Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C5/00—Separating dispersed particles from liquids by electrostatic effect
  • B03C5/02—Separators
  • B03C5/022—Non-uniform field separators
  • B03C5/026—Non-uniform field separators using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
  • B01L2400/0424—Dielectrophoretic forces

Definitions

• the present invention relates to a device for assaying one or more analytes in a sample, said device comprising an electrode, means for optical detection and means for electrochemical detection, wherein the device is configured such that the analyte is capable of attachment to the electrode, for example via a capture probe that is a component of the electrode.
• ITO Indium tin oxide
• Dry etching methods have also been investigated for patterning of ITO thin films ⁇ Mohri.M; KaUnuma,H., Sakamoto, M., & Sawai,H. Plasma-Etching oflto Thin-Films Using A Ch4/H2 Gasixture. Japanese Journal of Applied Physics Part 2-Letters 29, LI 932-L1935 (1990)).
• Electrodes to improve target binding have been described in the following documents:
• Siemens AG also focused on electrochemical impedance spectroscopy based on interdigitated electrodes in W02004057022.
• Slab optical waveguide spectroscopy is a technique to optically study electron transfer reactions on electrode surfaces.
• no electrodes, or devices containing them which are able to carry out the three functions of promoting transport of analytes in a sample, detecting their optical properties and detecting their electrochemical properties have been previously described.
• a problem with the prior art therefore is that separate devices are required in order to carry out all three functions.
• the prior art arrangements are less convenient but also more expensive and can be more time consuming since separate devices need to be operated to carry out all three functions.
• separate devices are required to carry out all these functions and therefore a more bulky arrangement as a whole, no device in a portable form able to carry out all these three functions has previously been described. So far nobody has been able to combine all three functions in a single device.
• a first aspect of the present invention is a device for assaying one or more analytes, said device comprising:
• a means for electrochemical detection wherein the device is configured such that the electrode is capable of promoting transport of an analyte when a field is applied to the analyte via the electrode; and wherein the means for electrochemical detection employs the electrode; and the means for optical detection employs the electrode, and wherein the device is configured to carry out dielectrophoresis.
• Another aspect of the present invention is the use of the device of the present invention for promoting transport of an analyte, detecting the optical properties of the analyte and detecting the electrochemical properties of the analyte.
• a further aspect of the present invention is a method for assaying one or more analytes, which method comprises the steps of: a) promoting transport of an analyte b) performing optical measurement of the analyte c) performing an electrochemical measurement of the analyte; wherein said method employs the device of the present invention.
• the present invention comprises the use of electrodes for electrochemical detection, optical detection, and accelerated binding in biomolecular interaction assays (e.g. DNA biosensors).
• the invention is comprised of an electrode configuration which can perform (1) electrochemical detection (e.g. electrochemical impedance spectroscopy), (2) optical detection (e.g. total internal reflection fluorescence), and (3) forced transport of target molecules (for example by dielectrophoresis) at the same time or sequentially.
• the present invention enables highly sensitive and rapid detection of analytes using probe target reactions by an enhanced signal to noise ratio (optical and electrochemical transduction) and enhanced binding rate (forced transport).
• a further advantage of the present invention is that it enables very high packing densities on microelectrode chips due to the incorporation of three functions in a single electrode. Higher packing densities lead to higher yields and thus to lower production costs. Furthermore, the reduction of size enables portable triple function detection devices.
• a device for assaying one or more analytes comprising: an electrode, means for optical detection and means for electrochemical detection, wherein the device is configured such that the electrode is capable of promoting transport of an analyte when a field is applied to the analyte via the electrode; and wherein the means for electrochemical detection employs the electrode; and the means for optical detection employs the electrode.
• the present device may be a fluidic device, such as a microfluidic or nanofluidic device.
• the present invention is preferably directed to analyte s that are bio-molecules, although any charged ionisable or polarisable analytes may be assayed, if desired.
• the analyte may comprise one or more compounds selected from a cell, a protein, a polypeptide, a peptide, a peptide fragment, an amino acid, a carbohydrate, a lipid, a natural or synthetic chemical or metabolite or nucleic acid such as DNA or RNA.
• the analyte is usually contained in a sample.
• the sample typically comprises a biological sample such as a cellular sample.
• the biological sample may or may not need to be pre- treated, depending on its structure.
• the electrode is composed of an optically transparent material. While the material is not especially limited, provided that it does not unduly hinder any of the electrode function, indium tin oxide (ITO) may be employed as the optically transparent material.
• ITO indium tin oxide
• the device is suitable for promoting transport of an analyte to the electrode (whether or not the analyte is labelled), detecting the optical properties of the analyte and detecting the electrochemical properties of the analyte.
• the optical detection and electrochemical detection may be carried out either sequentially or simultaneously.
• the electrode may further comprise a capture probe, which capture probe is capable of reacting with the analyte to capture the analyte on the electrode.
• the electrode comprises the capture probe the position and/or orientation of capture probe may be influenced to promote binding of the analyte to the capture probe. Orientation of the capture probe may also be employed to enhance the means for optical detection and/or the means for electrochemical detection.
• the analyte may bind directly to the electrode or the capture probe may react with the analyte to capture it on the electrode.
• Any capture probe known in the art could be suitable for use depending upon the analyte to be detected.
• a DNA probe may be used to capture a specific DNA target sequence by hybridisation. This embodiment is particularly suitable when the analyte is DNA wherein the DNA is collected at the region of high electric fields at the electrodes.
• the device according to the present invention is preferably configured to carry out an assay method for detecting the presence or absence of the analyte in the sample.
• the assay method may also comprise quantifying the sample.
• the analyte and/or the capture probe may be influenced to binding to the electrodes and/or between the electrodes.
• the plurality of electrodes are preferably in the form of an interdigitated electrode structure.
• the device is configured to carry out dielectrophoresis.
• the device and method according to the present invention are particularly advantageous for assaying analytes which have electrical properties which allow them to exhibit a strong dielectrophoretic activity in the presence of an electric field. Accordingly, analytes which exhibit effective polarizability in an electric field are particularly suited to the present invention.
• the device and assay method are particularly useful for detecting DNA or RNA which can be easily manipulated using electric fields.
• the device is configured to apply at least two alternating fields, wherein at least one alternating field is composed of a plurality of pulses to influence a sample and/or the electrode or capture probe capable of binding an analyte.
• alternating field means that an electric field which has a non-constant value which may be created, for example, by applying alternating current (AC) or an alternating voltage to a pair of electrodes. It should be noted here that the term AC can apply to both alternating current and alternating voltage.
• alternating field composed of a plurality of pulses means more than one application of the alternating field, typically in immediate succession, for example by switching the applied field on and off, or by reducing and then increasing the field (or vice versa). This includes single peak magnitudes along with varying peak magnitudes and varying frequencies for the first field.
• the wording "apply a second alternating field" and "apply one or more further alternating fields” means that a second or one or more further alternating fields are applied simultaneously with the first alternating field.
• the two or more alternating fields may be a series of superimposed fields, each having different frequencies and/or shapes, such as sinusoidal or square.
• a high frequency sinusoidal alternating field and low frequency sinusoidal alternating field may be superimposed and applied simultaneously.
• the second or one or more further alternating fields are applied sequentially after the first alternating field.
• the first alternating field controls movement of the analyte towards the electrode and the second alternating field promotes binding of the analyte to the electrode.
• the present inventors have surprisingly found that application of a first alternating field and a second alternating field to a medium comprising a sample reduces the time and increases the sensitivity for processing the sample.
• the present inventors have also surprisingly found that application of a first alternating field composed of a plurality of pulses and optionally a second alternating field to a medium comprising a sample reduces the time and increases the sensitivity for processing the sample.
• the second alternating field is composed of a plurality of pulses and has a second frequency, a second pulse duration and a second pulse rise time.
• the first alternating field and second alternating field are different.
• the first and second alternating field may differ by their frequency and/or pulse duration and/or pulse rise time and/or amplitude.
• the inventors have unexpectedly found that more than one alternating field, which may be pulsed and are preferably different, can be used to manipulate an analyte and improve the speed and efficiency of processing a sample.
• the alternating fields are able to control different events which occur during the method, including bulk events, such as movement of the analyte to the electrode (i.e. toward the detector), and surface confined events, such as binding of the analyte to the electrode.
• the first alternating field may be used to control movement of the analyte to the electrode, for example movement of DNA to the electrode.
• the first and/or the second alternating field may be used to control binding of the analyte to the electrode, for example DNA hybridisation.
• the first and/or second alternating field may be used to position and/or orientate the capture probes attached to the electrode, for example by elongation, to enhance the hybridization efficiency.
• the first and/or second alternating field may also be applied after the analyte has bound to the electrode to remove unspecifically bound analyte and any adsorbed analyte and improve the washing efficiency.
• an alternating field may be applied during washing with a buffer. If the buffer used for washing has a high ionic strength this induces negative dielectrophoresis and unspecifically bound analytes, such as DNA, would be driven to the region from the region of high electric fields near the electrodes to a region of lower electric fields away from electrodes. This is particularly useful because it is easy to remove unspecifically bound analytes and promote their movement away from electrodes.
• the present inventors have also found that if the alternating field applied comprises a plurality of pulses the manipulation of an analyte and/or a binding phase is improved and, therefore, the speed and efficiency of processing a sample is improved.
• the frequency and amplitude of the alternating fields is set at a suitable level which allows for optimal polarity of the analyte being processed thereby allowing selective manipulation and movement of the target analyte and/or the electrode or capture probe.
• the specific frequency and amplitude required for each alternating field will depend upon the type of sample being processed, the electrical properties, density, shape and size of the target analyte.
• the pulse rise time and frequency of the alternating field are set at a suitable level which allows for optimal movement of the analyte through the medium.
• the specific pulse rise time and frequency required for each alternating field composed of plurality of pulses will depend upon the type of sample being processed, the electrical properties, the density, shape and size of the target analyte. Without being bound by theory it may be that a large pulse rise time and low frequency may be required for larger analytes to allow sufficient force to be applied for sufficient time to cause them to move.
• the first and second alternating fields may be applied either simultaneously or sequentially depending upon the type of events to be controlled in the assay device.
• both the first and second alternating fields are composed of a plurality of pulses.
• the first and second alternating fields are applied sequentially the voltage, and/or frequency and/or pulse duration and/or pulse rise time of the first alternating field may be changed in order to produce the second pulsed alternating field.
• the first and second alternating field are applied simultaneously.
• the number of pulses applied is not particularly limited and may be in the range 1 to the total number of cycles possible in the time period of the alternating field application.
• Each alternating field is preferably applied for a period of time of 1 to 20 minutes, preferably 5 to 20 minutes, more preferably from 10 to 20 minutes.
• the first alternating field preferably has a frequency of 1 to 10 9 Hz more preferably 10 4 to 10 7 Hz. This range of frequency may improve analyte movement by inducing dipolar charge on the analyte throughout the medium, particularly for DNA. There may be a decreasing effect on analyte movement when higher frequencies than 10 7 Hz are used, as there is progressively less time for induced dipoles to form and for transport to occur.
• the first alternating field which may be pulsed, preferably has field strength of 10kV/m to 1000 MV/m.
• the first alternating field which may be pulsed, preferably has a frequency of 30 Hz and a voltage of 350 mV.
• the second alternating field which may be pulsed, preferably has a frequency of 10 2 to 10 9 Hz.
• the second alternating field which may be pulsed, preferably has a voltage of 10 mV to 5 V and even more preferably in the range from 1OmV to 2V.
• the second pulsed alternating field preferably has a pulse duration of 10 "2 s to 10 "8 s.
• the second pulsed alternating field also has a pulse rise time of 10 "8 s to 10 "10 s. This pulse duration and pulse rise time may improve surface confined events, particularly for DNA hybridisation.
• the first alternating field and second alternating field preferably have waveforms independently selected from sinusoidal, square, sawtooth and triangular.
• Further alternating fields preferably have a frequency of 10 2 to 10 9 Hz. Further preferred alternating fields preferably have a voltage range of 10 mV to 5 V. Further preferred alternating fields preferably have a pulse duration of 10 s to 10 ' s. Further preferred alternating fields preferably have a pulse rise time of 10 " s to 10 " s.
• the analyte binding function of the present invention is made on the basis that the application of two alternating fields or the application of one or more pulsed alternating fields may be used to control specific events when processing a sample including transport of the target analyte from the bulk solution to the electrode and binding of the analyte to the electrode. Accordingly, the processing of the sample is quicker and more sensitive.
• the present invention is particularly useful for nucleic acid (e.g. DNA) assays because DNA is polar isab Ie and, therefore, moves in an alternating field.
• the present invention may be employed for many different types of assays for different analytes well known to the person skilled in the art.
• the analyte is labelled with one or more labels to form the labelled analyte.
• the device and method may operate without labelling the analytes, provided that the analytes contain some moiety that may act as a label (and in the context of the present invention, such moieties are considered to be labels) to allow distinction between different analytes.
• the means for labelling the analyte are not particularly limited and many suitable methods are well known in the art.
• the analyte is DNA or RNA it may be labelled by enzymatic extension of label-bound primers, post-hybridization labelling at ligand or reactive sites or "sandwich” hybridization of unlabelled target and label-oligonucleotide conjugate probe (Fritzsche W, Taton T A, Nanotechnology 14 (2003) R63-R73 "Metal nanoparticles as labels for heterogeneous, chip-based DNA detection").
• oligonucleotides to nanoparticles
• thiol-modified and disulfide-modified oligonucleotides spontaneously bind to gold nanoparticles surfaces, di- and tri-sulphide modified conjugates, oligothiol-nanoparticle conjugates and oligonucleotide conjugates from Nanoprobes' phosphine-modified nanoparticles (see figure 2 of Fritzsche W, Taton T A, Nanotechnology 14 (2003) R63-R73 "Metal nanoparticles as labels for heterogeneous, chip-based DNA detection").
• Both DNA and RNA strands may be biotinylated.
• the biotinylated target strand may be hybridized to oligonucleotide probe-coated magnetic beads. Streptavidin-coated gold nanoparticles may then bind to the captured target strand (Wang J, Xu D, Kawde A, Poslky R, Analytical Chemistry (2001), 73, 5576-5581 "Metal Nanoparticle-Based Electrochemical Stripping Potentiometric Detection of DNA hybridization”).
• the magnetic beads allow magnetic removal of non-hybridized DNA.
• the one or more labels are preferably selected from nanoparticles, single molecules and chemiluminescent enzymes.
• Suitable chemiluminescent enzymes include HRP and alkaline phosphatase.
• the labels are nanoparticles.
• Nanoparticles are particularly advantageous in the embodiment of the present invention where the label(s) used in step (a) are the same as the label(s) used in step (b) because they operate successfully in both optical and electrical detection methods.
• the proximity of the nanoparticles to the surface is not especially important, which makes the assay more flexible.
• the nanoparticles comprise a collection of molecules because this gives rise to greater signal in optical and electrical detection methods than when single molecules are used.
• the nanoparticles are selected from metals, metal nanoshells, metal binary compounds and quantum dots.
• preferred metals or other elements are gold, silver, copper, cadmium, selenium, palladium and platinum.
• preferred metal binary and other compounds include CdSe, ZnS, CdTe, CdS, PbS, PbSe, HgI, ZnTe, GaAs, HgS, CdAs, CdP, ZnP, AgS 5 InP, GaP, GaInP, and InGaN.
• Metal nanoshells are sphere nanoparticles comprising a core nanoparticle surrounded by a thin metal shell.
• Examples of metal nanoshells are a core of gold sulphide or silica surrounded by a thin gold shell.
• Quantum dots are semiconductor nanocrystals, which are highly light-absorbing, luminescent nanoparticles (West J, Halas N, Annual Review of Biomedical Engineering, 2003, 5: 285-292 "Engineered Nanomaterials for Biophotonics Applications: Improving Sensing, Imaging and Therapeutics”).
• quantum dots are CdSe, ZnS, CdTe, CdS, PbS, PbSe, HgI, ZnTe, GaAs, HgS, CdAs, CdP, ZnP, AgS, InP, GaP, GaInP, and InGaN nanocrystals.
• the size of the labels is preferably less than 200 nm in diameter, more preferably less than 100 nm in diameter, still more preferably 2-50 nm in diameter, still more preferably 5-50 nm in diameter, still more preferably 10-30 nm in diameter, most preferably 15-25 nm.
• the present invention is for detecting a plurality of analytes, each different analyte is labelled with one or more different labels relatable to the analyte.
• the labels may be different due to their composition and/or type.
• the labels when the labels are nanoparticles the labels may be different metal nanoparticles.
• the nanoparticles are metal nanoshells, the dimensions of the core and shell layers may be varied to produce different labels.
• the labels have different physical properties, for example size, shape and surface roughness.
• the labels may have the same composition and/or type and different physical properties.
• the different labels for the different analytes are preferably distinguishable from one another in the optical detection and the electrochemical detection.
• the labels may have different frequencies of emission, different scattering signals and different oxidation potentials.
• the bound analyte may be detected at the electrode both optically and electrochemically.
• optical and electrochemical detection may either be simultaneous or sequential.
• the device and method of the present invention improve sensitivity and selectivity of the results.
• the device and method of the present invention increase the accuracy and number of the analytes detected.
• the sensitivity and selectivity of the device and method of the present invention are improved significantly compared to carrying out either an optical detection method or an electrical detection method.
• the device and method of the present invention are also quick, cheap and simple to carry out.
• the detection data comprises information on the effect of the frequency of the oscillating voltage on the intensity, changes in the emission lifetime and/or the frequency of light emitted or absorbed by the one or more labels.
• Changes in emission and absorption frequency can result from variation in the chemical or environmental nature of the label, for example brought about by alterations in the degree of protonation (e.g. from changes in pH) or brought about by alterations in the degree of complexation (e.g. from changes in complexant proximity and/or concentration).
• Changes in emission lifetime can be observed as a consequence of variation in the environment surrounding the label (e.g. changes in solvation, local dielectric constant, and alteration in energy transfer to neighbouring species due to changes in separation).
• the means for optical detection without being especially limited is configured to carry out optical emission detection, optical absorbance detection, optical scattering detection, spectral shift detection, surface plasmon resonance imaging, and surface-enhanced Raman scattering from adsorbed dyes.
• the means for optical detection is configured to carry out optical emission detection.
• the optical emission detection can comprise the steps of irradiating the analytes with light capable of exciting the analytes and detecting the frequency and intensity of light emissions from the analytes.
• the optical data of frequency and/or intensity can be used to provide information on the identity and/or quantity of analytes present.
• the light employed in the optical detection is not especially limited, provided that it is able to sufficiently excite the analytes.
• the light to which the embedded analyte is exposed is a laser light.
• the frequency of the light is also not especially limited, and UV, visible or infrared light may be employed.
• optical detection methods include optical absorbance detection, optical scattering detection, spectral shift detection, surface plasmon resonance imaging, and surface-enhanced Raman scattering from adsorbed dyes are well known in the art (Fritzsche W, Taton T A, Nanotechnology 14 (2003) R63-R73 "Metal nanoparticles as labels for heterogeneous, chip- based DNA detection”).
• the means for electrochemical detection is configured to carry out by electrochemical impedance spectroscopy.
• the identity and/or quantity of the analyte or plurality of analytes are determined from both the optical and electrochemical data obtained.
• the intensity of light emissions can be used to provide information on the identity and/or quantity of analytes present.
• the amount of analyte present can be quantified by voltammetry.
• Quantitative data can be obtained from the signal peaks by integration, i.e., determining the area under the graph for each signal peak produced.
• the device can be configured to carry out optical and electrochemical detection simultaneously or sequentially.
• optical and electrochemical measurements can be made simultaneously.
• An implementation of this embodiment of the present invention is as follows. An oscillating sinusoidal voltage is applied across a solution containing charged species. The species will behave differently depending upon the frequency of the oscillation, the composition of the solution and the surrounding conditions (temperature, pressure etc.). This is because they will affect the mobility of the species in the solution. High frequency and/or low mobility give rise to simple oscillation of the species which in fact looks like simple capacitance. Low frequency and/or high mobility allows the species to reach the electrodes and undergo redox reaction at the surface, causing a current to flow, which can be measured.
• the frequency of light emission is typically constant, but the intensity may change with the frequency of the applied electrochemical perturbation. This is because the reagents may be able to penetrate farther and cause more reaction at lower frequencies.
• the intensity and/or frequency of the emitted light can be measured, and the effect of the frequency of oscillation of the voltage on the intensity and/or frequency of the emitted light can also be measured.
• it may be the frequency of the emitted light that changes, or both frequency and intensity, depending on the nature of the system and species under investigation. The relationship generally depends on the speed of the differently charged molecules and/or ions in the solution.
• Some fluorescent labels which can be used as tags in biosystems are also redox active, being able to switch between fluorescent and less or non- fluorescent states. Those that do not can also have their fluorescence output reduced or eliminated by quenching species. Modulation of the voltage on an underlying electrode surface onto (or adjacent electrode surface near) which labelled species (such as oligonucleotides) have been immobilised by standard immobilisation procedures will produce a modulation in fluorescence output from the label (either through direct redox reaction or via reaction with a soluble redox mediator or quencher). This change in light output is typically measured through use of a suitable detector e.g. a photovoltaic or photomultiplier, which can measure the light intensity of the emitted light.
• a suitable detector e.g. a photovoltaic or photomultiplier, which can measure the light intensity of the emitted light.
• Both the light response and current response can be measured by analysing the ratio of photovoltaic voltage to applied voltage (this may be termed fluorescence impedance) as a function of frequency (fluorescence impedance spectroscopy) and the ratio of current to applied voltage as a function of frequency.
• the extent of light and current output modulation (both in-phase and out-of-phase) as a function of the frequency of the applied voltage will be determined by such factors as the rate of diffusion and/or migration of the mediator or quencher through the immobilised oligos, the rate of reaction (which may be affected by the availability of the label for reaction) and the distance of the label from the electrode surface.
• any or all of these factors may change significantly upon specific oligonucleotide hybridisation, as indeed they may with non-specific binding.
• the combination of all of these measurements may lead to a characteristic and distinct change in the measured light impedance and impedance spectral data either alone or in combination indicative of specific binding and distinct from nonspecific binding.
• the device can be configured to carry out optical and electrical detection sequentially. Without being especially limited as to the order of detection the device can be configured to carry out the optical detection first followed by the electrochemical detection.
• the inventors have also surprisingly discovered that after optical detection the labelled analytes are in a state that can be successfully used in electrochemical detection.
• the advantages of the present invention are that they improve sensitivity and selectivity of the results.
• the present method increases the accuracy and number of the analytes detected.
• the one or more labels that are suitable for optical and electrochemical detection which are used are the same. This more readily allows the data from both the optical and electrical methods to be used to determine the identity and/or quantity of analyte or plurality of analytes in one sample.
• the labels used for optical detection are different to those used in electrochemical detection. This is advantageous because it provides more data when the optical detection and electrochemical detection are carried out on separate labels. DESCRIPTION OF THE FIGURES
• Figure 1 shows the effect of an oscillating sinusoidal voltage applied across a solution containing charged species.
• Figure 2 shows biotin integrated into a DNA or RNA molecule.
• the duplex is labelled with an anti-biotin antibody which is tagged with a nanoparticle suitable for optical and electrical detection.
• Figure 3 shows an electrode configuration in accordance with the present invention which can perform (1) electrochemical detection (e.g. electrochemical impedance spectroscopy), (2) optical detection (e.g. total internal reflection fluorescence), and (3) forced transport of target molecules (e.g. by dielectrophoresis) at the same time or sequentially.
• electrochemical detection e.g. electrochemical impedance spectroscopy
• optical detection e.g. total internal reflection fluorescence
• forced transport of target molecules e.g. by dielectrophoresis
• Figure 4 shows a complete mask layout of the gold interdigitated microelectrode structures, including four device chips, alignment marks and dummy metal lines to speed lift-off processing.
• the number of digits (N) on each electrode is preferably from 5 to 10.
• the length of each digit (L) is preferably from 75 to 150 ⁇ m.
• the width of each digit (W) and the width of the gap between each digit (G) is each preferably from 1.5 to 10 ⁇ m and W and G are preferably the same.
• Figure 5 shows Nyquist plots of a gold interdigitated electrode used for dielectrophoresis (DEP) measurements.
• Figure 6 shows a gold electrode (IDE) in a solution of polystyrene beads.
• the IDE was connected to an AC field of 7 V at 50 MHz.
• Figure 7 shows a gold IDE electrode in a solution of polystyrene beads. Images were recorded with different applied voltages at a frequency of 100 kHz. (a) 2V, (b) 4V, (c) 6V & (d) 8V.
• Figure 8 shows a gold IDE electrode in a solution of polystyrene beads. Images were recorded with an applied voltage of 8 V at a frequency of 100 kV at different time interval.
• Figure 9 shows a gold IDE electrode in a solution of polystyrene beads. Images were recorded with an applied voltage of 5 V at a frequency of 20 kV at different time interval, (a) Field OFF reference time, (b) Field ON (15"), (c) Field ON (45") & (d) Field ON (2').
• Figure 10 shows a gold IDE electrode in a solution of polystyrene beads. Images were recorded with an applied voltage of 8V at a frequency of 2OkV at different time interval, (a) Field OFF reference time, (b) Field ON (12"), (c) Field ON (15"), (d) Field ON (1 '5"), (e) Field ON (2'), (f) Field ON (10') & (g) Field ON (30').
• Figure 1 1 shows a gold IDE electrode in a solution of 1 nM Qdots in distilled water, (a) Field off, (b) The IDE was connected to an AC field of 2V at 1 MHz for 6min, (c) same as (b) with reverse polarisation (d) the electrode was measured after being left 12hours in distilled water without field.
• Figure 12 shows three Gold IDE electrodes in a solution 1 nM Qdots in distilled water. Each electrode was connected for a duration of 6 min to an AC field of 2 V at 20 KHz (a), 100 kHz
• Figure 13 shows Transmission image of a damaged IDE. The image was recorded after applying an AC field of 5 V at 20 kHz.
• Figure 14 shows a gold IDE electrode immobilised with complementary probe, in a solution 1 nM Qdots in 3 mM HEPES buffer, pH 6.9.
• Figure 15 shows two Gold IDE electrodes immobilised with complementary probe connected to an AC field of 2.5 V at 100 kHz for 10 min.
• the electrodes were immersed in 3 mM HEPES buffer solution containing (a) 1 nM Qdots and 1 nM target, (b) 1 nM Qdots and 10 nM target (c) Shows (b) after washing in SSC+SDS solution.
• RNA is reverse transcribed, incorporating a nucleotide labelled with a nanoparticle, according to conventional techniques.
• Labels are excited with light of a given wavelength, and their emission is detected at a predetermined wavelength, according to conventional methods.
• Electrochemical detection is then carried out on the labelled analyte from the optical detection method.
• the labelled analyte is dissolved in an acidic solution. Electrodes are inserted into the solution and a deposition potential of -0.8 V is applied. After a deposition time of two minutes a second potential of +1.2 V is applied to oxidise the deposited nanoparticles. Electrochemical currents are recorded and integrated to give the charge passed in each process, which determines the amount of deposited nanoparticles.
• Example 3 effect on hybridization efficiency of applying the AC fields investigated by electrochemical impedance spectroscopy (EIS) and fluorescence detection.
• an electrode control was performed by measuring the impedance of the interdigitated electrodes (IDE). This was done in a solution of 10 mM [Fe (CN) 6] 3 -/4- by applying a 10 mV rms amplitude voltage at frequencies between 1 MHz and 0.1 Hz to the electrode with a potentiostat.
• the characteristic semi-circle observed ( Figure 5) confirmed that both IDE electrodes and connections were properly working.
• the solution of polystyrene beads was injected into the flow cell and the potentiostat replaced by a 50 MHz Pulse Generator (HP 8112A), The sample was excited with a 470 nm pulsed laser diode and the fluorescence collected through a 1OX objective and sent onto a cooled EMCCD camera (-70 0 C) via a 535 nm 40 nm bandpass filter.
• HP 8112A 50 MHz Pulse Generator
• Figure 7 shows a series of images corresponding to different applied voltages. At 6 V (figure (c)) one can observe that the vertical part of the top electrode becomes brighter. At 8 V a dark area starts to appear showing the field geometry across the IDE. The concentration of beads at the IDE becomes apparent within the first minute as shown ( Figure 8) although the response is still relatively weak at this frequency.
• Figure 10 shows a series of images recorded with an applied voltage of 8 V. As the spacing between electrodes is 10 ⁇ m, the root mean squared (rms) field across the electrode is approximately 5.7 x 105 V m "1 and even higher at the tip of the electrode due to local enhancement. The time series presented Figure 10 shows clearly that the beads start first to condensate on the tip fingers (top electrode) and gradually cover the length of the electrode fingers.
• FIG 11 shows an electrode immersed in InM of Qdots dissolved in distilled water.
• an AC field here 1 MHz, 2 V peak voltage
• the Qdot can be seen to concentrate at the periphery of the electrode fingers, clearly revealing the shape of the attractive electrode (b) in the IDE pair.
• the opposite electrode become attractive as expected and as shown in Figure l l(c). It was also observed that once the Qdots have been concentrated at the electrode, the latter tend to stay there as shown in Figure l l(d), where the electrode is displaying a strong signal even after 12 h in distilled water. This is consistent with high concentration Qdot coagulation and electrode adsorption.
• Figure 12 shows the result obtained with an applied AC field of 2 V at 20 kHz, 100 kHz and 1 MHz.
• the Qdots are mostly attracted to the tip of the electrode fingers where the field strength is the highest.
• the attraction of the nanoparticles was observed to be more homogeneous.
• best results were obtained at 100 kHz with an applied peak voltage between 2 and 3 volts (of the order of 2 x 105 V rms m "1 ).
• the formation of bubbles and ultimately damage to the electrode was observed, as shown in Figure 13. (The picture shows that only one electrode is present, the other one has completely detached.)
• Hybridization is usually conducted in a buffer solution such as SSC.
• a buffer solution such as SSC.
• HEPES 3 mM, with 1 mM NaOH, pH 6.9, which has a conductivity of approximately 20 ⁇ S cm "1 .
• the results presented ( Figure 14) show the successful build up of Qdots at the attractive electrode through DEP after 6 and 12 mins.
• the enhanced hybridization efficiency during AC field application might be caused by the re-orientation of the probe layer or the increase of the local target concentration by AC field-induced dielectrophoretic trapping of target oligonucleotides or by a combination of both of these phenomena.

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