductive bridge of the present invention is a conductive polymer. In that case the invention provides a system for assaying one or more targets in a sample, comprising:
(a) an assay device having one or more assay sets at least one for each target to be assayed; each of the assay sets comprising at least two electrodes and a recognition moiety immobilized either to one or more of the at least two electrodes or on a substrate between the at least two electrodes; the recognition moiety being capable of specific binding to one of the targets;
(b) an electric or electronic module for determining electric conductance between the at least two electrodes of each assay set; and
(c) reagents comprising monomers of a conducting polymer which can bind to a complex formed between said recognition moiety and said target, such that upon polymerization of the monomers a conducting bridge between the at least two electrodes of a set is formed.
In accordance with one embodiment the polymer is a priori conductive. In accordance with another embodiment, the polymers become conducting by a step of doping. (The manner of doping polymers to render them conductive is well known to the artisan).
An example of this embodiment is the use of polyanyline formation as bridge.
For example, biotin can be conjugated to target if present in sample. Then a peroxidase-streptavidin complex is added after hybridization so that this complex binds only to a detection assay which comprise a complex between a target and a recognition moiety. Later, anyline and peroxide source are added to solution to enable polymerization only where peroxidase exists and finally the treatment is completed by doping of the polymers to afford conductivity.
By another alternative the conductive polymer can be grown utilizing nucleation-center forming entities which can be either bound to the targets or to the complexes between the recognition moiety and the targets as explained above.
The targets may be one or more nucleic acid sequences and in that case the recognition moiety may also be oligonucleotides having a sequence complementary to at least a portion of the target sequences.
Where the targets are nucleic acid sequences, the assay device may be in the form of a nucleic acid sequence chip, for example, a DNA chip.
The targets may also be non-nucleic acid sequences, such as sugars, hormones, proteins, or proteinaceous substances. Where the target is a protein, the recognition moiety may be protein binding molecules such as antibodies, or antibody fragments which still maintain the proteinaceous binding properties of the full antibody. The assay device in that case may be in the form of an antibody chip.
The properties of the system of the present invention allow formation of a fully conductive bridge between the two electrodes, (utilizing either nucleation-center forming entities, and a layer of conductive substance grown thereon, or formation of a conductive polymer between the two electrodes), even if the complexes between the targets and the recognition moiety do not form a physical connection between the two electrodes. For example, if the recognition moiety is immobilized on the substrate present between two electrodes of a set, it is possible that a complex formed between a target (bearing a nucleation-center forming entity), and the recognition moiety, does not physically connect between the two electrodes. The nucleation-center forming entities may serve as a nucleus for growth of a conductive substance, which eventually connects between the two electrodes thereby forming a conductive bridge between the electrodes. Thus, the system of the present invention, is highly sensitive, allowing the formation of a conductive bridge even where few, or even a single complex between a recognition moiety and a target is formed between, or on the electrodes of an assay set.
The electrodes of the system or other electrodes which are not part of the system may be used to create an electric field for directing a charged target to the recognition moiety. Thus, in accordance with an embodiment of the invention the electric or electronic circuitry is adapted for creating of such an electric field either through the same or different electrodes than the measuring electrodes. The electric field may also be of assistance in the step of forming the bridge between electrode of an assay set.
The present invention further concerns methods for assaying a target in a sample. The methods of the invention may be based on the concept of the first aspect of the system of the invention, wherein the conductive layer is formed on nucleation-center forming entities, which are deposited or bound to the complexes between the recognition moiety and the target. Thus the invention provides a method for assaying one or more targets in a sample comprising:
(a) providing an assay device having one or more assay sets at least one for each target to be assayed; each of the assay sets comprising at least two electrodes and a recognition moiety immobilized either to one or more of the at least two electrodes or on a substrate between the at least two electrodes; the recognition moiety being capable of specific binding to one of the targets;
(b) contacting said assay device with said sample under conditions permitting binding of targets to specific recognition moieties;
(c) contacting said device with a first reagent solutions to form nucleation-center forming entities on complexes formed between a target and a recognition moiety;
(d) connecting said device with a second reagent solution to grow a conducting metal substance from said nucleation-center forming entities for a time sufficient to yield a conductive bridge between said at least two electrodes;
(e) contacting said at least two electrodes to an electric or electronic module to measure conductance between said at least two electrodes; and
(f) determining conductance between said at least two electrodes, conductance above a threshold conductance indicating the presence of a respective target in the sample.
Alternatively, the nucleation centers may be attached to or deposited on the free target while still present in the sample and in that case the present invention provides a method for assaying one or more targets in a sample, comprising:
(a) reacting the targets with a first reagent solution to bind nucleation-center forming entities to said targets;
(b) providing an assay device having one or more assay sets at least one for each target to be assayed; each of the assay sets comprising at least two electrodes and a recognition moiety immobilized either to one or more of the at least two electrodes or on a substrate between the at least two electrodes; the recognition moiety being capable of specific binding to one of the targets;
(c) contacting said assay device with said sample under conditions permitting binding of targets to specific recognition moieties;
(d) contacting said device with a second reagent solution to grow a conducting metal substance from said nucleation center for a time sufficient to yield a conductive bridge between said at least two electrodes;
(e) connecting said at least two electrodes to an electric or electronic module to measure conductance between said at least two electrodes; and
(f) determining conductance between said at least two electrodes, conductance above a threshold conductance indicating the presence of a respective target in the sample.
The method may also be based on the second aspect of the method of the invention and in that case the conductive bridge is composed of a conductive polymer. Thus the invention provides a method for assaying one or more targets in a sample, comprising:
(a) providing an assay device having one or more assay sets at least one for each target to be assayed; each of the assay sets comprising at least two electrodes and a recognition moiety immobilized either to one or more of theat least two electrodes and/or on a substrate between the at least two electrodes; the recognition moiety being capable of specific binding to one of the targets;
(b) contacting said assay device with said sample under conditions permitting binding of targets to specific recognition moieties;
(c) contacting said device with a first reagent solution comprising monomers of a conductive polymer such that said monomers can bind to complexes formed between the targets and recognition moieties;
(d) treating said device such that said monomers will polymerize to form a conducting polymer, and thereby forming a conducting bridge between at least two electrodes of at least one assay set; and
(e) determining a conductance between said at least two electrodes, conductance above a threshold conductance indicating the presence of a respective target in the sample.
The method may have step (a0) before step (a):
(a0) reacting the sample with a second reagent solution containing entities which can form nucleation centers for growing therefrom a conducting polymer from said monomers, such that said entities bind to said targets if present in the sample.
Alternatively, the method may have step (a1) after step (a):
(a1) contacting said assay device with a second reagent solution containing entities which can form nucleation centers for growing therefrom a conductive polymer from said monomers, such that said entities bind to said targets if bound to said recognition moieties.
As explained in connection with the system, in the context of the method, the nucleation-center forming entities may be attached directly to the target or complex or may be attached to the target or complex indirectly, for example, by use of moieties such as biotin-avidin antibodies, etc.
The measure of conductance in accordance with any of the methods of the invention can be used to determine concentration as will be explained hereinbelow. In addition, a plurality of detection sites may be used, where the proportion of conductive sites is indicative of concentrations as will be explained hereinbelow.
The term “determination” or “determine” should be understood as referring collectively both to a qualitative measurement in order to deform in formation of a bridge (to achieve a “yes” or “no” answer) as well as to a quantitative measurement intended also to determine the extent of bridging.
The assay device comprises one or more assay sets each with at least two electrodes and a recognition moiety. In accordance with one embodiment, at least one electrode, and preferably two, of each assay set, have a recognition moiety immobilized thereon. Other electrodes of each assay set may have a binding moiety (namely a moiety which can bind a target in a semi specific or non specific manner) immobilized thereon or having surface properties such which allow non specific binding of the target thereto. Where recognition moiety is immobilized on at least two electrodes of an assay set, these recognition units may be the same or may be different (although binding to the same target). For example, one recognition moiety may be capable of specific binding to one epitope of the target and the other recognition moiety to another epitope. A specific example is the case of a nucleic acid sequence, where one recognition moiety comprises a sequence complementary to that of one portion of the target nucleic acid sequence and the other recognition moiety comprises a sequence complementary to another portion of the target nucleic acid sequence.
In accordance with another embodiment, the recognition moiety is immobilized on a substrate which is present between the two electrodes.
The present invention also concerns an electric device for determining one or more targets in a sample, comprising:
an integrated circuit comprising the first group of N1 conductors and a second group of N2 conductors, defining between them N1XN2 junctions, each such junction being formed with an electronic module comprising two electrodes, each one linked to or defined as an integral portion of one of the conductors, and comprises a diode or non-linear component permitting current flow through the electronic module only in the direction from the first group of conductors to the second group of conductors, whereby a current flowing between one conductor of the first group to one conductor of the second group of conductors defines a single junction point between them; each pair of electrodes forming part of an assay set, each assay set having a recognition moiety bound either to at least one of the electrodes or to a non-conducting substance disposed between the electrodes.
The electronic device of the present invention may be used for determining a plurality of different targets in a sample, determining concentration of a single target in a sample or determining the concentrations of a plurality of different targets in a sample as will be explained hereinbelow.
The electronic device of the invention may also be used to optimize “signal-to-noise ratio”, and thus to expand a dynamic range when assaying simultaneously a plurality of targets, each being at a different concentration in the sample as will be explained hereinbelow.
Typically, the electronic device is in the form of an array when the first group of conductors is at the X axis and the second group at the Y axis and allowing multiplexing testing. The advantage of the electronic device of the invention is its small dimensions where the distance between the center of each assay set to the center of an adjacent assay set is 100 μM or less, thus allowing formation of a plurality of assay sets in a relatively small area.
The assay sets in the device of the invention may be formed as a two-dimensional array on a non-conductive substrate. Preferably, however, the electrodes of each assay set are open ends of conductive layers of a substrate which are separated from one another by non-conductive (i.e. insulating) layers. Such a device is formed from a multi-layer substrate consisting, as aforesaid from conductive layers separate by non-conductive layers, where the electrodes are formed by either boring holes or cutting openings in the substrates or cutting out portions of the substrates, in a direction essentially normal to the layers. The recognition moieties may be immobilized on the open ends of the conductive layers or in the open ends of the non-conductive layers between two open ends (electrodes) of conductive layers.
The formation of a conductive bridge when performing the assay, for detection of a target indicates the presence of the target in the sample. It will obviously be clear to the artisan, that a small degree of electric conductance, and hence electric current when electric potential is applied between the electrode, may exist between electrodes of an assay set also under control conditions (e.g. under conditions identical to the assay conditions without a target in the sample). Thus whenever the existence of currents is discussed herein, it should be understood that this means current which is larger than under control conditions. While the assay may be limited to detection of the target in an all-or-none fashion (to give a “yes” or “no” answer), according to some embodiments of the invention, the assay may also be performed for the purpose of quantitative determination of the content of the target in the sample, e.g. determining its quantity (amount or concentration).
Quantitative measurements may be carried out by using a plurality of assay sets, the recognition moieties of all are capable of binding to the same targets. All assay sets which are capable of binding to the same target are usually grouped together in the electronic device of the invention in one spatially distinct region, and the region in which they are grouped will be termed “hybridization site”. Each individual assay set in the hybridization site will be termed hereinafter “detection site”. The proportion of detection sites which conduct electricity (due to presence of target and consequence formation of conductive bridges) in a single hybridization site, will be proportional to the amount or concentration of the target in the sample.
If the electronic device comprises a plurality of hybridization sites (i.e. plurality of groups of assay sets, where the sets in each group all bear recognition moieties for binding to the same target) the system can simultaneously assay for a plurality of different targets and, by determining the proportion of detection sites which are conductive in one hybridization site, can also simultaneously give the amount of each target in the sample.
Furthermore, since as indicated above, some assay sets (i.e. detection sites) in a hybridization site may be conductive even in the absence of the targets, the fact that a plurality of detection sites is available for each hybridization site (suitable for the detection of one target) allows to improve the “noise-to-signal ratio”. Only when a predetermined proportion of detection sites, which proportion is above a certain threshold value, will conduct electric current, this will then be regarded as indicating a positive result (namely presence of the target in the sample). This threshold is not necessarily identical for all hybridization sites. For example for one hybridization site for assaying a target which is predicted to be abundant in a sample the threshold may be set to 50% of detection sites (assay sets) being conductive, while for another target which is predicted to be rare in the sample the threshold may be set to 15% of detection sites being conductive. Thus the“dynamic range” of the electronic device of the invention may be quite broad.
It is also possible to form the device with a plurality of hybridization sites all of which are designed for assaying the same target, although with different sensitivities: for example, one site may be formed with a high amount of recognition moieties, or with moieties with a higher affinity to the target which will give rise to a signal even in the presence of a low concentration of the target in the sample; while other sites may be formed with a lower amount of the recognition moieties or with recognition moieties having a lower affinity to the targets, thus allowing a quantitative determination of the targets over a concentration range as compared to the former sites. Such a device then enables the use of a single device for assaying many different samples where the concentration of the target may vary from one sample to the other by several folds.
A device in accordance with the of the invention may comprise one or a plurality of assay sets. In case of a plurality of assay sets all may have the same target specificity i.e. may have a single hybridization site with a plurality of detection sites (namely the recognition moieties of all detection sites will bind the same target) in which case, as pointed out above, the extent of bridging between electrodes in different assay sets (detection sites) may then serve as a quantitative measure for the concentration of the target in a sample. Alternatively, the device may comprise two or more groups of assay sets (hybridization sites) each characterized by having recognition moieties with a different target specificity, which render the device useful in a multiplexing assay for assaying a panel of corresponding two or more different target entities (e.g. different nucleic acid sequences).
For example, each assay set or a group of assay sets may be specific for a different nucleotide sequence. Such an electronic device may be useful in a variety of multiplexing diagnostic applications, i.e. simultaneous detection of a number of targets, detection of an unknown target which is then characterized by its binding (and hence bridge formation) to one of the recognition moieties in the different assay sets (e.g. randomly prepared nucleic acid sequences, each forming the recognition moiety in a different assay set). A specific example is the case of high throughput assays, for example, such aimed at finding a molecule, e.g. peptide, which specifically binds to another molecule, e.g. a receptor; assays for sequencing of unknown nucleotide strands (e.g. genome sequences); etc. In the case of a sequencing assay, a plurality of different sequences may be attached by linkers to electrodes of different assay sets and then a bridge formed in a specific assay set (where the specific nucleic acid sequence serves as the recognition moiety) identifies the specific sequence.
The target may be any one of a wide variety of entities including proteins, nucleic acid sequences, peptides, organic molecules, large macromolecular complexes, cell membranes, and many others. In case of a large entity such as a cell, cell membrane, viral particle, macromolecular complex, etc., the entire large entity, or only the portion thereof binding to the recognition moiety may be regarded as the target. For example, in the case of microorganism, the entire microorganism may be regarded as a target, or at times, where the recognition moiety binds to a specific antigen on the surface of the cell, such an antigen may then be regarded as the target.
Depending on the nature of the target, the recognition moiety may be a single-stranded nucleic acid sequence or a double-stranded sequence with a sticky end, an antibody, a receptor, a lectin, a sugar, an antigen, etc. The recognition moiety and the target are thus members of an affinity group: one member of the affinity group is the target, the other member of the group serves as the recognition moiety.
The recognition moiety may be immobilized on the electrode and/or on a non-electrode substrate present between the electrodes, by the use of linkers which may be selected from a wide variety of molecules capable of attachment to a solid substrate on the one hand, and covalently binding or complexing to the recognition moiety on the other hand. Examples are a variety of sulfur-containing molecules which can, through their sulfur-containing moiety, beam associated with a metal substrate such as gold, silver, platinum, etc. Such a linker may be covalently bound to the recognition moiety, or may be complexed thereto.
The measurement of electric conductance between an electrode of a set may be performed directly, by measuring current-potential relationship, or by performing other measurements indicative of the passage of current through the bridge. For example, the bridge may be treated in a manner that in addition to conducting current, it also emits light, in which case the electric connectivity of the bridge may be determined according to light emission.
The invention will now be illustrated in the following detailed description with occasional references to the annexed drawings. As will be appreciated, the description below is exemplary and should not be construed as limiting the scope of the invention as defined in the appended claims.
Reference is first being made to
When the assay device 102 is contacted with a sample comprising a target 114, a path 116 forms between the two electrodes 104 and 106. By subsequent steps (see below) a conductive bridge is formed and current can flow through the bridge between the two terminals of module 108, as represented by the bidirectional arrow 118. In the embodiment shown in
In
When the device in
In
Assay set 192 shown in
Assay set 210 shown in
In all the embodiments shown in
Each of the assay sets shown in
In all assay devices illustrated in
The recognition moieties 292A, 292B, 292C and 292D bind to respective epitopes 294A, 294B, 294C and 294D. Thus assay set 290AB will be specific for target 293AB having epitopes 294A and 294B, assay set 290BC will be specific for target 293BC having epitopes 294B and 294C and assay set 290CD will be specific for target 293CD having epitopes 294C and 294D. Consequently, when contacted with a sample, a bridge will form in an assay set, depending on the type of target in the sample as illustrated under
Reference is now being made to
For detection of the target in a sample, the assay device is contacted with a sample suspected of comprising a target nucleotide sequence, in this specific example a single or a double-stranded DNA sequence 310. The sample may be a blood sample, a food sample, a water sample, etc. In the case where the target is a double-stranded sequence, the target in the sample may be treated to form single-stranded sticky ends at the terminals of a double-stranded target DNA 310. These sticky ends are complementary to the sequences of the oligonucleotides in recognition moieties 306 and 308. Electrodes 300 are spaced from one another at a distance which should not exceed the combined length of the target DNA 310 and the recognition moieties 306 and 308. When the electrodes are contacted with a sample comprising target DNA sequence 310, the target terminal ends connect to their complementary oligonucleotide sequence in the − recognition moieties 306 and 308 to form a path 312 between the two electrodes 300 (step (b)). In the case of targets which are double-stranded, following hybridization, the binding of the recognition moieties 306 and 308 to the target nucleic acid sequence 310 may be strengthened by ligating the nicks to form covalent binding.
At times, particularly where sequence 310 is long, it may not be practical to rely on diffusion for hybridization of the target 310 to the recognition moieties 306 and 308. In such a case, the target 310 may be made to connect to one electrode and then, by a directional stream of fluid from the first electrode to the second, or by applying an electric field the nucleic acid bridge is made to extend so that its end reaches the second electrode.
It is also clear that in order to avoid folding of the nucleic acid molecules and to ensure proper binding, appropriate solutions may at times be needed. In addition, after hybridization, rinsing may at times be required in order to remove unbound nucleic acid strands.
The formation of a conductive bridge between the electrodes begins, according to the specifically illustrated embodiment, by an ion exchange step involving exposure of the nucleic acid fiber to an alkaline solution comprising silver ions (Ag+) as illustrated in
At a next step (step (d)), the sequence is exposed to a reducing agent, e.g. hydroquinone, or to an electromagnetic radiation, to reduce the metal ions in situ into metallic silver which forms nucleation sites 318. In a different embodiment metal nucleation centers may be formed by attaching a host of colloids or clusters to the DNA path in a sequence specific or non-specific manner. After rinsing a reagent solution comprising metal ions and a reducing agent, e.g. hydroquinone under acidic conditions, is added. Under these conditions, the ions are converted to metal only at nucleation sites and consequently the nucleation centers grow and merge with each other to form a conductive functionalized bridge 320 (step (e)).
The so formed functionalized bridge 320 may be subjected to a variety of post fabrication treatments, which may include, for example, thermal treatment intended to increase the bridge's thickness and homogeneity; passivation treatment for the purpose of forming an electrically insulating layer around the bridge, e.g. by exposure to alkane thiol; electrochemical or photochemical coating of the wire using polymers.
Reference is now being made to
A solution comprising pre-PPV molecules 416 is then contacted with path 414. By the virtue of being positively charged, pre-PPV 416 becomes complexed with the negatively charged DNA path 414 (step (d)). At a next step, conjugation is induced by removal of tetrahydrothiophene groups and hydrochloric acid from each repeat unit, yielding a luminescent PPV bridge (step (e)). This component is suitable for optical detection. Alternatively, the PPV may be doped with agents which either cause electron deficiency (holes) or give rise to extra electrons and thus converted into a conducting polyer. Doping may be performed by many known methods e.g. exposure to H2SO4 vapor. The extent of doping determines the conductivity of the PPVwire.
Many other conductive polymers may be used instead or in addition to PPV in accordance with the invention. This includes a variety of polymers with positively charged side groups as well as polymers with positively charged groups in the backbone, polymers with recognition groups capable of binding to nucleic acid fibers or polymers that complex with DNA. In addition in a similar manner, mutatis mutandis, other types of conducting substances (n-type or p-type) may be bound to the fiber.
Yet another embodiment of the invention is illustrated in
(a) Disulfide Based Linkers:
Controlled pore glass (CPG) derivatized with a disulfide group is used for the synthesis (starting from its 3′ side) of an oligonucleotide having a free 5′ site which serves as the recognition moiety. The oligonucleotide is prepared using a conventional DNA synthesizer (see scheme in
(b) Thiol-Based Linkers:
Linkers are being prepared according to (a) above and the disulfide bond is cleaved to obtain a free thiol.
(c) Biotin-Streptavidin Complex Based Linkers:
Biotin moiety is attached to an oligonucleotide having a specific sequence, as known per se which will serve as the recognition moiety. The biotin-oligonucleotide is coupled via a streptavidin molecule to another molecule containing a biotin moiety at one side (see also
(d) Repressor Based Linkers:
A nucleic acid binding protein, such as the lac repressor, is covalently attached to a thiol group. A DNA sequence, serving as the recognition moiety is synthesized containing also the specific sequence to which the repressor binds. The DNA is coupled to the repressor through said specific sequence.
(e) Thiophosphate Based Linkers:
The construction (starting from its 3′ side) of an oligonucleotide, serving as a recognition moiety, is carried out using a conventional DNA synthesizer wherein thiophosphates containing-nucleotides are used instead of standard nucleotides.
(f) Artificial Site Specific Based Linkers:
A synthetic site-specific moiety such as, for example Rh(Phen)2Phi, known to bind 5′-pyr-pyr-pur-3 sequence (pyr=pyrimidine, pur=purine), is covalently coupled to a thiol group.
(a) Micropipette Wetting:
Electrodes are exposed to solutions of the appropriate linkers, for example, by employing pipettes or micropipettes or by any liquid dispensers. Such liquid dispensers may be fixed onto a manipulator that may be computer controlled. Different types of linkers can be deposited on each electrode. Additionally, different types of linkers can be deposited simultaneously or sequentially on different electrodes.
(b) Jet Printing:
Ink-jet like printing techniques are used for the selective exposure of different electrodes to different linkers. By utilizing such a technique, it is possible to attain high precision, resolution, and to increase rates of production, facilitating large scale production.
Electrode-Linker Synthesis:
(c1) Using Selective Masking Techniques:
(c2) Using Photodeprotection Techniques:
(c3) Using Blockers:
(c4) Electrode Printing:
The above techniques may be used alone or in any combination with other techniques.
Attachment of a nucleic acid fiber to a substrate, which may be the electrode or the substrate on which the -device is formed is carried out using DNA binding proteins. For example, repressors from a bacterial origin (lac-repressor or λ repressor) which can bind to both the substrate (for example a plastic substrate) and the DNA thus joining-the two. Such connection may be later possessed, together with the nucleic acid fiber, to render the bridge conductive, for example, when it connects two electrodes. Alternatively such connection may merely serve to stabilize the conductive bridge to the carrier substrate without participating in the electric functionality.
The integrated circuit (IC) is composed of a substrate such as silicon, derivatized silicon, silica, derivatized silica, organic polymer or any other substance capable of acting as a support for the fabrication or mechanical fixation or stabilization of the functionalized bridge. The substrate may serve an electrical function.
A typical example for IC preparation is described in the following:
A glass substrate is immersed in fuming nitric acid (100% HNO3) for 10 min, rinsed with deionized (DI) water, then immersed in 1 N NaOH solution for an additional 10 min and rinsed with DI water. The cleaned glass is dried thoroughly, then immersed for c.a. 12 hrs in a solution of an alkyl tricholorosilane (octyl trichlorosilane, trimethyl trichlorosilane etc.) in tetrachloroethane (1:5 v/v). The glass plate is then rinsed carefully several times with tetrachloroethane and isopropanol, then dried thoroughly.
Electrodes are fabricated according to one of the following routes: (i) Standard photo, electron, or x-ray lithography on the substrate and subsequent deposition of a conductive substance (e.g. metal). Alternatively, the conductive substance may be deposited first and patterned next, (ii) Electrode assembly onto the surface: Patterning of the glass surface using polyelectrolytes such as polyetheyleneimine, polyalcoholes, polyacids, polypyridines etc. or other ligating agents such as a thiol monolayer (fabricated from organic compounds containing thiol and silane moieties at opposite sites on the molecular skeleton) followed by the fixation of electrically conducting components such as Gold colloids enabling the assembly of conducting electrodes onto the substrate.
(i) The path made of two complementary nucleic acid sequences is exposed to a solution containing the appropriate metal ion, thus, ion exchange occurs at the phosphate groups of the nucleic acid skeleton exposed to the solution. Intercalation of ions inside the nucleic acid may also take place under certain conditions;
(ii) The ion exchanged nucleic acid complex is then reduced by a reducing agent such as hydroquinone or by exposure to electomagnetic radiation.
Cycles (i) and (ii) can be repeated in a sequential order until a conducting bridge is achieved. Alternatively, the formation of conducting metal bridge includes the following steps as stand-alone processes or in conjunction with steps (i) and (ii) or combined with one or more of the following techniques.
(iii) The relevant part of the ion-exchanged bridge is exposed to a metastable mixture of the reducing agent and metal ions. Reduction takes place only at the surface of the metal clusters formed by steps (i) and (ii) thus, the gap between the metal clusters is bridged by the metal deposition process.
(iv) The ion exchanged nucleic acid sequence or the partially treated nucleic acid bridge is exposed to electrochemical processors, transforming the ions loaded on the nucleic acid polyelectrolyte into a metallic conductor. In addition, electrochemical processes along the nucleic acid molecule promote the vectorial growth of the metal wire along it.
(v) Photochemical deposition of the metal from its corresponding ions for the formation of the metallic wire.
(vi) Clusters or colloids are adsorbed onto the nucleic acid bridge using sequence selective components, for example, specific sequences which are capable of binding to specific sites on the nucleic acid sequence non-sequence-specific binding agents, e.g. polyelectrolytes undergoing electrostatic interactions with the DNA. These Clusters and/or colloids serve as catalysts for processes (iii)-(v) above.
(vii) Defects in granular wires fabricated by one or more of the above techniques may be annealed using diverse methods such as thermal annealing processes, electrodeposition, etc.
An example of the fabrication of a silver-functionalized bridge is as follows:
(i) A DNA fiber fixed on a substrate is exposed to a basic solution of silver ions (pH=10.5, NH4OH, 0.1 M AgNO3). After the DNA polyelectrolyte is exchanged by the silver ions, the substrate is rinsed carefully with deionized water (DI) and dried.
(ii) The silver loaded DNA bridge fixed on a substrate is exposed to a basic solution of hydroquinone (0.05 M, pH=5) as a reducing agent. Steps (i) and (ii) are repeated sequentially until an electrically conducting wire is formed.
Complementary Processes:
(a) Step (III) is Performed after One or More (i)+(ii) Cycles:
(iii) The DNA fiber loaded with silver metal clusters (after cycles (i) and (ii) have been performed) and after final rinsing with DI water is exposed to an acidic solution of hydroquinone (citrate buffer, pH=3.5, 0.05 M hydroquinone) and AgNO3 (0.1 M). Cycle (iii) is terminated when the wire width attains the desired dimension. The process can be made light sensitive and thus can also be controlled by the illumination conditions.
(b) Electrochemical Deposition for Improved Process:
(iv) In order to expedite and improve the metallic conductor, an electrochemical process is performed. For that purpose, pre-treatment with an alkane thiol is performed prior to the (i)+(ii) processes. This ensures the inertness of the metal electrodes against electrochemical metal deposition. After one or more of the (i)+(ii) cycles, the electrodes connected through the DNA-covered metal wire are connected to a current or bias controlled electrical source and the relevant part of the DNA fiber is exposed to a solution of the metal ion (different concentrations according to a specific protocol). The gaps between the conducting domains are filled via electrochemical metallic deposition.
(c) Photochemical deposition for an improved process:
(v) In order to improve the metallic conductor, a photochemical process is performed in a similar manner to the electrochemical process outlined above but using photochemical reaction as driving processes. For example, metalization of a DNA fiber may be obtained using an electron donor (triethanolamine, oxalic acid, DTT etc.), a photosensitizer (Ru-polypyridine complexes, xanthene dyes semiconductor particles such as TiO2, CdS etc.), an electron relay such as different bipyridinium salts and the relevant metal ion or metal complex. The photosensitizer transduces the absorbed light energy into a thermodynamic potential through electron transfer processes involving the electron donor and-41-electron acceptor in any of the possible sequences. The reduced electron acceptor acts as an electron relay and charges the metal clusters/colloids with electrons. The charged clusters/colloids act as catalysts for the reduction of the metal ions thus inducing the growth of the metal conductor.
(d) Gold Clusters and/or Colloids as Nucleation Centers:
(vi) Instead of performing the first (i)+(ü) cycles, the relevant part of the DNA bridge is exposed to a solution of gold colloids pre-coated (partially) with cationic thiols (such as pyridinium alkane thiol). The Gold colloids are being adsorbed to the DNA skeleton by ion pairing and the growth of the wire is attained using one or more of the above techniques. Alternatively, the gold colloids may be attached by various means such as biotin-streptavidin binding to modified nucleotides, e.g. modified with biotin.
(e) Curing Processes:
(vii) Defects in a granular wire obtained by one or a combination of the above techniques are annealed using diverse processes such as thermal annealing processes (hydrogen atmosphere (10% H2 in N2), 300 C over several hours).
(a) Device Preparation:
The detection of λ-DNA relies on the fact that such a molecule possesses two 12-base sticky ends.
(b) Detection:
A solution suspected of containing a 16 μm long λ-DNA, having two 12-base sticky ends that are complementary to the oligonucleotides attached to the gold electrodes is made to flow normal to the electrodes. The flow is induced to stretch the DNA, allowing its hybridization with the two distance surface-bound oligonucleotides. In case the sample contains λ-DNA molecules they bind and form a bridge connecting the electrodes.
Two-terminal measurements performed on these samples prove that the stretched DNA molecule is practically an insulator with a resistance higher than 1013Ω. To detect the presence of DNA paths they are first instilled to be electrically conductive, by vectorially depositing silver metal along the DNA molecule. The three-step chemical deposition process (
Atomic force microscope (AFM) images of a section of a 100 nm wide, 12 μm long wire are presented in
This example proves that λ-DNA molecules can indeed be detected using the present invention.
A schematic representation of the manner of production of organic conjugated polymers is shown in
(i) The relevant part of the path is exposed to a solution containing a cationic segment capable of forming a conjugated-polymer by a chemical transformation or a cationic non conjugated-polymer capable of undergoing conjugation by a chemical transformation or a cationic conjugated-polymer. Thus, ion exchange process occurs at the phosphate groups of the DNA skeleton exposed to the solution.
(ii) The ion exchanged DNA complex is treated according to the nature of the organic species that is bound to the polyanionic skeleton. Electrical conductance is achieved either by the former process or by a sequential doping process. Doping may be achieved via conventional redox processes, by protonation—deprotonation processes, by electrochemical means or by photochemical means. Additionally, sequence selective processes between the DNA skeleton and the building blocks of the above organic conjugated-polymer based conducting wires can be utilized for the production of wires.
I. The fabrication of a PPV (poly-p-phenylene vinylene) conducting wire is as follows:
(i) A DNA fiber fixed on a substrate (b) is exposed to a solution of a pre-PPV water soluble polymer. After the DNA polyelectrolyte is exchanged by the pre-PPV polymer, the substrate is rinsed carefully and dried.
(ii) The pre-PPV polymer loaded DNA fiber fixed on the substrate is reacted in a vacuum oven (e.g. 1e-6 bar, 300 C, 6 hr.).
(iii) The resulting luminescent PPV polymer is doped using conventional methods until displaying conductivity.
II. An alternative route for the fabrication of a PPV conductive wire is as follows:
(i) A DNA path fixed on a substrate (
(ii) The bis-(tetrahydrothiophenium)-p-xylilene dichloride loaded DNA sequences fixed on a substrate is polymerized in a basic solution to form a pre-PPV polymer attached to the DNA backbone (
(iii) The pre-PPV polymer loaded DNA sequences fixed on a substrate is reacted in a vacuum oven (1e-6 bar, 300 C, 6 hr.).
(iv) The resulting luminescent PPV polymer is doped using conventional methods until displaying desired conductivity.
III. The fabrication of a PANI (polyaniline) conducting wire is carried out as follows:
(i) A DNA bridge fixed on a substrate is exposed to a solution of an acid soluble PANI polymer. After the DNA polyelectrolyte is exchanged by PANI polymer, the substrate is rinsed carefully and dried.
(ii) The resulting PANI polymer is doped using conventional methods until displaying desired conductivity.
IV. An alternative route to the fabrication of a PANI conducting wire is as follows:
(i) A DNA bridge fixed on a substrate is exposed to a solution of anilinium ions. After the DNA polyelectrolyte is exchanged by the anilinium ion, the substrate is rinsed carefully and dried.
(ii) The anilinium ions loaded on the DNA sequences are oxidized using a solution of an oxidizing agent such as peroxidisulphate ions, yielding a polyaniline polymer. The resulting PANI polymer is doped using conventional methods until displaying desired conductivity.
V. An alternative route to the fabrication of a PANI conducting wire is as follows:
(i) A DNA bridge fixed on a substrate is exposed to a solution of a short oligomers of PANI (>1 repeat unit). After the DNA polyelectrolyte is exchanged by the PANI oligomer, the substrate is rinsed carefully and dried.
(ii) The PANI oligomer ions loaded on the DNA sequence are oxidized using a solution of an oxidizing agent such as peroxidisulphate ions, yielding a polyaniline polymer. The resulting PANI polymer is doped using conventional methods until displaying desired conductivity.
The process described in Example 9 may be followed up to and including step I(ii). The resulting PPV component is highly luminescent. Fabricating the PPV component between electrodes of appropriate work functions then forms an electroluminescent device.
N-(2-ethyl maleimido) pyrrole is attached to a 3′-thio modified specific sequence of monostranded oligonucleotide. Electro-oxidation of a solution containing the 3′-mercapto(N-(2-ethyl succine imido) pyrrole) oligonucleotide induces the formation of a polypyrrole eoated electrode bearing specific sequence oligonucleotides. The polymer is deposited exclusively at the anode side allowing the selective coating of a plurality of electrodes simply by dipping the electrode assay set into a series of solutions containing the desired sequences each time using a different electrode as the anode. Since polypyrrole is a conductor in its doped state, electric connectivity of the polymer layer is enabled upon electrodoping the layer.
In
In step (D), the assay device bearing the DNA duplex is contacted with a solution containing transferase and biotinylated bases which induces the elongation of the DNA skeleton at the 3′-deoxy site 506a.
In a subsequent step (step E), the assay device is exposed to a solution containing gold colloids coupled to streptavidin units 507. The resulting assay device bears DNA molecules with pendant gold colloids 508a.
In the step step (step F), said assay device is exposed to a solution containing hydroquinone and Au(SCN)2. Gold is deposited only on metal surfaces that act as catalysis centers. The colloids grow and merge to form a conductive path 509 bridging the two gold electrodes. The detection of current at applied bias signals the presence of the target DNA sequence in solution.
In the absence of the target DNA sequence, no recognition group is formed between the electrode pair and no gold particles bind between the electrodes. The absence of metal nucleation centers prevents the formation of a conductive path between the electrode pair. The absence of electric current upon induced bias signals the absence of the target in the sample.
The recognition moiety in this case is an antigen or an antibody selective to its antibody or antigen, respectively. The recognition moiety is attached to the electrode(s) by one of a variety of different methods, for example, by complexing it with another group that can bind to gold, by attaching a thiol group, etc; or it can be directly covalently linked to the electrode. In many cases van der Walls forces are sufficient to ensure binding of the recognition moiety to the electrode. The target may be attached to a modifier which eventually serves to bridge the gap between the electrodes. For example, an antigen can be attached to an end of a DNA fiber and bind to its antibody on the electrode with this modifier attached. The other side of the DNA fiber can selectively bind to another electrode in the assay set, or it can be non-selectively attached to it. Substrate passivation, electrode definition, metalization and detection follows the general principles outlined above, e.g. in Example 8.
The introduction of modified bases into DNA or RNA fibers may help in constructing the detection system. Examples of modified nucleotides are: biotin derivatized nucleotides or nucleotides with prime amine groups connected to them. There are different standard molecular biological techniques allowing the introduction of modified nucleotides in specific location along an existing DNA or RNA fiber or constructing copies of a nucleic acid template with a complete sequence of modified nucleotides. For example, the polymerase chain reaction (PCR) technique can be used to amplify a nucleic acid template with modified nucleotides. In this case the modified bases serve as the nucleotides in the PCR solution (mixed with unmodified bases or not) and together with the provided primers (the latter can be synthesized with the same modified nucleotides if necessary) facilitating the amplification process. Alternatively, the method of random priming allows the replacement of nucleotides with modified nucleotides. In some cases ligation of the polymerization products along the template is required to ensure a continuous fiber. Another possibility is to use one of the stranded polymerases (e.g. a Klenow fragment) to fill gaps along double-stranded DNA fibers or to fill sticky-ends with modified bases. Alternatively, DNA terminal transferase can be used to attach modified bases to the 3′ side of a nucleic acid fiber (single or double-stranded). In cases where the modified bases are localized on specific points on the nucleic acid fiber they can be used to attach the fiber specifically to other groups, e.g. a thiol group, a streptavidin or another nucleic acid fiber, etc.
Constructing a complete sequence of a nucleic acid fiber with modified nucleotides allows to achieve highly selective metalization of the bridges. By this method, the modified bases attach specifically groups or complexes that can serve as nucleation centers to catalyze metalization prior to detection. For example, nucleotides derivatized with amine groups can bind specifically tiny gold clusters or colloids that serve as well defined nucleation centers for gold or silver (and many other metals) deposition for yielding a conductive bridge. Another examples is to use bases derivatized with biotin and to attach colloids or gold clusters coated with streptavidin along the DNA fiber. These colloids or gold clusters again can serve as nucleation centers for the metalization process.
In situ polymerase chain reaction (PCR) is a relatively recent technique used usually to detect minute quantities of DNA or RNA in tissue sections or intact cells. It uses the high selectivity of hybridization techniques, allowing, for example, to correlate a specific sequence with a tissue section, with the amplification power of the PCR allowing the many fold increase in detection sensitivity (for a recent review of the technique see: In Situ Polymerase Chain Reaction and Related Technology, edited by J. Gu, Eaton Publishing, 1995). This technique can be employed for specific target amplification, on the substrate or electrodes of the assay device, for example, a DNA sequence present in a minute concentration.
In a first step, the target in front of the sample binds to the specific recognition moiety immobilized on a substrate member situated between an assay set of electrodes, e.g. by hybridization with a complementary DNA sequence fixed to the substrate by e.g. biotin-streptavidin, amine-thiol, etc. A solution containing appropriate primers, optimized concentration of bases and the appropriate buffers and one of the standard PCR polymerases (e.g. a Taq polymerase) is added and then a thermal cycle can be started. In that case the assay device is placed in a temperature control apparatus allowing to control and modify its temperature in fast and automatic way. The template is now specifically duplicated in each cycle to form new templates for the next cycle, hence exponential amplification is possible. The new templates are generated in situ, and may attach to the substrate to form a network with the original DNA target. This may be achieved by non specific binding of the long DNA fibers or by specific binding. For example, by photoactivating, at the end of each cycle, a moiety group attached to the newly formed templates leads to their binding to other complexes on the substrate or to other templates already attached to the substrate. This binding should not interfere with the possibility to bind the primers at the next cycle for further amplification. After sufficient cycles (e.g. 30) a network of DNA fibers, exact copies of the original target, attached to the substrate, fills the gap between an assay set of electrodes. Metalization process then follows, according to one of the techniques specified above, allowing eventually an electric detection of the formation of this network bridge.
One modification to the above is the possibility to carry the amplification procedures between two substrates (e.g. closely spaced glass slides or nylon membranes) that will force newly formed templates to stay in situ. For example, a filter membrane with the proper cutoff, allowing the passage of primers, bases, polymerase and buffers but not long templates can serve for that purpose. In this case all the necessary ingredients can be continuously fed through the membrane ensuring no escape of the templates. Such a semi-permeable membrane also enables efficient washing before metalization and detection.
The ligase chain reaction is a technique for amplifying a specific sequence by ligating at each cycle two subsections of a template. By using a thermocycler identical to the one used for the PCR technique, denaturization of the templates followed by annealing of subsections that exactly match the template to form a continuous nucleic acid fiber with a single or multiple nicks. These nicks are ligated by a special ligase that work at high temperatures (e.g. pfu ligase). This technique can be applied in situ, similar to Example 15 above. An example for a possible amplification is to ligate two short subsections, to a strand not long enough to bridge the gap between electrodes. Because of the amplification power of this technique (again in each cycle newly formed ligated fibers serve as templates for the next cycle), it allows to form a bridge across electrodes made specifically from copies of the target to be detected which is introduced originally in only a minute concentration.
To allow electrical detection of small molecules such as an enzyme (or its substrate) or a protein in some cases they may be attached to a modifier. For example, biotin can be attached to a nucleic acid fiber to enable the detection of avidin or streptavidin. In some cases the modifier can be a synthetic polymer. Another example is the use of a conducting polymer as a modifier facilitating electrical detection in later stages.
The target to be detected in this case is a bacterium. The recognition group can be, for example, an antibody to a specific antigen on the bacterium. Alternatively, biotin-avidin or other specific binding between a molecule or a supramolecular structure on the bacterium membrane and a proper recognition moiety on the electrodes or on the substrate between electrodes is possible. The bacterium will form a bridge across electrodes. Metalization then facilitates electrical detection. An electric field may assist in directing the bacterium to the proper location on the substrate or electrodes. In some cases, it is possible to use ionic currents (alone or in combination with electronic currents) through the bacterium (e.g. using the bacterium natural ionic channels) for electrical detection. Electric or magnetic fields or light can be used as tweezers for bacteria, trapping them between electrodes prior to their metalization and electrical detection.
Step 1: Passivation of Chip
The method is schematically represented in
Chip 700 was treated in an ozone chamber for one hour at room temperature to render it a chemically passivated, resulting in chip 702. Chip 702 is then placed in an oven at 120° C. for one hour and cooled to room temperature in a desiccator.
Step 2: Derivatizing of Chip with a Layer of Poly-Amonopropyl Siloxane
19 mL absolute ethanol, 1 mL water and 0.4 mL triethoxy aminopropyl silane were mixed and left to react for 5 minutes at room temperature and the resulting solution is designated 704. The oven-dried chip 702 was immersed into the reaction solution 704 for 2.5 minutes, then washed successively with several portions of ethanol and water, spin dried and placed in an oven for one hour at 120° C. for crosslinking of the surface attached polysiloxane. The chip coated with a layer of polyamonoproyp siloxane 706 serves for fixation of nucleic acid probes to selected locations by one of several methods such as electrostatic attachment, photoinduced crosslinking of nucleic acids to organic layer on surface, attachment of carboxy derivatives of nucleic acids, electrochemical attachment, etc. Alternatively, The organic layer on chip may be used as anchor to attach precursors for on-chip synthesis of oligonucleotides. Layers having different properties may be formed using different procedures known in the art.
Step 3: Attachment of Nucleic Acid Probes to Chip 706 of
The method of attachment is schematically shown in
Nucleic acids derivatized with carboxyalkyl groups 708 and EDAC used as coupling agent, were dissolved in phosphate buffer, pH=8.0-8.5, to form activated ester 710. The solution was contacted with chip 704 (prepared as described in Example 19) in a controlled manner so that the nucleic acids become bound to the chip at selected places. The solution is removed from derivatized chip 712, bearing probe nucleic acids 714, by successive washing with water.
Step 4: Attachment of Biotin to Nucleic Acids in a Sample
The process is shown schematically in
Procedure for Attachment of Biotin:
Sample 800 which may contain one or more molecules of one or more target nucleic sequences 802 is mixed with cis-platinum biotin complexes 804. The solution is heated to 85 C for 30 min. then cooled to room temperature, and mixed with a stop solution. Typical stop solutions contain one or more compounds that bind irreversibly to the agent thus rendering it inactive. Such solutions are solution containing tris-[hydroxymethyl]aminoethane (>40 mM), EDTA (>5 mM), magnesium acetate (>100 mM) etc. The nucleic acid 806 bears one or more biotin fragments. The resulting biotin-bearing sample, 808, which may contains one or more molecules of one or more biotin-containing target sequences 806, can be purified using different purification procedures such as size exclusion chromatography.
The hybridization procedure is shown schematically in
Step 5: Hybridization and Stringency Wash.
Hybridization solution: 28 mL Formamide, 7 mL SSC (20×), 8 mL, Denharts solution (50×), 0.4 mL salmon sperm solution and a solution of 10% SDS were mixed and heated to 42 C for 30 min before use.
Hybridization: Each derivatized chip, 820 prepared as described above was immersed in hybridization solution and shaken for 30 min at 42° C. Then, the biotin-containing sample, 822 (prepared as described in Example 21) and treated according to step 4, was added to the solution. The chip was shaken in solution for 12 hours at 42° C. in order to hybridize which serve as targets 824, with their complementary sequences, 826, (recognition moieties) present on surface of derivatized chip 820.
Stringency wash: Derivatized chip, 820, was removed from hybridization solution and immersed in a 0.2×SSC solution at 42° C. while shaking. The stringency process was repeated three times, yielding hybridized chip 828, wherein biotin-containing targets, if present in sample 822, are bound to the recognition moieties.
The attachment is shown schematically in
Step 6: Attachment of Avidin-Containing Nucleation Centers to Biotin-Containing Nucleic Acids that were Hybridized to Surface-Bound Recognition Moieties.
Chips after stringency wash, 840, were shaken for 40 min in a solution of a 1:4 ratio of milk blocking solution in distilled water at room temperature. Then, a solution of Steptavidin-gold conjugate (Streptavidin-nanogold, Nanoprobes), 842, was added. The chip was further shaken in solution at room temperature, for additional two hours after which it was washed three times with 0.1× membrane wash solution at room temperature (shaken in each wash solution for five minutes). After drying, chip, 840, bears a complex of the target and recognition moiety derivatized with Steptavidin-gold conjugate, 846 if biotin-containing target sequences to be detected were present in sample, 822, in previous step 5.
A. Solutions
The following stock solutions were used:
Solution a: 240 mg of KAuCl were dissolved in 10 mL distilled water and filtered through a 0.22μfilter.
Solution b: 600 mg of KSCN were dissolved in 10 mL distilled water and filtered through a 0.22μ filter.
Solution c: 550 mg of hydroquinone were dissolved in 10 mL distilled water and filtered through a 0.22μ filter.
Solution d: 320 mg of thiodipropionic acid were dissolved in distilled water and the pH adjusted to 5.50. The solution was adjusted to 10 mL and filtered through a 0.22μ filter.
Solution e: 1M phosphate buffer pH=5.50 was prepared from distilled water and filtered through a 0.22μfilter.
For gold deposition, the following solutions were used:
Solution I: 600 μL of solution a were mixed with 600 μL of solution b at room temperature. The resulting orange precipitate was collected and redissolved in 5000 ml of solution e, forming solution g. 1200 μL of solution c were added to solution g as soon as it turns fully transparent, forming solution h. Solution h was filtered through a 0.22μ filter and used immediately.
Solution II: 3000 μL of solution a were mixed with 3000 μL of solution d at room temperature, forming solution i. Solution i was filtered through a 0.22μ filter and used immediately.
B. Procedure
The application of the electronic device of the invention in a multisite array may require a more sophisticated reading scheme than direct connection to each electrode pair. For such arrays, a multiplexing scheme can be applied. In such scheme, the number of input-output lines is only twice the square root of the number of sites.
Electrical detection in a DNA array is performed at detection sites having minimal size set by microelectronics (areas as small as fraction of square micron). A hybridization site containing one type of DNA (or RNA) reagents may span a larger area and may therefore contain many detection sites. One should then distinguish between individual detection sites (shown schematically in
Consider now the figure, which shows n detection-sites per one hybridization site. The detection site is made small enough, such that for a given target sample, the probability to have a DNA (or RNA) molecule hybridized to it is less than unity. After gold deposition process, in each hybridization site we will find m<n conductive detection sites due to hybridization and formation of a metal bridge. A quantitative measurement of the amount of target molecules can be performed by counting the fraction of positive conducting sites, m/n within each hybridization site. One can also lump together p different detection sites to create a meta-site. Setting a detection threshold such that only for p>pcritical a meta-signal is considered positive detection, one can suppress false positives exponentially by choosing a proper p. Thus, the signal-to-noise ratio can also be significantly enhanced. This method allows the increase of dynamic range of the measurement as the detection sites at different hybridization sites can be lumped to different meta-site sizes, allowing different sensitivities and thresholds. The electronic multiplexing measurement allows easy control of such manipulation by, for example, computer control and proper software.
In some applications the ability of working with small sample volumes is of great advantage. For such purposes, embodiments making use of microfluidics techniques may be used.
| Number | Date | Country | Kind |
|---|---|---|---|
| 124322 | May 1998 | IL | national |
This application is a continuation of U.S. patent application Ser. No. 09/674,090, which has a 371 (c) date of Mar. 1, 2001 and is a 35 U.S.C. 371 national phase application of PCT/IL1999/000232, filed May 4, 1999, which claimed priority to IL 124322, filed May 4, 1998, all of which are incorporated herein by reference as if fully set forth.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09674090 | Mar 2001 | US |
| Child | 13197294 | US |
