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US20100234234A1 - Methods and Devices for Detecting Structural Changes in a Molecule Measuring Electrochemical Impedance - Google Patents

Methods and Devices for Detecting Structural Changes in a Molecule Measuring Electrochemical Impedance Download PDF

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Publication number
US20100234234A1
US20100234234A1 US12/440,832 US44083207A US2010234234A1 US 20100234234 A1 US20100234234 A1 US 20100234234A1 US 44083207 A US44083207 A US 44083207A US 2010234234 A1 US2010234234 A1 US 2010234234A1
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Prior art keywords
electrodes
electrode
molecule
array
protein
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Paul Ko Ferrigno
Christoph Walti
David Evans
Steven Johnson
Alexander Giles Davies
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Medical Research Council
University of Leeds
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Medical Research Council
University of Leeds
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Priority claimed from GB0620808A external-priority patent/GB0620808D0/en
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Assigned to MEDICAL RESEARCH COUNCIL, UNIVERSITY OF LEEDS reassignment MEDICAL RESEARCH COUNCIL ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DAVIES, ALEXANDER GILES, EVANS, DAVID, JOHNSON, STEVENS, WALTI, CHRISTOPH, KO FERRIGNO, PAUL
Publication of US20100234234A1 publication Critical patent/US20100234234A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00704—Processes involving means for analysing and characterising the products integrated with the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides

Definitions

  • the present invention relates to arrays of biological molecules and their construction.
  • the invention also relates to methods of monitoring structural changes of biological molecules, in particular conformational changes or changes in their molecular association(s).
  • High-throughput, high-sensitivity detection or identification of molecules and other nanoscale objects is an important concept not only for medical diagnosis but also for drug-discovery, security, forensic and other applications.
  • the most prominent tools for biological or chemical target detection or identification in a highly parallel fashion are microarrays.
  • DNA microarrays have been used extensively in genomic research, where they enabled massively-parallel interrogation of genetic code. However they are only capable of detecting or identifying complementary single stranded DNA or RNA molecules.
  • protein-arrays sometimes also known in the art as protein microarrays, are required.
  • Protein microarrays present a significantly more difficult challenge than nucleic acid arrays, for example because of the complex nature of the proteome.
  • Prospective protein arrays face several difficulties, in particular the identification of specific, high-affinity robust probe molecules that can bind to native proteins; the development of label-free sensing strategies for the detection of low abundance proteins in complex biological solutions; and the use of micron- or sub-micron-sized array features to enable high array densities of probe molecules.
  • methods of producing protein microarrays typically use surface immobilized antibodies and optical sensing of interactions with fluorescently labelled proteins.
  • antibodies tend to lose their specificity and/or affinity when attached to surfaces.
  • antibodies are most often selected for binding to denatured, prokaryotically-expressed proteins either in animals or in vitro from phage display libraries.
  • they predominantly recognize epitopes comprising conformationally constrained amino acid side chains in linear sequences.
  • RNA-aptamers have been employed in protein arrays and these also suffer from the drawback that they usually have been selected for binding to prokaryotically expressed proteins that may not be correctly folded, and will not be post-translationally modified.
  • the fluorescent dyes that are used to label proteins for subsequent detection of probe-target interactions are typically hydrophobic, and are likely to lead to conformational changes in labelled protein that may mask or destroy biologically relevant conformations.
  • US Patent Application. No. 2005/023155 describes an apparatus and methods for the electrical detection of molecular interactions between a probe molecule and a protein or peptide target molecule, but without requiring the use of electrochemical or other reporters to obtain measurable signals.
  • the invention described is a label-free detection system based on an electrochemical cell and conventional electrochemical impedance spectroscopy (EIS).
  • EIS electrochemical impedance spectroscopy
  • the system is based on a glass capillary which closely resembles a conventional electrochemical cell.
  • a problem with this approach is that array fabrication such as integrated array fabrication is extremely difficult.
  • the present invention seeks to overcome problem(s) associated with the prior art.
  • Electrochemical Impedance Spectroscopy has been used for label-free detection of interactions between certain molecules in the prior art.
  • the usual way in which this is employed is by measurement of the impedance changes which take place depending on whether molecules are bound or unbound from one another. It is these impedance measurements which represent the readout in such systems.
  • the present inventors have surprisingly discovered that the interactions of or conformation changes in biological molecules can actually lead to a dramatic phase shifting effect in the context of electrochemical impedance spectroscopy. Specifically, by using an alternating electrical field in an EIS type application, and by then analysing the phase shift effect, conformational changes or changes in the association between biological molecules can be very clearly detected. This is a dramatic departure from prior art techniques which have relied solely on the direct measurement of changes in impedance for their readout.
  • the new techniques taught herein offer a number of technical benefits. One of the most important of these is that the analysis is rendered independent of the surface area of the particular probes or the particular biological molecules of interest. Thus, differences in electrode surface area or differences in electrode volumes which arise due to manufacturing tolerances or other factors are advantageously controlled for and do not confound the analysis. Furthermore, by monitoring the phase shifting as taught herein, an advantageously sensitive detection system is created.
  • the invention is based upon these surprising findings.
  • the present inventors have developed techniques allowing the functionalising of extremely closed spaced electrodes. Indeed, individual electrodes within an array can be differently functionalised at spacings of only a few micrometers apart.
  • the techniques described herein are based on the controlled activation or de-protection of the individual electrodes in the array. Specifically, the present inventors have shown that an electrical field can be used to control the protection and de-protection (masking/unmasking) of individual electrodes within the array. Furthermore, they have shown that by simultaneously controlling the electrical fields surrounding neighbouring electrodes by use of further potentiostats, that “spread” effects of particular localised electric fields used in de-protection can be prevented from causing undesirable de-protection of neighbouring electrodes.
  • the present invention is based upon these novel techniques for attachment of biological molecules of interest to very small and very close spaced arrayed electrodes.
  • the invention provides a method of detecting a structural change in a molecule, said molecule being attached to a surface, said surface being electrically conductive, wherein the phase of the electrochemical impedance at said surface is monitored, and wherein a change in the phase in the electrochemical impedance at said surface indicates a change in the structure of said molecule.
  • structural change is meant any structural variation in the compound of interest.
  • the structural change may refer to the three-dimensional structure or conformation of the molecule.
  • a structural change may refer to the binding of another entity. Such binding may be covalent or may be hydrogen bonding or may be any other kind of binding or bonding such as polymerisation or other such event.
  • a structural change may also be considered as a modification of the molecule of interest, for example by enzymatic actions such as glycosylation, phosphorylation, de-phosphorylation or other such biologically relevant change in the chemical structure of a molecule of interest.
  • Other examples of a structural change which might be monitored include cleavage or chopping of a molecule, for example by the action of proteases or peptidases or nucleases thereon.
  • the molecule is attached to the surface by a chemical bond, most suitably the molecule is attached to the surface by tethering via a thiol linkage.
  • attachment to the surface may be in a sandwich or layered type arrangement.
  • a peptide may be attached to the surface via a thiol linkage.
  • the molecule of interest may then be attached by virtue of its interaction with said first peptide.
  • chemical modification of the biological molecule of interest is advantageously avoided since the thiol group bond is mediated by the initial peptide joined directly to the electrode and the molecule of interest may then simply interact with that peptide without the need for any chemical modification thereof.
  • the surface comprises an electrode.
  • the invention in another aspect, relates to a method of making an apparatus for studying a molecule of biological interest, said method comprising providing a substrate comprising one or more electrodes wherein at least one of said electrodes further comprises a masking agent, removing said masking agent from said at least one electrode by application of an electrochemical or potential to said electrode, and attaching to said electrode a molecule of biological interest.
  • said apparatus comprises at least two or more electrodes, wherein during removal of said masking agent from said at least one electrode, the electrochemical potential of at least one further electrode is controlled to prevent removal of the masking agent therefrom.
  • said apparatus comprises an array comprising at least ten individually addressable electrodes.
  • the invention relates to an array comprising at least one electrically conductive electrode, said electrode having attached thereto a molecule of biological interest, said molecule comprising a polypeptide.
  • the invention relates to an array comprising at least two electrically conductive electrodes, at least one of said electrodes having attached thereto a molecule of biological interest, said molecule comprising a polypeptide, wherein said electrodes have one or more of:
  • the invention relates to a method of detecting a structural change in a polypeptide, said method comprising
  • step (c) indicates a structural change in said polypeptide.
  • the phase of the electrochemical impedance at said surface is monitored, and wherein a change in the phase in the electrochemical impedance at said surface indicates a change in the structure of said molecule.
  • the invention relates to an array or a method as described above wherein said array comprises at least 10 individually addressable electrodes.
  • the invention relates to an array or a method as described above wherein said electrodes have one or more of:
  • the invention relates to an array or a method as described above wherein said electrodes have both (i) and (ii).
  • the invention relates to a method as described above wherein said structural change is selected from the group consisting of:
  • the invention relates to a method or array as described above wherein the molecule comprises a polypeptide.
  • the molecule comprises a scaffold protein.
  • the molecule comprises a peptide aptamer.
  • the molecule comprises a scaffold protein, said scaffold protein comprising the peptide aptamer.
  • said electrode comprises metal.
  • said metal comprises gold.
  • the invention relates to a method or an array as described above wherein said molecule is a polypeptide and wherein said polypeptide is attached to said surface by a thiol linkage.
  • said molecule of biological interest is selected from the group consisting of aptamers, peptide aptamers, unlabelled peptide aptamers, label-free peptide aptamers, unlabelled scaffold protein comprising one or more peptide aptamers.
  • the term “functionalising” as used herein typically refers to the attaching of a molecule of biological interest to the entity being “functionalised”.
  • the molecule of biological interest is sometimes referred to herein as “probe-molecule”.
  • various electrical parameters are typically monitored by supplying a constant current and measuring the voltage, or conversely by supplying a constant voltage and measuring the current.
  • DC direct current
  • AC current alternating or AC current
  • detection is usually measured at the same frequency as the input current.
  • the tuning or synchronisation of the detection to the same frequency to the input current is called “locking in”. This is important for the elimination of noise or spurious signals.
  • the voltage and the current would oscillate or fluctuate in harmony.
  • certain electrical properties such as capacitance can introduce a phase difference between the input frequency and the output frequency, whereby the peaks observed in the measured output occur with a certain time delay or lag relative to the peaks in the input.
  • phase lag or ‘phase shift’
  • phase shift the phenomenon which is exploited in the present invention.
  • the invention teaches the monitoring of the phase lag.
  • the prior art typically deals only with the measurement of the impedance (resistance) of the system.
  • the approach taught herein focuses instead on looking at the phase shift between the current and voltage observed in the systems of the invention.
  • electrode separations may be reduced.
  • electrode separation is meant the distance between electrodes or the gap between the nearest surfaces of neighbouring or adjacent electrodes.
  • separation of the various patches or electrodes on the array had to be quite large in order to avoid complications and problems for example the bleeding together of individual samples.
  • electrically controlled de-protection or unmasking of the different electrodes during manufacture of the array that such problems are advantageously avoided thereby permitting a closer electrode spacing than has been possible in the prior art.
  • electrodes of the invention are no larger than 20 micrometers in diameter.
  • the spacing of electrodes of the invention is no more than 15 micrometers between neighbouring or adjacent electrodes. More preferably, the spacing is 20 nanometers, more preferably the spacing is 10 nanometers, more preferably the spacing is 5 nanometers.
  • the molecules in the array are built up by the use of an actively controlled surface.
  • the masking/de-protection is actively controlled by the application or suppression of electric fields around the individual electrodes being functionalised. This is in contrast to prior art techniques which are built up on inert or inactive surfaces whose properties do not change.
  • the present invention is based upon the construction of arrays using a dynamically changing surface, the properties of which are manipulated by the use of localised electric fields which are produced or suppressed on individual electrodes according to operator choice.
  • Nucleic acids have typically been attached to solid substrates by the use of a short alkane chain together with a sulphur group.
  • the implementation for proteins disclosed herein is different.
  • the inventors had the realisation that it is possible to provide a sulphur group as an integral part of the protein molecule. This can be described as an alteration to permit attachment, such as a mutation or addition to the polypeptide sequence to introduce one or more thiol groups e.g introduction of cysteine residue. This can then be exploited as the way of binding to the surface of the solid electrode.
  • the sulphur group it is advantageous for the sulphur group to be provided in the polypeptide in the form of a cysteine residue.
  • the probe molecule or biological molecule of interest comprises a scaffold protein. More preferably, the probe molecule or biological molecule of interest comprises a scaffold protein comprising a peptide aptamer.
  • the arrays may be operated in “label-free” mode. This advantage flows from the use of phase shift measurements in EIS detection to alleviate the need for the labelling of individual molecules in the analysis.
  • the target or targets for all aspects of the invention can be selected from, but are not limited to, one or more of proteins, polypeptides, antibodies, nanoparticles, drugs, toxins, harmful gases, hazardous chemicals, explosives, viral particles, cells, multi-cellular organisms, cytokines and chemokines, gametocyte, organelles, lipids, nucleic acid sequences, oligosaccharides, chemical intermediates of metabolic pathways and macromolecules.
  • the target comprises, or consists of, a biological molecule, more suitably a biological macromolecule, most suitably a polypeptide.
  • antibodies can be selected from one or more of the classes IgA, IgD, IgE, IgG and IgM.
  • nanoparticles can be selected from, but are not limited to, one or more of insulating, metallic or semiconducting nanoparticles; nanoparticle by-products of manufacturing processes; and nanoparticles of industrial, medical, or research value.
  • the invention may also involve candidate drugs, e.g. chemical entities which may be tested or screened for a particular activity or property using the arrays or methods of the invention.
  • candidate drugs e.g. chemical entities which may be tested or screened for a particular activity or property using the arrays or methods of the invention.
  • toxins can be selected from, but are not limited to, one or more toxins originating from animals, plants, or bacteria.
  • viral particles can be selected from, but are not limited to, one or more viral particles with and without a genome.
  • multi-cellular organisms can be selected from, but are not limited to, one or more of helminths, nematodes, schistosomes and trypanosomes.
  • organelles can be selected from, but are not limited to, one or more of nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, lysosome, phagosome, intracellular membranes, extracellular membranes, cytoskeleton, nuclear membrane, chromatin, nuclear matrix and chloroplasts.
  • lipids can be selected from, but are not limited to, one or more of signalling lipids, structural lipids, phospholipids, glycolipids and fatty acids.
  • nucleic acid sequences can be selected from, but are not limited to, one or more of DNA, cDNA, RNA, rRNA, mRNA, miRNA and tRNA.
  • a particular aspect of this invention is a method for the detection or identification of native proteins, and/or to detection of their conformation or their binding or association with one or more other molecule(s).
  • the molecule comprises a scaffold protein
  • said scaffold protein comprises a peptide aptamer. Attachment of polypeptides to solid phase substrates can perturb their structure and/or behaviour.
  • the peptide of interest is constrained to its desired conformation or spatial arrangement.
  • a more versatile system for presentation of the biological molecule of interest on the solid support is provided, and greater control over the proper conformation of the aptamer is afforded.
  • Suitably attachment to the substrate/support/electrode is via the scaffold protein.
  • the chemistry of attachment need not be individually designed for each different aptamer of interest but can advantageously be performed identically for each since attachment is suitably via the common scaffold protein (rather than the individual peptide aptamer(s)).
  • Peptide aptamers are protein binding species that have been engineered to bind to various molecular targets such as small molecules, proteins, nucleic acids, or organisms and other nanoscale targets such as metal or semiconductor nanoparticles.
  • Peptide aptamers offer utility for biotechnological and therapeutic applications as well as security and forensic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies.
  • peptide aptamers offer advantages over antibodies as they can be engineered completely in vitro, possess desirable storage properties, and can be designed to elicit little or no immunogenicity in therapeutic applications.
  • the structural constraint imposed by the scaffold which may be due to (1) simple restraint of the amino acid residues at either end of the inserted peptide, thus minimising freedom of movement by the remaining amino acids and their side chains and allowing them to adopt a stable configuration relative to one another, or (2) may involve conformational constraints driven by main chain or side chain interactions between amino acids of the peptide moiety and amino acids of the scaffold, or (3) a combination of (1) and (2), greatly increases the binding affinity of the peptide aptamer for its target when compared to the affinity displayed by the free peptide, to levels comparable to that of an antibody.
  • peptide aptamers are selected in vivo in eukaryotic cells for their ability to recognise native protein structures.
  • the methods of the invention are applicable to electrodes of any scale, it is particularly beneficial to arrays of small electrodes.
  • the electrodes have a diameter (largest dimension) of not more than 50 ⁇ m, more preferably not more than 20 ⁇ m, more preferably not more than 1 ⁇ m, more preferably not more than 500 nm, more preferably not more than 200 nm, more preferably not more than 50 nm.
  • Diameter means largest dimension since the electrodes may not always assume geometrically recognised shapes due to manufacturing tolerances or other considerations.
  • ‘diameter’ should be interpreted accordingly to mean ‘width’ or ‘largest dimension’, suitably the largest dimension perpendicular to the plane of protrusion from the underlying substrate (so as not to take the length as the ‘largest dimension’—the diameter is a measure of width).
  • the probe-molecule can be immobilised on the surface using a peptide-aptamer that is specific to the probe-molecule.
  • This particular peptide-aptamer is itself immobilised on the surface through an appropriate linker, preferably a thiol-linker, and more preferably the thiol is effected through a cysteine-residue in the scaffold of the peptide-aptamer.
  • CDK2 and CDK4 cyclin-dependent kinase 2
  • CDK2 and CDK4 belong to a group of proteins involved in the regulation of the cell cycle; they are functionally related, yet share less than 50% sequence identity.
  • the scaffold is stefin A based such as STM.
  • the peptide aptamer may have an affinity for cyclin-dependent kinase, especially where the cyclin-dependent kinase is selected from CDK2 and CDK4; the scaffold attached peptide aptamers may be selected from STM pep2 and STM pep9 .
  • the invention is not restricted to the use of these two peptide aptamers as probe-molecules; in fact the methods for the fabrication of devices to detect molecular interactions as well as the methods for the detection and the methods for the controlled release are generally applicable and hence can be used in conjunction with a wide range of probe-molecules.
  • the target shall not be limited to proteins, but would also include other substance such as, inter alia, drugs, explosives, nanoparticles.
  • the resolution of the method disclosed in the present invention is only limited by the resolution of the electrode structure or the electrode array structure.
  • the preferred spacing between the individual electrodes as well as the density and the diameter of the electrodes are described above. In particular, spacings of 20 nm or less, 10 nm or less, or 5 nm or even less are embraced. In some embodiments such small spacings are particularly applicable to nucleic acid applications such as functionalising the electrode(s) with DNA.
  • the electrode structures or electrode array structures preferably comprising multiple, individually addressable Au electrodes, are first coated With masking molecules (masking agent) which results in a masking layer that prevents non-specific binding of probe-molecules such as peptide-aptamers to the electrode structure during electrode functionalisation.
  • the masking molecules are preferably thiol-modified to facilitate an orientation-controlled and reversible immobilization on the surface.
  • Electrochemical cleavage of gold-thiol bonds is well known in the art. Selective removal of the masking layer from an individual micro-electrode is preferably achieved in a three-electrode electrochemical cell (comprising one or more working electrodes, one or more counter electrodes, and one or more reference electrodes) by, for example, applying an electrochemical potential of ⁇ 1.4 V vs Ag/AgCl for 120 seconds using a potentiostat ( FIG. 3( b )).
  • a potentiostat FIG. 3( b )
  • electrochemical cells e.g. two-electrode electrochemical cells (comprising one or more working electrodes, and one or more counter electrodes) can be used as well. It is well understood by the person skilled in the art that electrochemical parameters depend on the environment (e.g. buffer) and hence the protocol given in the present invention is given as an example only.
  • a second potentiostat can be used to hold the neighbouring micro-electrodes' potential at, for example, ⁇ 0.2 V vs Ag/AgCl during the desorption process.
  • the bare micro-electrode(s) can be functionalised with the desired probe-molecule, for example a cysteine-modified peptide aptamer, by exploiting the thiol group to form a Au—S bond, for example by incubating the device in a solution containing the probe-molecule overnight in a sealed, humid environment ( FIG. 3( c )).
  • the desired probe-molecule for example a cysteine-modified peptide aptamer
  • An alternative way of functionalising different micro-electrodes with different probe-molecules is a variation of the method discussed above.
  • the entire micro-electrode array is first coated with the first probe-molecule (PM1) using the thiol-modification of the probe-molecule as described above (for example cysteine-modified peptide aptamers), and subsequently PM1 is released from all micro-electrodes which, in the final device, should not be functionalised with this particular probe-molecule, by electrochemically breaking the S—Au bond (see above).
  • the electric fields generated during this desorption can influence the electrochemical potential of neighbouring micro-electrodes, potentially disturbing the probe-molecule-layer on them.
  • the present invention enables the label-free electronic detection or identification of biological or chemical targets by detecting (bio)recognition events occurring between target-proteins in solution and probe-molecules immobilized on electrode structures or electrode array structures comprising one or more electrodes, preferably at a high-density.
  • the methods of the invention comprise label-free detection.
  • detection is based on detecting changes in one or more electrochemical (e/c) properties such as impedance. Most suitably detection is based on detecting a change in phase of the etc impedance at one or more frequencies.
  • Electronic, label-free, on-chip detection of the probe-molecule-target interactions is based on monitoring local changes in the impedance of the electrochemical double layer which forms above the surface of a metal electrode when it is submerged in an electrolyte. Any perturbation of this double-layer, for instance by attachment of proteins to the electrode, alters the double-layer's electrical properties.
  • the complex electrical impedance Z( ⁇ ) which is determined from the response of the system, i.e. the electrochemical current I( ⁇ ), upon applying an ac electrochemical potential ⁇ of frequency ⁇ to the electrode, is a measure of the extent to which the charge transfer to and from the electrode is impeded by the surface-immobilized proteins.
  • Z( ⁇ ) depends on the density, thickness and internal structure of the protein layer, and any alteration of this layer, for example the binding of a molecular target, potentially results in a measurable change of Z( ⁇ ).
  • Changes in Z( ⁇ ) manifest themselves in changes of the absolute impedance
  • scales with the electrode surface
  • ⁇ ( ⁇ ) is independent on the electrode area, and changes in ⁇ , ⁇ ( ⁇ ), therefore provide a reliable and reproducible measure of changes in the protein-layer properties.
  • a further advantage offered by the present invention is that the method of detecting target molecules is scaleable.
  • EIS electrochemical impedance spectroscopy
  • the resistance and/or capacitance is measured.
  • the measured quantity is proportional to the surface area of the electrode.
  • the phase of the electrochemical impedance is surface-area-independent and hence a change of the phase owing to, for example, a binding event occurring between the probe- and the target-molecule, is surface-area-independent.
  • proteins of interest are only present at very low abundance and in complex biological mixtures. It is a particular advantage of the present invention that proteins in such solutions can be detected or identified, even if they are only present at very low concentrations.
  • the electrodes of the electrode structure or electrode array structure can be functionalised with probe-molecules that are able to detect changes in bio-molecules in biological samples from cells or organisms that have been treated with a given drug. This is particularly useful for both assessing the likely efficacy or toxicity of a drug, and also for determining whether a drug is working in a particular patient by the production of a typical signature of responsiveness.
  • the probe-molecules are immobilised on a surface and in a particular aspect of the present invention, we provide a method to attach the probe-molecules, reversibly to the surface, which permits the controlled release of the probe-molecules from the surface.
  • the method is based on using a controlled-release-probe-molecule, which is a probe-molecule that has been modified with a linker-moiety to be attached reversibly to a surface.
  • the modification with the linker-moiety of the probe-molecule can be achieved by either integrating the linker-moiety directly into the probe-molecule or by adding it to the probe-molecule via a linker.
  • the controlled-release-probe-molecule can consist of more than one part and does not necessarily has to be attached to the electrode in one step, but can equally well be assembled from its individual parts directly on the electrode.
  • the preferred method is to assemble the controlled-release-probe-molecule, i.e. modifying it with a linker-moiety, first, and then immobilise it onto the surface in a subsequent step.
  • the controlled-release-probe-molecule is modified with a thiol, and preferably the thiol is affected through a cysteine-residue.
  • the S—Au-bond with which the controlled-release-probe-molecules are attached to the electrodes of the array is electrochemically active in the same way as the thiol-Au bonds of the masking-molecules used in the selective functionalisation process.
  • This offers the unique advantage that these bonds can be cleaved on selected electrodes only and hence the probe molecules are released back into the surrounding electrolyte (for example a protein-friendly buffer). This can obviously be done subsequent to binding a target (for example from a cell lysate), and hence the probe plus the target can be released, allowing subsequent identification of the target by, for example, mass spectrometry.
  • a peptide-aptamer array with multiple individually addressable electrodes functionalised with different peptide aptamers may be fabricated and the response of the lysate from a cancerous cell is compared with the response of a second, identical array to the exposure to the lysate of a healthy cell.
  • the proteins on the electrodes of the arrays where the response is different can be released off the electrode and the released proteins including the bound targets can be collected and analysed.
  • Current technologies do not offer this option and the analysis of such systems may prove to be of great importance in diagnostics, the discovery of new biomarkers or drug targets, of markers of clinical efficacy of experimental treatments and in drug development, for example.
  • a further particular aspect of the present invention is a method for the controlled release of target molecules by electric means.
  • Many biological molecules are known to alter their conformation quite dramatically when exposed to AC or DC electric fields.
  • surface-immobilized DNA molecules change from a random-coil-conformation to an elongated conformation when electric fields of several hundred kV/m at frequencies of around 300 kHz are applied.
  • This invention discloses a method for releasing the captured target-molecule off a probe-molecule immobilised on an electrode surface.
  • this electrode is not limited to be a single, isolated electrode, but can be, for example, also a set of electrodes or be part of a micro-array.
  • the invention is described by way of example using peptide-aptamers as probe-molecules. However, it is well understood by the person skilled in the art that the invention is not restricted to peptide aptamers and can be used in conjunction with other probe-molecules as well.
  • an electric field is applied to a peptide-aptamer, the electric field causes a conformational change in the scaffold.
  • This conformational change of the scaffold in turn alters the conformation of the peptide-insert of the peptide aptamer, as the three-dimensional conformation of the peptide insert is partially governed by the constraint applied by the scaffold.
  • the affinity of the target-probe binding changes dramatically upon a conformational change of the scaffold and thus the peptide insert, and the target-molecule is released.
  • Electrode structures or electrode array structures are functionalised with small chemical compounds that are candidate drugs and the response to patients or pathological specimens is compared to the appropriate control.
  • the candidate-drugs-bound-target complexes on the electrodes where the response to the specimen and the control is different (in a positive or negative way) can be released off the electrode.
  • the controlled release candidate drug molecules immobilized on electrode structures or electrode array structures can be used to identify patients or pathological specimens where a particular drug binds a target molecule that is absent in a normal sample from the same patient or from another appropriate control. This would allow the identification of bio-molecules whose function is being affected by the drug treatment, potentially revolutionising medicinal chemistry and drug optimisation efforts.
  • immobilise variations of the word, for example “immobilising”, means “attaching a moiety to a surface using a specific linker”.
  • the invention relates to the fabrication of devices for detection or identification of biological or chemical targets, methods of biological or chemical target detection or identification using probe-molecules immobilised onto an electrode structure or electrode array structure, and methods for the controlled release of molecules from such devices.
  • the scaffold is not STM.
  • the scaffold is not TrxA.
  • an array device for the detection or identification of a protein or multiple proteins from a single sample.
  • Our protein array employs peptide aptamers as the probe molecules, selected in vivo with high and very specific affinities for eukaryotically-expressed proteins.
  • Electronic transduction of biorecognition events is achieved by monitoring changes in the complex impedance characteristics of a protein film bound to a microfabricated electrode array, and we use this strategy to probe interactions between the surface-immobilized probe-proteins and target-proteins in solution.
  • the methods as described herein for protein-arrays are applicable to other methods or devices for the detection or identification of biological or chemical targets using probe-molecules immobilised on a surface.
  • the methods and devices of this invention shall not be limited to the detection or identification of proteins using a protein-array, but shall also be suitable for the detection or identification of targets using alternative probe-molecules.
  • the molecular mask may be released from the electrode by applying an electronic signal to the electrode, for example by electrochemical cleavage of a thiol-linkage.
  • an electrode neighbouring the electrode that binds the molecular masking agent to be released is protected by keeping its potential at a level where the induced release-reaction does not happen.
  • an electrochemical potential of between ⁇ 0.9 V and ⁇ 1.5 V vs Ag/AgCl is applied to achieve this.
  • a redox-probe may be used such as a K 3 Fe(CN) 6 4 ⁇ /3 ⁇ redox probe.
  • the electrolyte may consist of a buffer and a redox-probe.
  • the invention in another aspect, relates to a method to release a captured-target-probe-molecule complex which comprises the use of one or more probe-molecules immobilised onto an electrode structure comprising one or more electrodes.
  • FIG. 1( d ) is the same as FIG. 1( c ) but following exposure of STM to recombinant CDK2;
  • FIG. 2( a ) illustrates EIS ⁇ ( ⁇ ) data for an Au surface functionalised with STM pep9 and following exposure to CDK2-expressing yeast lysate;
  • FIG. 2( c ) illustrates ⁇ ( ⁇ ) of STM pep9 formed on independent electrodes following exposure to a CDK2-expressing yeast lysate and a CDK2-free lysate and ⁇ ( ⁇ ) of STM layer following exposure to CDK2-expressing yeast lysate.
  • FIG. 3 is a set of schematic diagrams showing the use of a molecular mask for selective functionalisation of a micro-electrode array:
  • FIG. 3( b ) the molecular mask can be released by electrochemical means
  • FIG. 3( c ) the bare Au micro-electrode surface is subsequently functionalised with the required protein
  • FIG. 3( d ) by repeating this cycle it is possible to functionalise independently multiple electrodes within a single device
  • FIG. 3( e ) the formation of a protein-protein complex occurring following exposure to a complex biological solution, results in a measurable shift in ⁇ ( ⁇ ) (central electrode).
  • FIG. 4( a ) is a cyclic voltammogram of an individual micro-electrode protected with mPEG inhibiting layer, following electrochemical desorption of the mPEG monolayer and after functionalisation with peptide aptamer STM pep2 ;
  • FIG. 4( b ) is a FRET analysis of STM pep2 , STM pep9 and STM upon exposure to CDK2, CDK4 and CDK-free lysate;
  • FIG. 4( c ) ⁇ ( ⁇ ) of the complex impedance for mPEG-, STM pep2 - and STM pep9 -functionalised micro-electrodes following exposure to a lysate containing CDK2;
  • FIG. 4( d ) is the same as FIG. 4( c ) but following exposure to cell lysate containing CDK4.
  • FIG. 5 shows a diagram
  • CDK2 and CDK4 Two different peptide aptamers were employed displayed by cysteine-modified STM with affinities for cyclin-dependent kinase 2 (CDK2) and CDK4. Both CDK2 and CDK4 belong to a group of proteins involved in the regulation of the cell cycle; they are functionally related, yet share less than 50% sequence identity.
  • the two CDK-interacting peptide aptamers (named STM pep2 and STM pep9 , where the subscripts pep2 and pep9 refer to two different peptide sequences) were generated by insertion of oligonucleotides encoding the CDK-interacting peptide sequence derived from the thioredoxin-based peptide aptamers of Colas et al.
  • peptide-insert region is predicted to be far from the surface when the STM-scaffold is bound to the electrode via the cysteine-residue, we confirmed that there is no adverse impact on the performance of the peptide aptamer caused by surface-protein interactions.
  • DPI dual polarisation interferometry
  • the STM pep9 layer was exposed to 150 ⁇ l of recombinant purified CDK2 (30 ⁇ g/ml in PBS) expressed in E. coli .
  • a second waveguide functionalised with cysteine modified STM protein but without any peptide aptamer insert was simultaneously exposed to recombinant CDK2.
  • FIG. 1 ( b ) shows the real-time increase in mass resulting from immobilisation of STM pep9 and STM on the two maleimide functionalised waveguides, and following subsequent exposure to CDK2. While the mass of immobilised material is similar for both STM pep9 and STM, only the STM pep9 functionalised waveguide displayed a significant increase in mass upon exposure to CDK2.
  • EIS Electrochemical impedance spectroscopy
  • the electrolyte consisted of 100 mM phosphate buffer pH 7.7 containing 10 mM K 3 Fe(CN) 6 4 ⁇ /3 ⁇ redox probe. All electrochemical potentials are reported against an Ag/AgCl reference electrode.
  • the gold working-electrodes were functionalised with peptide aptamers (here either STM pep9 or STM) by exposure of the electrode to 35 ⁇ l of protein in a PBS buffer pH 7 for 18 hours at room temperature.
  • the devices were subsequently exposed to 45 ⁇ l of a solution containing about 200 ng/ ⁇ l recombinant, purified CDK2 expressed in E. coli . Following exposure, the devices were rinsed with deionised water (18.2 ⁇ cm, Millipore) to remove any excess CDK2.
  • FIG. 1( c ) and ( d ) show the measured phase of the complex impedance, ⁇ , for STM pep9 and STM, respectively, both before and after exposing the system to recombinant CDK2.
  • a shift in ⁇ is observed upon CDK2 binding to STM pep9 , while no change was detected in the case of STM. This shift is more obvious when plotting the difference in the phase, ⁇ , before and after exposure to CDK2 ( FIG. 1( e )). While no change in ⁇ is observed for STM, a pronounced peak is measured for STM pep9 .
  • FIG. 2 The phases ⁇ of the complex impedances measured for the different devices are shown in FIG. 2 . While a distinct shift in ⁇ ( ⁇ ) is observed between 1 and 10 3 Hz for STM pep9 exposed to CDK2 lysate ( FIG. 2( a )), the phase for STM exposed to the lysate does not change across the whole frequency range investigated. Again, this phase-shift can be seen more clearly in FIG. 2( c ) which plots the absolute change in phase following exposure of the STM pep9 and STM functionalised electrode to the CDK2-expressing yeast lysate. The magnitude of ⁇ for the STM pep9 -CDK2 lysate reaches a maximum of about 12° at a drive frequency of 300 Hz.
  • Bait proteins such as CDK2 are typically expressed at around 10 2 -10 4 molecules per cell, giving an estimated maximum concentration of CDK2 in the yeast lysate of 15 ng/ml (440 pM), which is in the clinically relevant range. Low concentration of the target protein and highly contaminated samples are typical of many biological specimens and these results demonstrate the ability of our sensor to detect unambiguously target-aptamer binding from such samples.
  • Electrode array devices consisting of ten individually addressable Au micro-electrodes separated by 15 ⁇ m, were fabricated on n-doped silicon ⁇ 100> substrates capped with a 500 nm thermal oxide using a bi-layer resist process. The electrodes were of 20 ⁇ m width. Following fabrication, each device was mounted in a header package and wire bonded to provide electrical connection to each micro-electrode. To demonstrate the suitability of our technique for array format sensing, we functionalised different closely-spaced electrodes of the array with two different peptide aptamers, STM pep9 and STM pep2 .
  • the thiol-modification of the mPEG not only allows the spontaneous formation of a molecular monolayer on the Au micro-electrode through the Au—S bond but also provides a means for removal of the masking layer from a single individual electrode through reductive cleavage of the Au—S bond.
  • the quality of the resulting mPEG layers were verified using water contact-angle measurements and X-ray photo-electron spectroscopy, and the effectiveness of protein-inhibition was confirmed by fluorescence spectroscopy. After formation of the mPEG layer, the chips were soaked for 1 hr in deionised water to remove residual ethanol and form a water layer around the PEG, believed to be crucial to inhibiting protein binding.
  • the efficacy of the desorption is monitored with cyclic voltammetry (see FIG. 4 ( a )).
  • the peak separation on the voltammogram is seen to decrease from 425 mV to 100 mV, typical of a Au surface with this redox probe.
  • the bare Au micro-electrode can be functionalised with the desired protein by incubating the device in 35 ⁇ l of protein solution overnight in a sealed, humid environment ( FIG. 3( c )).
  • the adsorption of the protein, and the effectiveness of the mPEG monolayers for masking deposition on protected micro-electrodes is confirmed using cyclic voltammetry and EIS. This process has been repeated to functionalise a second electrode with a different protein ( FIG. 3( d )).
  • An exemplary arrangement that we have demonstrated with advantageously small features comprises 10 metal electrodes, divided into two sets of five.
  • Each electrode is of width 20 microns with a separation between adjacent electrodes, both in the x and y directions in the plane, of 15 microns.
  • the electrodes are finger-shaped (see FIG. 5 , which shows part of the array of 10 electrodes) and are not round, so the ‘diameter’ means largest dimension perpendicular to the plane of protrusion.
  • the electrodes are extended over the substrate surface in order to make electrical contact from the exterior.
  • the electrodes may be brought up vertically up through the surface to produce a 2D array of pixels in the plane of the surface.

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US9382578B2 (en) 2011-10-18 2016-07-05 Scanogen Inc. Detection units and methods for detecting a target analyte
US9382580B2 (en) 2011-10-18 2016-07-05 Scanogen Inc. Detection units and methods for detecting a target analyte
US10545113B2 (en) 2012-05-01 2020-01-28 Isis Innovation Limited Electrochemical detection method
US10179930B2 (en) 2014-02-06 2019-01-15 Scanogen Inc. Detection units and methods for detecting a target analyte
US11505818B2 (en) 2014-02-06 2022-11-22 Scanogen Inc. Detection units and methods for detecting a target analyte
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US12474330B2 (en) 2018-05-25 2025-11-18 Scanogen Inc. Detection units and methods for detecting a target analyte

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