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WO2024161144A1 - Biocapteur électrochimique - Google Patents

Biocapteur électrochimique Download PDF

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Publication number
WO2024161144A1
WO2024161144A1 PCT/GB2024/050281 GB2024050281W WO2024161144A1 WO 2024161144 A1 WO2024161144 A1 WO 2024161144A1 GB 2024050281 W GB2024050281 W GB 2024050281W WO 2024161144 A1 WO2024161144 A1 WO 2024161144A1
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WIPO (PCT)
Prior art keywords
target
nucleic acid
moieties
electrode
origami
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English (en)
Inventor
Petteri PISKUNEN
Viekko LINKO
Heini IJÄS
Damion K CORRIGAN
Paul Murray
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University of Strathclyde
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University of Strathclyde
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Priority to EP24704542.0A priority Critical patent/EP4658810A1/fr
Publication of WO2024161144A1 publication Critical patent/WO2024161144A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the present disclosure relates to a method of detecting the presence or absence of a target in a sample and a structure suitable for use in such a method.
  • the structure comprises: a nucleic acid origami molecule and one or more capture moieties suitable for binding to a first position on a target. When present, the target is able to bind to the one or more capture moieties.
  • the method comprises contacting (i) the structure; (ii) a working electrode comprising one or more probe moieties, which extend outwardly from a surface of the working electrode and are suitable for binding to a second position on the target; and (iii) the sample, in order to allow the target, when present, to interact with the one or more capture and one or more probe moieties.
  • An electrical stimulus is applied to the working electrode; and an electrical response is detected.
  • Electrochemical nucleic acid biosensors hold significant promise for the monitoring of various diseases. Central to their efficacy is the inherent strict base pair binding of nucleic acids, such as DNA, allowing for highly efficient hybridization between complementary sequences.
  • a transducer surface such as planar gold or carbon electrodes
  • a transducer surface can be functionalized to capture targets with high selectivity.
  • a supportive background electrolyte and redox species in solution it is possible to measure variations in electrochemical processes, associated with nucleic acid binding events across an electrode surface.
  • the potential applications are vast, with target analytes ranging, for example, from bacterial nucleic acids associated with AMR, circulating tumor DNA sequences (ctDNA), single nucleotide polymorphisms and recently detection of clinically relevant concentrations of biomarkers for SARS COV-2 with aptasensors (see, for example, Corrigan, D. K.
  • DNA origami lie in their modular nature and the addressability of each individual nucleobase in their structures, which enable accurate and reliable sub- nanometer positioning of functional elements like target molecules, proteins, or optically active particles (see, for example, Voigt, N. V. et al., Nat. Nanotechnol.2010, 5, 200– 203; Stephanopoulos, N., Chem., 2020, 6 (2), 364–405; Kuzyk, A. et al., ACS Photonics, 2018, 5 (4), 1151–1163; and Shen, B. et al., Langmuir 2018, 34 (49), 14911–14920).
  • DNA origami in biosensing primarily focus on the optimization of capture element positioning for the electrochemical detection of simple nucleic acids (see, for example, Han, S. et aL, Anal. Chem. 2020, 92 (7), 4780-4787), large synthetic mesoscale targets (see, for example, Arroyo-Curras, N. et aL, Nanoscale, 2020, 12 (27), 13907-1391 1 ), or the voltage driven, single molecule capture of proteins in a nanopore (see, for example, Raveendran, M. et aL, Nat. Common., 2020, 11 , 4384).
  • nucleic acid biosensors with sensitivities that are practically useful, but that need not involve complex chemistries, electrochemical labelling, technically challenging materials, or multistep processing.
  • nucleic acid origami molecules may be employed to amplify a signal from a captured target.
  • these nanostructure assemblies which comprise a nucleic acid origami molecule, such as a DNA origami molecule, to a conventional faradaicelectrochemical biosensor methodology, it is possible to achieve significant improvements in detection capabilities and shift the linear working range of a sensor to the low pM range, with a high degree of selectivity.
  • nanostructure assemblies which comprise a nucleic acid (e.g.DNA) origami molecule, as described herein, may be used as part of a sandwich assay for the detection of biological targets by electrochemical biosensor methodology and, when used, increase the sensitivity of target detection.
  • a nucleic acid e.g.DNA
  • origami molecule as described herein, may be used as part of a sandwich assay for the detection of biological targets by electrochemical biosensor methodology and, when used, increase the sensitivity of target detection.
  • the inventors have found that connecting one or more capture moieties, suitable for binding to a first position on a target, to a nucleic acid/DNA origami molecule, and contacting the resultant structure with a working electrode comprising one or more probe moieties suitable for binding to a second position on the target, enables the presence of a target, in a sample, to be detected at low concentrations. Detection is based on a change in electrical response from the working electrode, detected following the application of an electrical stimulus.
  • nucleic acid/DNA origami molecule amplifies a signal which is generated through capture of a target and enables far lower concentrations of target to be detected, as compared to a system not employing such nucleic acid/DNA origami molecules.
  • the present disclosure provides a method of detecting the presence or absence of a target in a sample comprising: (i) contacting: (a) a structure comprising: a nucleic acid, such as a DNA, origami molecule comprising a nucleic acid (such as a DNA) scaffold strand and a plurality of nucleic acid (such as DNA) staple strands; and one or more capture moieties suitable for binding to a first position on a target, wherein the one or more capture moieties are each bonded to a staple strand and the capture moieties extend outwardly from the surface of the nucleic acid (such as the DNA) origami molecule; (b) a working electrode comprising one or more probe moieties, which extend outwardly from a surface of the working electrode and are suitable for binding to a second position on the target; and (c) the sample; to allow the target, when present, to interact with the one or more capture and one or more probe moieties,
  • a structure as defined in the first aspect for use in a method of detecting a target in a sample.
  • a structure of the second aspect to detect the presence or absence of a target in a sample and/or to amplify the target signal in an electrochemical biosensor. The inventors have found that the structure of the second aspect may be used as part of a sandwich assay for the detection of biological targets by electrochemical biosensor methodology and, when used, unexpectedly increases the sensitivity of target detection.
  • the structure and working electrode of the first aspect may be provided to the user as separate components, which may then be contacted with a sample (which may or may not comprise a target suitable for binding to the capture and probe moieties).
  • a sample which may or may not comprise a target suitable for binding to the capture and probe moieties.
  • a kit comprising: (1) the nucleic acid (e.g. DNA) structure defined in the second aspect; and (2) a working electrode comprising one or more probe moieties, which extend outwardly from the surface of the working electrode and are suitable for binding to a second position on a target molecule.
  • the kit may further comprise: (3) a liquid suitable for electrical conductance; (4) at least one further electrode; and/or (5) a potentiostat for detecting or sensing an electrical response or change in electrical response.
  • kits of the fourth aspect to detect the presence or absence of a target in a sample and/or to amplify the target signal in an electrochemical biosensor.
  • DETAILED DESCRIPTION In the discussion that follows, reference is made to a number of terms, which have the meanings provided below, unless a context indicates to the contrary.
  • the nomenclature used herein for defining compounds, in particular the compounds according to the invention is in general based on the rules of the IUPAC organisation for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)”. For the avoidance of doubt, if a rule of the IUPAC organisation is in conflict with a definition provided herein, the definition herein is to prevail.
  • nucleic acid sequence refers to a polynucleotide having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.
  • sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.
  • nucleotides refers to both natural nucleotides and non- natural nucleotides, which are capable of being incorporated into an oligonucleotide, such as a splice-switching oligonucleotide. Nucleotides may differ from natural nucleotides by having a different phosphate moiety, sugar moiety and/or base moiety. Nucleotides may accordingly be bound to their respective neighbour(s) in a template or a complementing template by a natural bond in the form of a phosphodiester bond, or in the form of a non-natural bond, such as e.g. a peptide bond as in the case of PNA (peptide nucleic acids).
  • PNA peptide nucleic acids
  • the present invention provides a method of detecting the presence or absence of a target in a sample comprising: (i) contacting: (a) a structure comprising: a nucleic acid (e.g. a DNA) origami molecule comprising a nucleic acid scaffold strand and a plurality of nucleic acid staple strands; and one or more capture moieties suitable for binding to a first position on a target, wherein the one or more capture moieties are each bonded to a nucleic acid staple strand and the capture moieties extend outwardly from the surface of the origami molecule; (b) a working electrode comprising one or more probe moieties, which extend outwardly from a surface of the working electrode and are suitable for binding to a second position on the target; and (c) the sample; to allow the target, when present in the sample, to interact with the one or more capture and one or more probe moieties, wherein the working electrode is part of an electrode system comprising at least
  • the structure, working electrode and sample are contacted with one another.
  • contacting may be achieved in a variety of ways.
  • the sample is added to a mixture comprising the structure, the working electrode and the liquid.
  • the method of application may be any method that results in the contacting of the mixture and the sample.
  • the sample is applied to the mixture as a solution, a foam or a suspension.
  • the sample may be applied to the mixture by pouring, dropping or inserting it, for example when attached to a swab.
  • a mixture comprising the structure, the working electrode and the liquid may be applied to the sample, or the sample may be applied initially to either or two or more of the structure, the working electrode and the liquid, followed by contacting with the other components.
  • the contacting of the method of the invention is carried out in a liquid suitable for electrical conductance.
  • the structure comprises a nucleic acid origami molecule, such as a DNA origami molecule, which itself comprises a nucleic acid scaffold strand and a plurality of nucleic acid staple strands.
  • nucleic acid scaffold strand is a long single strand of nucleic acid that may be linear or circular.
  • a nucleic acid origami molecule is formed by binding nucleic acid staple strands, which are smaller in length than scaffold strands, to various pre-determined positions on one or more scaffold strands, thereby enabling folding of the scaffold strand into a two- or three-dimensional nucleic acid origami molecule.
  • Such nucleic acid origami molecules typically have dimensions in the 20 to 100 nm range. However, larger structures can also be prepared.
  • the nucleic acid origami molecule is substantially rigid. By “substantially rigid” is meant that the shape of the origami molecule is substantially the same both before and after the one or more capture moieties bind to a first position on a target.
  • the major axis, minor axis and convex hull of the origami molecule may fluctuate by no more than ⁇ 5%, ⁇ 2% or ⁇ 1%, where the major axis is the longest line that can be drawn through the molecule, the minor axis is the longest line that can be drawn through the molecule perpendicularly to the major axis and the convex hull is the smallest convex polytope containing the molecule.
  • the nucleic acid origami molecule has at least one substantially flat surface. By substantially flat is meant a flatness or tolerance of ⁇ 5, ⁇ 3 or ⁇ 1 nm.
  • the nucleic acid origami molecule may have at least two substantially flat surfaces. In some cases, each surface of the nucleic acid origami molecule is substantially flat.
  • the nucleic acid origami molecule can be a bundle of tubes or pipes, e.g.3-, 6-, or 12-helix bundles, or can have a substantially cuboidal shape (comprising three pairs of parallel faces, each of which is substantially flat).
  • the substantially cuboidal shape may comprise six faces of substantially equal surface area.
  • the three pairs of parallel faces of the substantially cuboidal shape may each have a different surface area, i.e. the substantially cuboidal shape may comprise two parallel faces of one surface area, two parallel faces of a different surface area, and two parallel faces of a third different surface area.
  • the substantially cuboidal shape may be substantially flat, i.e. may be much smaller in height than in width or length.
  • the height may be at least five, eight or ten times smaller than the width and the length.
  • the nucleic acid origami may be substantially flat and substantially rectangular in shape, i.e. it may be much smaller in height than in width or length and the width and length may be different in size.
  • Substantially flat, substantially rectangular nucleic acid origami structures can have surface areas of 10 4 to 10 6 nm 2 . However, smaller or larger structures can also be used.
  • the nucleic acid origami structure is a two-layered honeycomb nucleic acid lattice. Methods useful in the making of nucleic acid origami structures can be found, for example, in Rothemund, P. W., Nature 440:297-302 (2006); Douglas et al., Nature 459:414-418 (2009); Douglas et al., Nucleic Acid Research 37, 5001-5006 (2009); Dietz et al., Science 325:725-730 (2009); and U.S. Pat. App. Pub. Nos.
  • nucleic acid origami molecules can be further modified with customizable binding sites, coatings or other components as desired.
  • the nucleic acid origami molecule need not be limited to a particular shape.
  • Each nucleic acid within the origami comprises a negatively charged phosphate group.
  • the tethering of the nucleic acid origami structure to the target results in the accumulation of a local negative charge situated relatively close to the working electrode.
  • the change in electrical response on binding a target tethered to a nucleic acid origami structure is much greater than the response generated in the absence of the nucleic acid origami structure (where the resistance and impedance increase is due only to the steric bulk of the target bound to the working electrode surface).
  • the scaffold strand can have any sufficiently non-repetitive sequence.
  • the scaffold strand has an M13-derived sequence, such as a M13mp18-derived sequence.
  • the scaffold strand is a circular M13mp18 scaffold strand such as the M13mp18-derived sequence as described in Y. Xin et al., Chem. Eur. J.2021, 27, 8564.
  • design-specific scaffold strands with user-defined sequences may be synthesised (see, for example F. Engelhardt et al., CS Nano 2019, 13, 5, 5015–5027).
  • the nucleic acid origami structure is a DNA origami structure comprising a DNA scaffold strand and a plurality of DNA staple strands.
  • the structure of the invention further comprises one or more capture moieties suitable for binding to a first position on a target, wherein the one or more capture moieties are each bonded to a staple strand and the capture moieties extend outwardly from the surface of the origami molecule.
  • the capture moieties may extend from one or more surfaces of the nucleic acid origami molecule. In some embodiments, the capture moieties extend only from one surface of the origami molecule.
  • the capture moiety includes at least one of: a nucleic acid, such as a single stranded DNA, RNA, PNA or LNA molecule, an antibody, or an antigen binding fragment thereof, a cell surface receptor or its ligand, or a biologically active fragment of a cell surface receptor or its ligand, an aptamer, a molecular imprinted polymer, an enzyme, a lipid, a glycan, a glycoprotein, a glycolipid, or a proteoglycan.
  • a nucleic acid such as a single stranded DNA, RNA, PNA or LNA molecule
  • an antibody or an antigen binding fragment thereof
  • a cell surface receptor or its ligand or a biologically active fragment of a cell surface receptor or its ligand
  • an aptamer a molecular imprinted polymer
  • an enzyme a lipid, a glycan, a glycoprotein, a glycolipid, or a proteoglycan.
  • the capture moiety includes a fusion of one or more of the above moiety types.
  • the capture moieties may be fused or otherwise bound to the staple strand molecules, using techniques well known in the art.
  • the capture moiety comprises an antibody or an antigen binding fragment thereof
  • the capture moiety could be synthesised by tagging the antibody or antigen binding fragment thereof with a nucleic acid sequence and binding the complementary nucleic acid sequence to the staple strand.
  • the capture moiety would then self-organise and functionalise the staple strand with the capture moiety of choice. Tagging with nucleic acid sequences is well-known in the art and may be carried out using standard bio-conjugation chemistries (see, for example O. Koniev and A.
  • N-hydroxysuccinimidyl (NHS) ester coupling with amines N-hydroxysuccinimidyl (NHS) ester coupling with amines, carbodiimide (such as 1-ethyl-3-(3-dimethylaminopropyl)cabodiimide (EDC), dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC)) coupling with amines, biotin coupling with Streptavidin (see, for example, C. M. Dundas et al., Appl. Microbiol.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)cabodiimide
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • the phrase “antigen-binding fragment thereof”, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • binding fragments encompassed within the term “antigen- binding fragment thereof” include (i) a Fab fragment, a monovalent fragment consisting of the V H , V L , CL and CH1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and CH1 domains; (iv) a Fv fragment consisting of the V H and V L domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544 546), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.
  • a Fab fragment a monovalent fragment consisting of the V H , V L , CL and CH
  • the two domains of the Fv fragment, VH and VL are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VH and VL regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:58795883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment thereof”.
  • nucleic acid based capture moieties may be particularly attractive in certain embodiments, as they can easily be attached to a staple strand sequence, using conventional nucleic acid synthesis techniques and as described in further detail herein.
  • nucleic acid aptamer-modified staple strands can be generated and employed in generating suitable staple/capture moiety molecules.
  • Methods for generating nucleic acid origami molecules having single-stranded nucleic acid (ssNA) capture moieties protruding from the molecule are not particularly limited and are known in the art.
  • ssNA strands can be incorporated into the design of the structure from the very beginning.
  • ssNA strands can be appended to nucleic acid origami structures after assembly of the latter, e.g. by using conventional linker pairs known in the art such as Streptavidin-biotin or the like.
  • the one or more capture moieties of the invention may extend from the nucleic acid origami structure from any location at the surface of the origami structure. In cases where the nucleic acid origami structure is substantially cuboidal, the capture moieties may extend from at least one of the faces of the cuboid. In some embodiments, the capture moieties extend from at least one of the two faces of the cuboid with the largest surface area.
  • the nucleic acid origami structure may comprise any number of capture moieties, provided at least one capture moiety is present. Typically, however, the origami structure comprises two or more capture moieties. In some embodiments, the origami structure comprises three or more capture moieties, and the capture moieties are evenly spaced on the surface of the origami. The spacing of the capture moieties may be controlled by replacing individual staple strands at desired sites with staple strands modified to comprise capture moieties. In particular embodiments, the one or more capture moieties extend from one substantially flat surface, i.e.
  • each of the one or more capture moieties is bonded to the same substantially flat surface such that each of the one or more capture moieties extends in substantially the same direction away from the nucleic acid origami structure.
  • the nucleic acid origami structure is substantially cuboidal and each of the one or more capture moieties extend from the same face of the cuboid, such as one of the two faces of the cuboid with the largest surface area. The inventors have found that, where two or more capture moieties are used, connecting the two or more capture moieties to the same side of the nucleic acid origami structure increases the change in electrical signal generated when the capture moieties bind to targets, which themselves binds to probe moieties on an electrode surface.
  • the presence of a target, in a sample may be detected at even lower concentrations.
  • capture moieties are bonded to the nucleic acid origami on different faces
  • only the targets bonded to faces that are aligned with the probe moieties will bind to the probes. Accordingly, some of the targets will not bind to probe moieties and will not contribute to a change in electrical signal. Therefore, when the capture moieties do not extend in substantially the same direction away from the nucleic acid origami structure, a smaller electrical signal change results.
  • the capture moieties comprise ssNA strands extending from the nucleic acid origami structure from only particular regions of the surface of the structure, so that said ssNA strands protrude in substantially the same direction.
  • the ssNA strands can protrude form one face of said structure.
  • the capture moieties comprise DNA.
  • the capture moieties may be capture ssDNA strands protruding from the nucleic acid origami molecule.
  • the capture moieties are of are of SEQ ID NO.1 (ttttttTTGTCTTCGTACCGAGCTTTCATCGAATTTTTA).
  • the one or more probe moieties may be ssNA probe strands on the electrode surface, and are preferably ssDNA strands.
  • the capture and/or probe moieties are ssNA strands, they can be unmodified or modified ssNA strands. Suitable modifications are not particularly limited and are known in the art. They include for example Azo-modifications known in the art which can be used to realize photoresponsive and reversible binding and unbinding of the ssNA strands protruding from the origami molecule. Further modifications include modifications that modulate the strength of the interaction between the strands and target, such as backbone modifications that alter the surface charge (e.g. PNA) or structural flexibility to induce hybridization efficiency (e.g. LNA).
  • nucleic acid origami molecules can have at least 1 or at least 2, from 2 to 20, from 5 to 15, 6 or 12 protruding ssNA capture moieties.
  • the number of ssNA strands positively correlates with the binding strength of the nucleic acid origami structures to the working electrode, and also correlates with the efficiency of binding to the working electrode.
  • the number of ssNA strands can be chosen freely to accomplish a desired binding strength and/or a desired hybridization efficiency depending from the particular application and/or target of interest.
  • Aptamers are short oligonucleotides screened, using SELEX technology, from an expansive nucleic acid library based on their specific affinity to target molecules (see, for example S. Liang et al., Nat. Commun.10, 852 (2019); and M. Mie et al., Appl. Biochem. Biotechnol., 169, 250–255 (2013)).
  • target molecules can be highly diverse, ranging from small molecules, amino acids, peptides, and proteins to cells, viruses, and tissues.
  • aptamers By folding into specific secondary/tertiary conformations, aptamers can bind to their targets through noncovalent interactions, such as hydrogen bonding, hydrophobic effects, and van der Waals force. This molecular recognition process is similar to that of antibody-antigen interactions; therefore, aptamers are also regarded as ‘‘chemical antibodies.’’
  • aptamers can be automatically and reproducibly synthesized on a DNA/RNA synthesiser, be conveniently and site-specifically modified with various functional groups, and are generally 5 to 10 times smaller in comparison with antibodies.
  • the nucleic acid origami structure comprises labelled entities such as enzymes, e.g.
  • Electrode herein is meant a structure, which, when connected to an electronic device, is able to sense a current or charge and convert it to a signal.
  • an electrode can be defined as a structure which can apply a potential to and/or pass electrons to or from species in the solution.
  • Electrodes are known in the art and include, but are not limited to, certain metals and their oxides, including gold; platinum; palladium; silicon; aluminum; metal oxide electrodes including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (MO2O6), tungsten oxide (WO3) and ruthenium oxides; and carbon (including glassy carbon electrodes, graphite and carbon paste). Electrodes include gold, silicon, platinum, carbon and metal oxide electrodes.
  • the electrode system of the invention may be any system suitable for the application of a potential difference.
  • the electrode system comprises a minimum of two electrodes.
  • a working electrode applies the desired potential to the liquid suitable for electrical conductance and transports charge to (thereby acting as an anode and reducing the analyte) or from (thereby acting as a cathode and oxidising the analyte) the liquid.
  • a second electrode is required with a known potential, with which to use as a reference, and to complete the circuit and balance the charge, i.e. if the working electrode acts as an anode and reduces the analyte, then the second electrode balances the charge by oxidising a component of the liquid, thereby acting as a cathode.
  • the second electrode acts as both a reference and a counter electrode.
  • the electrode system comprises 2 to 4 electrodes, often 3 or 4.
  • the electrode system comprises 3 electrodes.
  • the electrode system comprises 3 electrodes, it comprises a working electrode, a reference electrode (of a known potential and with which to use as a reference) and a counter electrode (to complete the circuit and balance the charge).
  • changes in the potential difference at the working electrode are measured independently to the changes in the potential difference at the counter electrode. This set-up is advantageous over a 2 electrode system because analysis of the electrical response is simpler.
  • the electrode system comprises 4 electrodes, it comprises a working electrode, a reference electrode, a counter electrode and a working sense electrode.
  • the potential difference is measured at the working sense electrode (relative to the reference electrode), and is independent to the electrochemical reaction occurring at the working electrode, i.e. the effect of an applied current on the first substance itself is being measured.
  • Such a set-up is useful for the measure of impedance across the first substance (discussed below).
  • the electrodes of the electrode system may be made of any material suitable for conducting electrons such as the materials described above. It is preferable that the material is resistant to corrosion, and is able to conduct a suitable current load.
  • the current load required is in the range of ⁇ 1 nA to ⁇ 1 mA; ⁇ 10 nA to ⁇ 0.1 mA; or ⁇ 100 nA to ⁇ 0.01 mA (with an error of ⁇ 1%).
  • the electrodes comprise any one or a selection from the group consisting of gold, silver, platinum, palladium, titanium, graphite, carbon, brass, tungsten, ruthenium, iridium, titanium, nickel, aluminium, tin, or one or a selection of their oxides or salts.
  • the electrodes of the system comprise any one or a selection from the group consisting of gold, silver, platinum, palladium, titanium, graphite and carbon.
  • the electrodes comprise any one or a selection from the group consisting of gold, silver, platinum and palladium, preferably gold and silver.
  • the electrodes are made of one type of material, i.e. they are not made of a selection of materials.
  • the working electrode comprises polycrystalline gold.
  • the electrode system may be produced via additive printing processes, such as 3D-printing or screen-printing. These are suitable approaches for the production of cost- effective electrode systems and sensors (Tan, C., Nasir, M. Z. M., Ambrosi, A., Pumera, M., 2017. Anal. Chem.89, 8995-9001).
  • the electrode system may be microfabricated and may be produced by depositing the desired material, patterning the material with the desired micro features (e.g. by UV photolithography), and if necessary, removing or etching material.
  • the electrode system is screen-printed.
  • Screen printed electrodes feature many advantages over more traditional electrodes such as ease of fabrication and cleaning procedures, reliability, low-cost, repeatability and provide rapid time to result. SPEs are amenable to mass production, whereby a large volume of electrodes can be produced at relatively low-cost compared to traditional macro or microelectrodes (Hayat, A., Marty, J. L., 2014. Sensors.14, 10432-10453).
  • the conformation of the electrode will vary with the detection method used.
  • substantially flat planar electrodes can be used.
  • the electrode may be in the form of a tube, coil or rod, for example.
  • the one or more probe moieties may be any of the type of moieties as described above in respect of the capture moieties. However, as described, the probe moieties are capable of binding to a different, or second position/portion of the target, as compared to the capture moieties. In this manner, when a capture moiety and probe moiety bind a target, a sandwich is formed with the target in the middle.
  • the capture moieties and probe moieties may comprise the same or different material.
  • both the capture and probe moieties may be nucleic acid, antibodies, or antigen binding fragments thereof, or aptamers; or the capture moiety may be an aptamer and the probe moiety may be an antibody, for example.
  • the one or more probe moieties comprise plasmid DNA.
  • the one or more probe moieties may be of SEQ ID NO.2.
  • the one or more probe moieties of the invention may extend from the working electrode from any location at the surface of the working electrode.
  • the working electrode comprises two or more probe moieties, for example the working electrode may comprise three or more probe moieties.
  • the probe moieties may be evenly spaced on the surface of the electrode, and the surface of the electrode may be substantially flat.
  • the working electrode is formed on a substrate.
  • the substrate can comprise a wide variety of materials, as will be appreciated by those in the art, such as printed circuit board (PCB) materials.
  • the suitable substrates include, but are not limited to, fiberglass, teflon, ceramics, glass, silicon, mica, plastic (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes, TeflonTM, and derivatives thereof, etc.), GETEK (a blend of polypropylene oxide and fiberglass), etc.
  • glass may not be preferred as a substrate.
  • the present structures and methods may provide electrodes in single or array formats, e.g. wherein there is a matrix of addressable working electrodes.
  • array herein is meant a plurality of working electrodes in an array format; the size of the array will depend on the application and end use of the array. Arrays containing from about 2 different probe moieties to many thousands can be made.
  • the substrates can be part of a larger device comprising a detection chamber that exposes a given volume of sample to the working electrode. Generally, the detection chamber ranges from about 1 nL to 1 ml, such as about 10 ⁇ L to 500 ⁇ L. As will be appreciated by those in the art, depending on the experimental conditions and assay, smaller or larger volumes may be used.
  • Methods for functionalizing the working electrode with the probe moieties, such as ssNA strands are not particularly limited and are known in the art.
  • a nucleic acid self-assembled monolayer may be adsorbed directly onto a gold electrode surface using thiolated nucleic acids and alkane thiols.
  • Amine-modified probe moieties may be attached to the electrode by N- hydroxysuccinimidyl (NHS) ester coupling with amine-modified probe moieties, carbodiimide (such as 1-ethyl-3-(3-dimethylaminopropyl)cabodiimide (EDC), dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC)) coupling (both described above).
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)cabodiimide
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • the working electrode may comprise monolayers, which can include self-assembled monolayers (SAMs).
  • SAMs self-assembled monolayers
  • the working electrode may comprise a self-assembled monolayer (SAM) that serves to shield the electrode from non-specific target and/or DNA origami binding to the electrode surface.
  • SAM self-assembled monolayer
  • a monolayer facilitates the maintenance of the target away from the electrode surface.
  • a monolayer serves to keep charged species away from the surface of the electrode. Accordingly, the monolayer is preferably tightly packed in a uniform layer on the electrode surface, such that a minimum of “holes” exist. The monolayer thus serves as a physical barrier to block solvent accessibility to the electrode.
  • monolayer or “self-assembled monolayer” or “SAM” herein is meant a relatively ordered assembly of molecules spontaneously chemisorbed on a surface, in which the molecules are oriented approximately parallel to each other and roughly perpendicular to the surface.
  • a majority of the molecules include a functional group that adheres to the surface, and a portion that interacts with neighboring molecules in the monolayer to form the relatively ordered array.
  • a “mixed” monolayer comprises a heterogeneous monolayer, that is, where at least two different molecules make up the monolayer. The length of the species making up the monolayer will vary as needed. As outlined above, hybridization may be more efficient at a distance from the surface.
  • the probe moieties to which the target is able to bind may be substantially the same length as the monolayer forming species or longer than them, resulting in the nucleic acids being more accessible to a target in a sample.
  • the sample of the invention may or may not comprise a target.
  • the target of the invention may be any molecule, but typically comprises any one or more selected from the group consisting of proteins (such as blood based protein biomarkers), peptides, polysaccharides, lipids, and nucleic acids.
  • the target may be a characteristic component of a microorganism such as a bacteria or virus.
  • the target comprises bacterial, viral or animal nucleic acids, bacterial, viral or animal ribonucleic acids, antibodies or antigens.
  • the target may comprise bacterial, viral or animal nucleic acids, bacterial, viral or animal ribonucleic acids.
  • animal includes human.
  • the contacting of the method of the invention is carried out in a liquid suitable for electrical conductance.
  • the liquid suitable for electrical conductance comprises an electrolyte.
  • Electrolytes are substances that, when dissolved in polar solvents (for example, water), produce an electrically conducting solution. Electrolytes are salts that, on dissolution, separate into their constituent cations and anions. In the presence of an electrode system, and on the application of an electric potential, the cations of the electrolyte are attracted to the electrode acting as the anode, i.e. that with a surplus of electrons, and the anions of the electrolyte are attracted to the electrode acting as the cathode, i.e. that with a deficit of electrons. The resulting movement of ions gives rise to a current.
  • Suitable electrolytes comprise a metal cation (such as an alkali or alkaline earth metal cation) and a counterion.
  • the counterion is selected from any one or a combination of the group consisting of halide, sulfate, carbonate, bicarbonate, phosphate, nitrate, citrate, gluconate, acetate, oxide, lactate, glubionate, aspartate and picolinate.
  • the counterion is selected from any one or a combination of the group consisting of halide, sulfate, carbonate, bicarbonate, phosphate and nitrate.
  • the counterion is a halide, preferably chloride.
  • the metal cation of the electrolyte of the first substance is selected from any one or a combination of the group consisting of potassium, sodium, magnesium, calcium, zinc and chromium.
  • the metal cation is selected from any one or a combination of the group consisting of potassium, sodium, magnesium, calcium, zinc and chromium, and a counterion selected from any one or a combination of the group consisting of halide, sulfate, carbonate, bicarbonate, phosphate, nitrate, citrate, gluconate, acetate, oxide, lactate, glubionate, aspartate and picolinate, preferably potassium.
  • the electrolyte of the first substance is potassium chloride.
  • the liquid suitable for electrical conductance comprises a redox mediator.
  • Redox mediators are chemical compounds which act as electron shuttles between oxidising species and reducing species.
  • Transition metal salts or methylene blue are suitable redox mediators.
  • the transition metal of the transition metal salt is selected from any one or a combination of the group consisting of iron, ruthenium, iridium, chromium, vanadium, cerium, cobalt, osmium and manganese. Commonly, the transition metal is not a combination of metals.
  • the transition metal is selected from any one of the group consisting of iron, ruthenium and iridium, such as iron and ruthenium.
  • the transition metal iron When the transition metal iron is iron, it is often in an oxidation state of III or II; when the transition metal iron is iridium, it is often in an oxidation state of III or IV, when the transition metal iron is ruthenium, it is often in an oxidation state of II or III.
  • the redox mediator selected from any one of the group consisting of iron hexacyanide, ferrocene, ruthenium hexamine chloride, iridium chloride and methylene blue. Often, the redox mediator is [Fe III (CN) 6 ] 3- /[Fe II (CN) 6 ] 4- .
  • the concentration of redox mediator in the liquid suitable for electrical conductance may range from about 0.02 to about 10 mM, such as about 0.5 to about 10 mM, sometimes from about 0.7 to about 5 mM, such as about 1 to about 2 mM.
  • the method of the invention comprises applying an electrical stimulus to the working electrode and sensing an electrical response.
  • the electrical stimulus may be a potential and the electrical response may be a current.
  • differential pulse voltammetry, electrochemical impedance spectroscopy, amperometry and/or cyclic voltammetry are used to apply an electrical stimulus and sense an electrical response.
  • the electrode system may be electronically connected to a potentiostat.
  • the electronic connections may be any suitable connections to carry an electronic signal between the potentiostat and the electrode system.
  • each electrode of the electrode system is electronically connected to the potentiostat to allow the application and detection of electrical signals to and from each electrode.
  • the potentiostat is suitable for at least EIS measurements.
  • the potentiostat of the system is suitable for at least EIS and DPV measurements.
  • the potentiostat of the system is suitable for at least EIS, DPV, cyclic voltammetry and open circuit potentiometry measurements.
  • the potentiostat is capable of analysing electrical impedance as a function of test frequency, i.e. the potentiostat comprises an impedance analyser.
  • an impedance analyser may be used separately to the potentiostat for EIS measurements.
  • the potentiostat of the system is suitable for square wave voltammetry (SWV) and chronoamperometry measurements.
  • the potentiostat of the system is suitable for at least EIS, DPV, cyclic voltammetry, open circuit potentiometry, SWV and chronoamperometry measurements.
  • EIS is capable of real-time data capture.
  • the impedance of the working electrode-structure interface may be studied using an alternating potential difference across a range of frequencies to establish information regarding the interface, its electron transfer properties and surrounding diffusional behaviour.
  • impedance is a measure of the frequency dependant resistance of the first substance to a current flow of a circuit, and is calculated according to the formula below, where ⁇ ⁇ is equal to the frequency-dependant potential and I ⁇ is equal to the frequency-dependent current. Changes in impedance are reflective of changes at the electrode surface as a function of time: as the number of targets sandwiched between the probe moieties on the working electrode and the capture moieties on the origami structure increases, the frequency dependent resistance at the surface of the working electrode to a current flow (i.e. the impedance) increases. DPV can be used to sensitively investigate electron transfer to and from an electrode surface.
  • An electric potential is measured between the working electrode and the reference electrode, while the current is measured between the working electrode and the counter electrode.
  • the electric potential is increased or decreased linearly with time to a set potential (a potential linear sweep), or is incrementally increased or decreased with time (a staircase waveform).
  • a series of regular voltage pulses are superimposed upon the potential linear sweep or staircase waveform.
  • the current is measured before (initial current) and after (final current) the voltage pulse, and the difference between the final and initial current is plotted as a function of the applied electric potential. In this way, the effect of the non-Faradaic charging current is minimised, i.e.
  • the peak current ( I pk ) measured in DPV plots corresponds to the current generated on oxidation of a component of the liquid capable of electrical conductance. It is dependent on the resistance at the surface of the working electrode. Therefore, changes in the peak current are reflective of changes at the surface of the working electrode (e.g. binding of targets) as a function of time: as the number of targets moieties on the origami structure increases, the peak current decreases. Thus, measuring the peak current over time gives an indication of the presence or absence of a target.
  • the method of the invention may further comprise the measurement of a background signal in the absence of the sample.
  • “Absence” refers to the case where no sample has been intentionally contacted with the structure or working electrode. Therefore, a background electrical response may be measured before the sample is contacted with the structure or working electrode, i.e. the structure and working electrode may be first contacted in the absence of the sample, and the background signal may be measured by: (iia) applying an electrical stimulus to the working electrode; and (iiia) sensing an electrical response from the electrodes. The background electrical response may be subtracted from the electrical response. If the time periods used to measure the background electrical response are identical to those used to measure the electrical response then the background electrical response may be subtracted from the electrical response for each time period measured.
  • the second aspect of the invention provides a structure as defined in the first aspect, i.e.
  • a structure comprising: a nucleic acid origami molecule comprising a nucleic acid scaffold strand and a plurality of nucleic acid staple strands; and one or more capture moieties suitable for binding to a first position on a target, wherein the one or more capture moieties are each bonded to a nucleic acid staple strand and the capture moieties extend outwardly from the surface of the origami molecule.
  • the nucleic acid origami structure may be substantially cuboidal and each of the one or more capture moieties may extend from the same face of the cuboid.
  • the third aspect of the invention provides use of the structure of the second aspect to detect the presence or absence of a target in a sample and/or to amplify the target signal in an electrochemical biosensor.
  • each embodiment or example described in relation to the structure of the first aspect applies mutatis mutandis to the structure of the third aspect.
  • the fourth aspect of the invention provides a kit, comprising: (1) structure defined in the first aspect; and (2) a working electrode comprising one or more probe moieties, which extend outwardly from the surface of the working electrode and are suitable for binding to second position on the target molecule.
  • the kit further comprises (3) a liquid suitable for electrical conductance; (4) at least one further electrode; and/or (5) a potentiostat.
  • a liquid suitable for electrical conductance (4) at least one further electrode; and/or (5) a potentiostat.
  • each embodiment or example described in relation to the liquid suitable for electrical conductance, further electrode(s) and potentiostat of the first aspect applies mutatis mutandis to the liquid suitable for electrical conductance, further electrode and potentiostat of the fourth aspect.
  • the fifth aspect of the invention provides use of a kit of the fourth aspect to detect the presence or absence of a target in a sample and/or to amplify the target signal in an electrochemical biosensor.
  • kit of the fourth aspect applies mutatis mutandis to kit of the fifth aspect.
  • a method of detecting the presence or absence of a target in a sample comprising: (i) contacting: (a) a structure comprising: a nucleic acid origami molecule comprising a nucleic acid scaffold strand and a plurality of nucleic acid staple strands; and one or more capture moieties suitable for binding to a first position on a target, wherein the one or more capture moieties are each bonded to a nucleic acid staple strand and the capture moieties extend outwardly from the surface of the origami molecule; (b) a working electrode comprising one or more probe moieties, which extend outwardly from a surface of the working electrode and are suitable for binding to a second position on the target; and (c) the sample; to allow the target, when present, to interact with the one or more capture and one or more probe moieties, wherein the working electrode is part of an electrode system comprising at least one further electrode; (ii) applying an electrical stimulus to the working electrode; and (iii) sensing
  • nucleic acid origami molecule is substantially rigid. 3. The method of clause 1 or clause 2, wherein the nucleic acid origami molecule has at least one substantially flat surface from which the capture moieties extend. 4. The method of any one preceding clause, wherein the nucleic acid origami molecule is substantially cuboidal. 5. The method of clause 4, wherein the capture moieties extend from at least one of the faces of the cuboid. 6. The method of clause 5, wherein the capture moieties extend from at least one of the two faces of the cuboid with the largest surface area. 7. The method of any one preceding clause, wherein the origami is a two-layered honeycomb nucleic acid lattice. 8.
  • nucleic acid origami comprises two or more capture moieties.
  • nucleic acid origami comprises three or more capture moieties and the capture moieties are evenly spaced on the surface of the origami.
  • the capture moieties extend from one substantially flat surface.
  • nucleic acid origami molecule is a DNA origami molecule.
  • the capture moieties comprise DNA.
  • the capture moieties are of SEQ ID NO.1 14.
  • the scaffold strand is a circular M13mp18 scaffold strand.
  • the target is a molecule.
  • the target comprises any one or more selected from the group consisting of proteins, peptides, polysaccharides, lipids and nucleic acids.
  • the target comprises bacterial, viral or animal nucleic acids, bacterial, viral or animal ribonucleic acids, antibodies or antigens.
  • the probe moieties comprise plasmid DNA. 19. The method of clause 18, wherein the probe moieties are of SEQ ID NO.2. 20.
  • the working electrode comprises two or more probe moieties. 21. The method of any one preceding clause, wherein the working electrode comprises three or more probe moieties, the probe moieties are evenly spaced on the surface of the electrode, and the surface of the electrode is substantially flat. 22. The method of any one preceding clause, wherein the probe moieties are part of a self-assembled monolayer. 23. The method of any one preceding clause, wherein the working electrode comprises polycrystalline gold. 24. The method of any one preceding clause, wherein the contacting is in a liquid suitable for electrical conductance comprising a redox mediator. 25.
  • the electrolyte comprises a metal cation selected from any one or a combination of the group consisting of potassium, sodium, magnesium, calcium, zinc and chromium, and a counterion selected from any one or a combination of the group consisting of halide, sulfate, carbonate, bicarbonate, phosphate, nitrate, citrate, gluconate, acetate, oxide, lactate, glubionate, aspartate and picolinate.
  • the electrical stimulus is a potential and the electrical response is a current.
  • differential pulse voltammetry, electrochemical impedance spectroscopy, amperometry and/or cyclic voltammetry are used to apply an electrical stimulus and sense an electrical response.
  • the electrode system is electronically connected to a potentiostat.
  • a background signal is measured in the absence of the sample.
  • the structure and working electrode are first contacted in the absence of the sample, and the background signal is measured by: (iia) applying an electrical stimulus to the working electrode; and (iiia) sensing an electrical response from the electrodes. 33.
  • any one preceding clause which is for detecting the presence of 10 pM or more of a target in a sample.
  • 34 A structure as defined in any one of clauses 1 to 14.
  • 35 Use of the structure of clause 34 to detect the presence or absence of a target in a sample and/or to amplify the target signal in an electrochemical biosensor.
  • 36 A kit, comprising: (1) the nucleic acid structure defined in any one of clauses 1 to 13; and (2) a working electrode comprising one or more probe moieties, which extend outwardly from the surface of the working electrode and are suitable for binding to a second position on a target molecule.
  • kit of clause 36 further comprising: (3) a liquid suitable for electrical conductance; (4) at least one further electrode; and/or (5) a potentiostat. 38.
  • the nucleic acid origami molecules are exemplified as DNA origami molecules, but this should not be construed as limiting, unless the context dictates otherwise.
  • the present disclosure will now be further defined by way of example and with reference to the Figures, which show: Fig. 1. Design and characterization of DNA origami tiles and their use in signal amplification in biosensors.
  • Top panel DNA origami with capture strands bind to the target strands and the formed complex further attaches to the ssDNA-probe -functionalized gold electrode thus modulating the distribution of the redox species.
  • Bottom panel Schematic electrochemical impedance spectroscopy (EIS) responses. EIS can be used to monitor the drastic increase in the charge transfer resistance (R CT ) as the target-capturing DNA origami tile is present.
  • Fig. Electrochemical characterization of SAM assembly, and sensing performance without DNA origami tile amplification.
  • ⁇ A mean peak current
  • FE functionalized electrodes
  • FIG. 5 Electrochemical response to a non-complementary target.
  • (a) Mean peak current response to varying concentrations of non-complementary target (115-nt Junk Fragment) and Tile B at a fixed concentration of 50 pM.
  • (b) Box plot of DPV mean peak current plotted against a varying concentration of non-complementary target. Dash line denotes the division of the data set into two distinct populations. Grey shaded region corresponds to an estimated threshold of non-specific interactions contributing to electrochemical signal change. n 4 PGE.
  • Fig. 6. Electrochemical response of sensor design to complementary and non- complementary targets in a complex media.
  • DNA Origami Tile Design and Assembly Materials and Methods DNA Origami Tile Design and Assembly Materials All staple strands constituting the DNA origami tiles used were purchased from Integrated DNA Technologies. The employed M13mp18 scaffold strand was obtained from Tilibit Nanosystems. 50 ⁇ stock TAE (Tris/acetic acid/ethylenediaminetetraacetic acid (EDTA)) buffer was purchased from Thermo Fisher Scientific (Finland) and molecular grade agarose from Meridian Bioscience (Ohio, US). All other chemicals required in the DNA origami assembly, purification and characterization were sourced from Merck/Sigma-Aldrich (Finland). Milli-Q deionized water was used in all procedures. DNA origami annealing was carried out in a Biometra T-Gradient thermocycler.
  • TAE Tris/acetic acid/ethylenediaminetetraacetic acid (EDTA)
  • EDTA ethylenediaminetetraacetic acid
  • TEM Transmission electron microscopy
  • the DNA origami tile was designed using caDNAno, and is based on a previously published two-layered honeycomb-lattice DNA origami pegboard (see S. Julin et al., Angew. Chem. Int. Ed.2021, 60 (2), 827–832.
  • the plate-like design features 66 evenly spaced modification sites with 3.9 nm ⁇ 7.5 nm separations on both sides in identical positions (in total 132 binding sites). For this study, 0, 6, or 12 sites were used for creating extended capture strands. In other words, three versions of the tile design were used with either 0 (Tile A), 6 (Tile B, strands on one side), or 12 capture strands (Tile C, 6 strands per each side).
  • the DNA origami tiles were assembled by first mixing a ⁇ 10 ⁇ molar excess of synthetic staple strands with a circular 7,249-nt long M13mp18 scaffold strand in 2.5 ⁇ folding buffer (FOB: TAE buffer supplemented with MgCl 2 and NaCl).
  • FOB TAE buffer supplemented with MgCl 2 and NaCl.
  • the resulting solution contained 20 nM of scaffold and ⁇ 200 nM of each staple strand in 1 ⁇ FOB (1 ⁇ TAE (40 mM Tris, 19 mM acetic acid, 1 mM EDTA) with 20 nM MgCl 2 and 5 nM NaCl, pH ⁇ 8.5).
  • the now folded DNA origami were purified using polyethylene glycol (PEG) precipitation.
  • PEG polyethylene glycol
  • the unpurified origami were diluted to ⁇ 5 nM concentration with 1 ⁇ FOB and mixed 1:1 (v/v) with PEG precipitation buffer (1 ⁇ TAE, 15% (w/v) PEG 8000, 505 mM NaCl).
  • PEG precipitation buffer (1 ⁇ TAE, 15% (w/v) PEG 8000, 505 mM NaCl).
  • the mixture was then centrifuged at 14,000 ⁇ g for 30 min at room temperature. After centrifugation, the supernatant was removed by pipetting and the remaining DNA origami pellet was dissolved in its original volume of 1 ⁇ FOB. The solution was then incubated overnight at room temperature to resuspend the DNA origami tiles.
  • the concentrations of the purified DNA origami solutions were determined with an UV/Vis spectrophotometer.
  • Agarose Gel Electrophoresis (AGE) Agarose gel electrophoresis was used to verify the integrity of the DNA origami concentration and a 0.46 ⁇ g/mL ethidium bromide staining.10 ⁇ L aliquots were prepared from each of the investigated DNA origami samples by diluting them to a uniform 15 nM concentration with 1 ⁇ FOB. Then, 2 ⁇ L of 6 ⁇ gel loading solution was added to each aliquot and the samples were loaded into the gel. Similarly prepared 15 nM M13mp18 scaffold was used as the reference band.
  • the gel was run for 45 min at 90 V in an ice bath with 1 ⁇ TAE containing 11 mM of MgCl 2 as the running buffer.
  • a Bio-Rad ChemiDoc MP Imaging System was used to image the gel under ultraviolet light.
  • Transmission Electron Microscopy (TEM) The fabricated DNA origami tiles were also imaged with TEM (Fig.1b).
  • a 3 ⁇ L droplet of ⁇ 20 nM origami solution was deposited on an O 2 plasma cleaned (20 s flash) formvar carbon-coated copper TEM grid and incubated for 1 min. After incubation, the droplet was drained with a piece of filter paper and sequentially negatively stained with 2% (w/v) uranyl formate that contained 25 mM of NaOH.
  • the grid was first immersed in a 5 ⁇ L uranyl formate droplet, immediately drained with filter paper and then immersed in a 20 ⁇ L droplet before incubating for 45 s. After incubation, the grid was once more blotted with filter paper and left to completely dry in ambient conditions for at least 30 min before imaging with TEM. For imaging, a 120 kV acceleration voltage was used. Sensor Construction and Electrochemistry Materials All electrochemical measurements were undertaken using an Autolab PGSTAT128N potentiostat with the additional FRA32M electrochemical impedance spectroscopy module, by scripts written in the Nova 2.1 software package (Metrohm Autolab).
  • Polycrystalline gold electrodes (PGEs) of a 2 mm diameter were purchased from IJ Cambria Scientific Ltd (Llanelli, UK).
  • An external platinum counter electrode (Metrohm, Runcorn, UK) and Ag/AgCl 3 M KCl reference electrode (Cole-Parmer, UK) completed the electrochemical cell.
  • Oligonucleotides for sensor construction were sourced from Sigma Aldrich (Dorset, UK) (given in Table 1). 3-Mercapto-1-propanol (MCP) and all other chemicals required in this study were obtained from Sigma Aldrich (Dorset, UK). Buffers required in this work are detailed in Table 2.
  • Electrode Preparation and Electrochemical Measurement Appropriate cleaning is required to achieve conformity in PGE surfaces, and the removal of immobilized organics and contaminants.
  • Mechanical polishing was first undertaken to produce a near mirror finish via a series of decreasing alumina slurry diameters from 1 ⁇ m to 0.03 ⁇ m, on microcloths of varying roughness, with sonication in isopropanol (IPA) for 2 min between each polishing step. Polishing occurred in a figure of eight motion for a duration of two min per electrode. Stripping of organics was attained by immersion of the gold surfaces in hot piranha (H2SO4 and H2O23:1 (v/v)) for 15 min.
  • hot piranha H2SO4 and H2O23:1 (v/v)
  • Electrochemical circuit fitting of Nyquist Data from EIS measurements is required to extract analytical parameters of solution resistance (R s ), charge transfer resistance (R CT ), and capacitance (C).
  • R s solution resistance
  • R CT charge transfer resistance
  • C capacitance
  • Electrode Functionalization After cleaning, the electrodes were immersed in ethanol for 3 min, rinsed in Deionised (DI) H 2 O, and then dried under a steady Argon stream. A mixed SAM of pDNA and MCP was formed by overnight incubation (18 h) at 37 °C, with electrodes immersed in a solution of 1 ⁇ M probe : 10 ⁇ M MCP, in excess 50 ⁇ M TCEP (Tris (2- carboxyethyl)phosphine hydrochloride). The primary solvent throughout was TM buffer at pH 8 (10 mM Tris-HCl + 50 mM MgCl 2 ⁇ 6H 2 O). Following this step, electrodes were named as functionalized electrodes (FE).
  • DI Deionised
  • bacterial DNA sequences central to antimicrobial resistance were employed in assay development.
  • immobilised probes, and capture arms were primer sequences for the amplification of a region of an artificial plasmid attributing to the blaOXA-1 ⁇ -lactamase gene; encoding extended- spectrum ⁇ -lactamases (ESBLs) and resistance to Oxacillin, across a host of gram- negative species.
  • This blaOXA-1 ⁇ -lactamase gene sequence served as the complementary target sequence in this study.
  • Potassium ferri/ferrocyanide (Fe(CN)6 (-3/-4) ), is a commonly employed redox couple for the measurement of DNA immobilization on electrode surfaces.
  • the ferri- and ferrocyanide species possess trivalent and quadrivalent anions, meaning that interaction with immobilized DNA (a polyanion) is governed by electrostatic repulsion at an electrode surface.
  • Fig. 2a details the functionalization process with comparisons drawn between the immobilized ssDNA- probe as part of mixed pDNA/MCP SAM, and a pristine electrode surface. Thereafter, Fig. 2b and 2c report on the capability of functionalized electrodes to monitor the hybridization of free targets without amplification by an origami tile complex.
  • Fig.2a highlights the reproducibility of both the cleaning and functionalization methodologies for PGE.
  • Mean peak currents from a high sample size of PGE exist with a high degree of significance between them, representative of the SAM forming process.
  • Probe surface densities have been estimated for the functionalization protocol by chronocoulometric methods (see, for example A. B. Steel et al., Anal. Chem., 1998, 70 (22), 4670–4677; and S. D. Keighley et al., Biosens. Bioelectron., 2008, 23 (8),1291– 1297). Adoption of these methods produces a surface coverage of 4.62 ⁇ 2.28 ⁇ 10 12 molecules/cm 2 .
  • the Hill equation employed is as follows: ( 1) Where V max is maximum binding obtained, x is the concentration of the target, k is the dissociation coefficient and ⁇ is the Hill slope describing cooperativity.
  • the limit of detection can then be estimated by the following equation: (2) where ⁇ is the standard deviation of the blank (FE condition) and slope the hill slope from the fitting function (see P. Jolly et al., Biosens.
  • Tile B reported highly significant signal changes for both i PC and R CT . This supports the theory that large nucleic acid origami structures contribute to dramatic manipulation of the interfacial properties for functionalized electrodes via direct tethering through complementary targets present in solution. The impact of amplification by an origami tile is clear when contrasting the mean signal change of a conventional DNA biosensor design to a complementary target, against that of this novel sensor design. Tile C matched the level of significance in signal change for that of Tile B, however the magnitude of change is lesser.
  • Tile B was chosen for further interrogation. Signal change, and level of significance, for each tile design are reported in Table 4. Table 4. Tabulated electrochemical data for various DNA origami tile designs. Mean % change is provided for FE following incubation with a complementary target : tile complex of matched concentrations. Electrochemical Performance of a DNA Origami Tile-Enhanced Biosensor With an appropriate design confirmed, it was next necessary to assess the performance of this approach by investigating the response to complementary targets.
  • the working range is tighter. We theorize this to be a function of the size of the tile. As such, the electrode surface is quickly saturated, as low target concentrations are sufficient to effectively cross-link these Inter-device variability in the FE condition is high, though this is a common observation in SAM formation (see, for example L. S. Ho, ACS Omega, 2019, 4 (3), 5839–5847) The notion of low probe DNA coverage contributing to uniform monolayers is perhaps an oversimplification. This is increasingly apparent with reporting of heterogeneous SAM formation (see for example E. A.
  • Fig. 5a and 5b mean peak current is reported in response to incubation with increasing concentrations of the non-complementary target, and a fixed concentration of Tile B.
  • the scatter plot in Fig.5a allows for the fitting of the experimental data to assess whether a linear region is present that could be attributed to concentration dependent non-specific DNA interactions, or the reorganization effects reported by Piper et al (see A. Piper et al., Electrochem. Sci. Adv., 2021. Fitting of the data is poor with a coefficient correlation of 0.78 across the experimental range, and indicative of no sporadic layer organization that contributes solely to significant decline in peak current. This is better reflected in Fig.5b, where peak current data for each condition is provided as a box plot.
  • the system was interrogated in a complex media containing a high DNA load.
  • Specificity of the DNA Biosensor Design To validate the hypothesized sensor mechanism of action, an experiment was undertaken subjecting functionalized electrodes to Tile B at a concentration of 50 pM, and either of the two target sequences used previously in this study. The complementary sequence, 115-nt OXA fragment, and a randomly generated non-complementary sequence of 115-nt in length. Additionally, the sensing apparatus was challenged by undertaking the assay in a complex media.
  • a complex media is established by spiking components of a commercially available DNA Origami kit (Tillibit Nanosystems) to each sample, thus producing a high background non-specific DNA load on the sensor.
  • Confirmation of the sensor mechanism is provided in Fig.6.
  • Fig.6a displays peak amplitude depression for both the complementary and non- complementary target incubations. However, the magnitude of peak reduction is significantly larger for the complementary target. This is documented in Fig.6b with the respective percentage change of mean peak currents contrasted between both targets, with a high degree of significant difference noted (**** p ⁇ 0.0001). This is furthered by the data of impedimetric measurements presented in the bottom panel of Fig.6.
  • 6c shows the characteristic growth of the semicircle region associated with increasing impedance. Again, this is common to both complementary and non-complementary targets, however the magnitude of signal change is significantly greater for the complementary target.
  • Constituent components of the sample solution included either the complementary or non-complementary targets at 100 pM, Tile B at 50 pM, and the necessary concentrations of all reagents required for the assembly of a commercially available DNA origami nanostructure provided by Tilibit Nanosystems.
  • reaction mix from Tilibit Nanosystems The details of the reaction mix from Tilibit Nanosystems are provided in Table 6.
  • the sensor was therefore interrogated with a working concentration of the Tilibit type P7249 scaffold at a concentration of 1.5 nM ( ⁇ 15 greater than the target concentration), and the Tilibit staple mixture in the sample at a concentration of 76 nM ( ⁇ 760 greater than the target concentration).
  • Table 6 Reaction mixture for Tilibit Nanosystems assemblies. Reaction mixtures were purchased from Tilibit Nanosystems in order to challenge the sensing apparatus.

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Abstract

La présente divulgation concerne un procédé pour détecter la présence ou l'absence d'une cible dans un échantillon et une structure adaptée à l'utilisation d'un tel procédé. La structure comprend une molécule d'origami d'acide nucléique et une ou plusieurs fractions de capture aptes à se lier à une première position sur une cible. Lorsqu'elle est présente, la cible peut se lier à une ou plusieurs fractions de capture. Le procédé comprend la mise en contact (i) de la structure; (ii) une électrode de travail comprenant une ou plusieurs fractions de sonde, qui s'étendent vers l'extérieur à partir d'une surface de l'électrode de travail et sont appropriées pour se lier à une seconde position sur la cible; et (iii) l'échantillon, afin de permettre à la cible, lorsqu'elle est présente, d'interagir avec la ou les capture (s) et une ou plusieurs fractions de sonde. Un stimulus électrique est appliqué à l'électrode de travail et une réponse électrique est détectée.
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