Detecting analytes

Abstract

Provided is a method for detecting analyte in a sample, which method comprises: (a) contacting the sample with a peptide nucleic acid (PNA) probe; (b) performing an electrochemical impedance spectrometry (EIS) measurement on the sample; (c) determining the presence, absence, quantity and/or identity of the analyte from the EIS measurement;
wherein the analyte comprises nucleic acid;
and wherein the quantity of analyte in the sample when the sample is taken is substantially the same as the quantity of analyte in the sample when the sample is subjected to the EIS measurement.

Claims

1. A method for detecting an analyte in a sample, wherein the analyte comprises a nucleic acid, which method comprises: (a) subjecting the sample to a sample preparation step to fragment the nucleic acid; (b) contacting the sample with a peptide nucleic acid (PNA) probe; and (c) performing an electrochemical impedance spectrometry (EIS) measurement on the sample comprising: (i) applying an alternating voltage to the analyte; (ii) determining the rate of change of EIS measurements across the analyte, and (iii) determining the presence, absence, quantity and/or identity of the analyte from the rate of change data.

2. A method according to claim 1, wherein no amplification step and/or no concentration step is performed on the sample prior to step (c) and wherein the quantity of analyte in the sample when the sample is taken, is substantially the same as the quantity of analyte in the sample when the sample is subjected to the EIS measurement.

3. A method according to claim 1, wherein the analyte comprises ribosomal RNA and/or genomic DNA.

4. A method according to claim 1, wherein the analyte nucleic acid comprises 1000 bases (1 kb) or more.

5. A method according to claim 1, wherein prior to step (c) no PCR step is performed.

6. A method according to claim 1, wherein prior to step (c) no RTPCR step is performed.

7. A method according to claim 1, wherein the EIS measurements are measurements of electron transfer resistance, Ret.

8. A method according to claim 1, wherein the EIS measurements are measurements calculated from finding the width of the semicircular feature in a Nyquist plot.

9. A method according to claim 1, wherein step (c) comprises a step of performing a Fourier transform on EIS data.

10. A method according to claim 1, wherein an electrolyte is added to the system to aid in EIS measurement.

11. A method according to claim 10, wherein the electrolyte is a transition metal complex.

12. A method according to claim 11, wherein the transition metal complex comprises the [Fe(CN).sub.6].sup.3−/4− system.

13. A method according to claim 1, wherein a liquid medium is employed in the system to aid in EIS measurement.

14. A method according to claim 13, wherein the liquid medium is acidic or basic.

15. A method according to claim 1, wherein the method is for analysing two or more analytes, and further comprises the step of labelling each analyte with one or more labels to form labelled analytes distinguishable from each other by their labels.

16. A method according to claim 15, wherein the one or more labels are suitable for optical and/or electrical detection.

17. A method according to claim 16, wherein the labels are selected from nanoparticles, single molecules, chemiluminescent enzymes and fluorophores.

18. A method according to claim 17, wherein the labels are nanoparticles comprising a collection of molecules and/or atoms.

19. A method according to claim 18, wherein the nanoparticles are selected from metals, metal nanoshells, metal binary compounds and quantum dots.

20. A method according to claim 16, wherein the optical detection method is selected from optical emission detection, optical absorbance detection, optical scattering detection, spectral shift detection, surface plasmon resonance imaging, and surface-enhanced Raman scattering from adsorbed dyes.

21. A method according to claim 16, wherein the optical detection is optical emission detection and comprises the steps of irradiating the labelled analytes with light capable of exciting the labels and detecting the frequency and intensity of light emissions from the labels.

22. A method according to claim 21, wherein the light is laser light.

23. A method according to claim 21, wherein the light is selected from infra-red light, visible light and UV light.

24. A method according to claim 23, wherein the light is white light.

25. A method according to claim 1, wherein the PNA probe comprises a spacer portion and a PNA portion.

26. A method according to claim 25, wherein the spacer portion comprises two or more groups selected from: a terminal group for attaching the spacer to a surface; an alkyl group; an ether group; and a carbonyl group, and/or wherein the spacer portion comprises 3 or more atoms in its backbone.

27. A method according to claim 25, wherein the spacer portion comprises the following formula: ##STR00007## where T is a terminal group capable of attaching to a surface; R.sub.1, R.sub.2, R.sub.3 R.sub.4, R.sub.5 and R.sub.6 are each independently organic groups; w is an integer of 0 or 1; x is an integer of from 0-15; y is an integer of from 0-15; z is an integer of 0 or 1; n is an integer of from 0-10; m is an integer of from 0-15; and wherein [n.Math.m+w+x+y+z] is at least 3.

28. A method according to claim 1, wherein the sample preparation step comprises heating the sample, and/or sonicating the sample.

29. A method according to claim 1, wherein the sample preparation step fragments the nucleic acid in the sample such that the average length of the nucleic acid sequences less than 1000 bp (bp=base pairs), less than 800 bp, less than 500 bp, 20 bp or more, 30 bp or more, 40 bp or more, 50 bp or more 60 bp or more 70 bp or more, 80 bp or more, 90 bp or more, 100 bp or more, 110 bp or more, 120 bp or more, 130 bp or more, 140 bp or more, or 150 bp or more, from 20-800 bp, 30-600 bp, 40-500 bp, 50-400 bp, 60-350 bp, 70-300 bp, 80-250 bp, 90-200 bp, 100-180 bp, or about 120 bp.

30. A method of detecting a pathogen in a wound in a subject, which method comprises detecting a nucleic acid characteristic of the pathogen by performing a method as defined in claim 1.

31. A method according to claim 30, wherein the sample is a sample taken from a wound in the subject.

32. A method according to claim 30, wherein the subject is a human.

33. A method according to claim 30, wherein the pathogen is a pathogen which is resistant to treatment.

34. A method according to claim 30, wherein the pathogen is selected from an E. coli and an MRSA.

35. A method for detecting an analyte in a sample, wherein the analyte comprises nucleic acids, the method comprising: (a) subjecting the sample to a sample preparation step to fragment the nucleic acids; (b) contacting the sample with a peptide nucleic acid (PNA) probe; and (c) performing an electrochemic impedance spectrometry (EIS) measurement on the sample, comprising the steps of: (i) applying an alternating voltage to the analyte, wherein the alternating voltage comprises a plurality of superimposed frequencies sufficient to distinguish the presence of the analyte by electrochemical impedance spectrometry (EIS); and (ii) determining the presence, absence, identity and/or quantity of the analyte from EIS data.

36. A method according to claim 35, wherein the EIS data comprises data parameters derived from the complex impedance (x+iy), which parameters are selected from one or more of the following: Real component (x) Imaginary component (y) Modulus or absolute value [r=|z|=(x.sup.2+y.sup.2).sup.1/2] Angle [θ=tan−1(y/x)].

37. A method according to claim 35, wherein the plurality of frequencies is determined prior to step (b) by statistical analysis, and/or by empirical methods.

38. A method according to claim 35, wherein the minimum number of superimposed frequencies is from 2-20.

39. A method according to claim 38, wherein the number of superimposed frequencies is at least 3-10.

40. A method according to claim 35, wherein the number of superimposed frequencies is at least 7.

Description

(1) The present invention will be described in further detail with reference to the accompanying Figures, in which:

(2) FIG. 1 shows typical Nyquist plots of EIS data from Macro gold (small Z values) and interdigitated micro (IME) electrodes.

(3) FIG. 2 shows plots of real component (x), imaginary component (y), modulus (r), angle (θ), Principal component 1, and Principal component 2 against frequencies for the data for positive controls and immobilised probes for both macro and interdigitated electrodes.

(4) FIG. 3 shows the EIS response of gold protein macroelectrodes (6700 pM antibody) from normal single sine sequential EIS measurement with approximately 23 seconds simultaneous FFT analysis (black—recording time over two minutes; red—5 multisine EIS measurement over 9 seconds; blue—15 multisine EIS measurement every 9 seconds).

(5) FIG. 4 shows a comparison of the Nyquist plots of modified gold electrode with 69 mer HCV DNA probe and blocked with 1 mM MCH (diamonds), and hybridization with 1 μM of complementary target (ITI 025) (squares). The impedance measurements were carried out in 2×SSC containing 10 mM [Fe(CN).sub.6].sup.3− and 10 mM [Fe(CN).sub.6].sup.4− (plus probe or target) at an applied dc potential between the electrodes in the IDE pair of 0 V.

(6) FIG. 5 shows a comparison of the Nyquist plots of modified gold electrode with 69 mer HCV DNA probe and blocked with 1 mM MCH (diamonds), hybridization with 1 μM of non-complementary target (ITI 012) (squares), hybridization with 1 nM (triangles) and 50 nM (circles) complementary target (ITI 025). The impedance measurements were done in 2×SSC containing 10 mM [Fe(CN).sub.6].sup.3− and 10 mM [Fe(CN).sub.6].sup.4− (plus probe or target) at an applied DC potential between the electrodes in the IDE pair of 0 V.

(7) FIG. 6 shows Ret versus time EIS measurements during probe (thiol-DNA) layer formation (diamonds), after blocking with MCH (squares), during hybridization with 1 μM complementary target (triangles) and washing after hybridization (circles).

(8) FIG. 7 shows fluorescence measurement after EIS measurement of complementary target (50 nM) binding and 20 nM QD incubation; PMT setting 180.

(9) FIG. 8 shows a mask layout of 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 nm. The width of each digit (W) and the width of the gap between each digit (G) is each preferably from 1.5 to 10 nm and W and G are preferably the same.

(10) FIG. 9 shows EIS response (electron transfer resistance value (Rct)) with DNA probes before and after hybridisation with 1 μM target, whilst FIG. 10 shows EIS response (Rct) of PNA probes before and after hybridisation with 1 μM target. This big difference in the sensitivity of PNA and DNA probes is caused by the fact that the PNA is a neutral molecule without the negative charged phosphate backbone of DNA molecules. The hybridisation of negatively charged nucleic acid targets cause a big change of the overall charge of the electrode surface. This leads to a large degree of repulsion of the negatively charged electroactive species (ferri/ferrocyanide) used for the faradaic EIS detection, which in turn causes a large increase of the electron transfer resistance value (Rct).

(11) FIG. 11 shows a dose response curve for impedimetric E. coli species identification using an E. coli specific PNA probe (P51) hybridised with different concentrations of E. coli 16S rRNA without prior amplification.

(12) FIG. 12 shows EIS response (electron transfer resistance value (Rct)) before and after hybridisation with genomic DNA extracted from different amounts of methicillin-resistant staphylococcus aureus (MRSA) cells (10.sup.5-10.sup.8 cells/ml mock wound fluid (MWF)) and as control from 10.sup.8 cells/ml methiccillin-susceptible S. aureus (MSSA) spiked into MWF and a buffer control incubation (2×SSC).

(13) FIG. 13 shows qPCR results from tests conducted with a DNA template recovered from MRSA. The plot shows cell/mL concentrations from WF vs cycle threshold.

(14) FIG. 14 shows qPCR data demonstrating variation in gDNA yield upon extraction from 108 cells/mL MRSA in wound fluid.

(15) FIG. 15 shows (A) an image of the prototype potentiostat, and (B) a Nyquist plot pre and 10 minutes post introduction of a 23 bp fully complementary oligonucleotide (1 μM).

(16) FIGS. 16 A, B & C show bioanalyser data of MRSA gDNA following heat treatment at 95° C. for 0, 1 and 5 mins respectively.

(17) FIG. 17 shows agarose gel electrophoresis of MRSA samples following no heat treatment, 1 min at 95° C. and 5 mins at 95° C.

(18) FIG. 18. shows a Nyquist plot of EIS measurements made on a PNA modified gold electrode before and after hybridisation with MRSA gDNA extracted from a suspension of 107 cells/mL and a Randles circuit—RS=solution resistance, CDL=double layer capacitance, RCT=charge transfer resistance and ZW=Warburg diffusion element.

(19) FIG. 19 shows signal Increase Ratio in response to incubating MRSA genomic DNA extracted from suspensions of cells with concentrations of 107 cells/mL with probes containing 4 different spacers; n=3 and error bars=standard deviation.

(20) FIG. 20 shows impact of target size on EIS signal: Agilent Bioanalyzer analysis of (A) extracted MRSA gDNA and (B) observable DNA fragmentation when MRSA gDNA was incubated at 95° C. for 5 mins in 2×SSC using DNA 12000 kit; (C) EIS response to incubation with MRSA gDNA following denaturation at 95° C. in 2×SSC for 0, 1 or 5 mins; n=3 and error bars=standard deviation.

(21) FIG. 21 shows (A) dose response curve for MRSA genomic DNA (n=5 and error bars=standard deviation; line=0 MRSA cells/mL+3 standard deviations); and (B) specific and non-specific signal changes from MSSA, E. coli and MRSA (n=3 and error bars=standard deviation).

(22) FIG. 22 shows Signal Increase Ratios caused by incubation with gDNA extracted from MRSA cells spiked into human wound fluid and uninnoculated human wound fluid; Signal Increase Ratio measured 10 mins after sample addition; n=3 and error bars=standard deviation.

(23) FIG. 23 shows sensor behaviour following exposure to MRSA and E. coli gDNA following extraction from suspensions of cells from 106 cells/mL; n=3 and error bars=standard deviation.

(24) The methods of all aspects of the invention have a number of specific advantages over known methods: fast time to result (TTR) in seconds to minutes compatible with near patient environment requirements; wide applicability of approach to different probe-target systems; compatibility with rapid multisine EIS for enhanced data collection; EIS detection compatibility with electronic control and measurement; and label-free detection.

(25) The method of the present invention may be used to detect either a single analyte or a plurality of different analytes simultaneously.

(26) Preferably, the method of the present invention is a label free method, i.e. there is no requirement to label the analyte in order to aid in detection. However, in some circumstances labels may be employed. For example, when the method is used to detect a plurality of different analytes simultaneously, each different analyte may be labelled with one or more different labels relatable to the analyte. Alternatively, multiple analytes may be detected by spatial separation, such as by arraying a set of probes for the analytes on a surface. Detection of a plurality of different analytes is also known as multiplexing.

(27) In the electrochemical detection methods of the invention, the analyte is investigated in solution or suspension in a liquid medium. The liquid medium is not particularly limited provided that it is suitable for analysis using EIS. Preferably the liquid medium comprises an electrolyte to facilitate the EIS measurement. The electrolyte is a solvent or buffer containing inert ions e.g. PBS; typically redox active species are then added at much lower concentrations. The electrolyte is not particularly limited, and may include any electrolyte known in the art. However electrolytes containing transition metal redox systems are preferred, such as Fe(II)/Fe(III) electrolyte systems. [Fe(CN).sub.6].sup.3−/4− is particularly preferred.

(28) If a plurality of different labels is used to label different analytes, they may be introduced with biotinylated detection probes. Preferably each label has a different oxidation potential for the electrochemical detection method and, therefore, produces different signal peaks in the data obtained. For example, when metal nanoparticles are used as labels for different analytes (see below) different metals with different oxidation potentials may be used for each analyte.

(29) In preferred embodiments the alternating potential applied to the electrode is not especially limited, and depends upon the medium employed. Thus, in practice, the largest possible amplitude for EIS is fixed by the solvent limits (for water around 2 V, giving a rms amplitude of around 1-2 V). Accordingly, in aqueous media the potential may be from +1.0 to +2.0 V, and preferably from +1.2 V to +1.8 V. When using redox species in the system, both oxidised and reduced species are present and this typically results in the use of less than 250 mV amplitude. In more preferred embodiments, the alternating voltage applied between electrodes is of amplitude about 10 mV root mean squared (rms). This enables the response to be linearised for e.g. equivalent circuit analysis. Higher amplitude responses can be used (and if statistical methods are to be employed to extract characteristic signals, they could be different/advantageous).

(30) In a preferred embodiment, the electrical detection method is carried out on a chip. In the multiplexing embodiment of the present invention, where label(s) are used for optical detection, the optical and electrical detection may be carried on one chip when the analyte(s) have been labelled with the different labels simultaneously. Alternatively, where the analyte(s) have been separated into two aliquots and labelled separately they may then be combined after labelling for optical and electrical detection on one chip or optical and electrical detection may be carried out separately on two separate chips.

(31) Using EIS, the amount of analyte present can be quantified. Quantitative data can be obtained from the signal peaks by integration, i.e. determining the area under the graph for each signal peak produced.

(32) Embodiments Employing Labelling

(33) In some preferred embodiments of the present invention, labels are employed, in particular when multiplexing is desirable. The labels referred to are not especially limited, but are preferably selected from nanoparticles, single molecules, intrinsic components of the target such as specific nucleotides or amino acids, and chemiluminescent enzymes. Suitable chemiluminescent enzymes include HRP and alkaline phosphatise. Fluorescent labels are particularly preferred, since optical detection of the labels is readily combined with the electrochemical methods of the invention.

(34) Preferably, the labels are nanoparticles. Nanoparticles are particularly advantageous in these embodiments of the present invention because they operate successfully in electrical detection methods. The proximity of the nanoparticles to the surface is not especially important, which makes the assay more flexible. In a preferred embodiment 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.

(35) Preferably the nanoparticles are selected from metals, metal nanoshells, metal binary compounds and quantum dots. Examples of preferred metals or other elements are gold, silver, copper, cadmium, selenium, palladium and platinum. Examples of preferred metal binary and other compounds include CdSe, ZnS, CdTe, CdS, PbS, PbSe, HgI, ZnTe, GaAs, HgS, CdAs, CdP, ZnP, AgS, InP, GaP, GalnP, and InGaN.

(36) 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.

(37) 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”). Examples of quantum dots are CdSe, ZnS, CdTe, CdS, PbS, PbSe, HgI, ZnTe, GaAs, HgS, CdAs, CdP, ZnP, AgS, InP, GaP, GalnP, and InGaN nanocrystals.

(38) Any of the above labels may be attached to an antibody.

(39) 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.

(40) When the method of 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. In this aspect of the invention, the labels may be different due to their composition and/or type. For example, when the labels are nanoparticles the labels may be different metal nanoparticles. When the nanoparticles are metal nanoshells, the dimensions of the core and shell layers may be varied to produce different labels. Alternatively or in addition, the labels have different physical properties, for example size, shape and surface roughness. In one embodiment, the labels may have the same composition and/or type and different physical properties.

(41) The different labels for the different analytes are preferably distinguishable from one another in the optical detection method and the electrical detection method. For example, the labels may have different frequencies of emission, different scattering signals and different oxidation potentials.

(42) In embodiments of the present invention where labelling is employed, such as in multiplexing, the method typically comprises a further initial step of labelling the analyte with one or more labels to form the labelled analyte.

(43) The means for labelling the analyte are not particularly limited and many suitable methods are well known in the art. For example, when the analyte is DNA or RNA it may be labelled by 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”).

(44) Many different methods are known in the art for conjugating oligonucleotides to nanoparticles, for example 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 FIG. 2 of Fritzsche W, Taton T A, Nanotechnology 14 (2003) R63-R73 “Metal nanoparticles as labels for heterogeneous, chip-based DNA detection”).

(45) In one embodiment, both DNA or 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.

(46) To perform the EIS step, a pair of electrodes must be used. These are not especially limited, but in typical embodiments they are screen-printed or macro gold electrodes, or alternatively interdigitated electrodes (LEDs). The material of the electrodes is not especially limited, provided that it does not interfere with the chemical processes taking place when the nucleic acid analyte binds to the PNA probes on the electrode surface. Typically the electrodes are formed from an inert metal, such as gold. A mask layout of gold interdigitated microelectrode structures, including four device chips, alignment marks and dummy metal lines to speed lift-off processing is shown in FIG. 8. 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 μm. The width of each digit (W) and the width of the gap between each digit (G) is each preferably from 1.5 to 10 μm and W and G are preferably the same.

(47) The present invention will be described further by way of example only.

EXAMPLES

Example 1—Investigating EIS Parameters for Multiple Frequency Analysis

(48) In order to investigate the optimum parameters to use in the method one aspect of the invention, any EIS set-up may be employed. However, typically the electrodes, electrolytes, liquid medium, analytes (and probes if they are to be used) that will be involved in the final analysis will be employed to ensure that the parameters are as close to optimal as possible.

(49) In this Example, probe-target hybridisation on commercial gold interdigitated electrodes (IDEs) from Abtech was studied. An electrochemical cleaning cycle was utilised, applying to both electrodes in the IDE pair a linear potential sweep between −0.6 V and +1.65 V versus Ag/AgCl in 50 mM aqueous H.sub.2SO.sub.4 solution at a sweep rate of 50 mVs for 30-40 complete cycles, until a stable cyclic voltammogram (CV) characteristic of clean gold electrodes was seen. Before preparing the DNA (69 mer ITI 021) solution, the DNA probes were purified by passing them through a MicroSpin™ G-25 column (Amersham Biosciences, Buckinghamshire, UK) after cleavage of the disulfide protected nucleotides with 5 mM of TCEP solution.

(50) Nyquist plots of a large frequency range for EIS for both macro gold and interdigitated micro (IME) electrodes were plotted, and these are shown in FIG. 1; each shows distinct signals for complementary target binding.

(51) The differences between the positive control (probe with complementary target bound) and negative control (probe only or probe with non-complementary target) were compared in terms of parameters derived from the complex impedance, which can be written as x+iy, where i is (−1).sup.1/2%. These are: Real component (x) Imaginary component (y) Modulus or absolute value [r=|z|=(x.sup.2+y.sup.2).sup.1/2] Angle [θ=tan−1(y/x)] Principal component 1 Principal component 2

(52) These differences were investigated in terms of each of these quantities by plotting them against the logarithm of frequency (see FIG. 2).

(53) FIG. 2 shows that for both large (macro) and small (interdigitated micro) electrodes, the real component and modulus provide similar information and best discriminate the EIS signal from the positive controls and immobilised probes, particularly at the lower end of the frequency range. The imaginary component best discriminates the EIS signal in the middle of the frequency range.

(54) For optimising the time to result (TTR), the present invention selects the most useful range of frequency and smallest number of measurements that best discriminates between the different EIS data for all experimental conditions, and does not require employing fitting models such as equivalent circuits. Statistical analysis in this Example determined a 7-point optimal frequency range for both macro gold and interdigitated micro electrodes (IME) using the fold change between the EIS signal of the positive control and the immobilised probes.

(55) The results are summarised in Table 1.

(56) TABLE-US-00001 TABLE 1 Summary results for 7-point optimal frequency range (in Hz) for Macro Electrode and Interdigitated Micro Electrode based on complementary hybridisation vs. immobilised probe without target comparison. Optimum Range for Optimum Range for Signal Type No. of Points Macro Electrode IME Electrodes Modulus 7 [4, 44] [3, 30] Real 7 [3, 44] [3, 30] component Imaginary 7 [30, 338] [13, 150] Component

(57) It is notable that, for both types of electrodes, the modulus data and real component give a very similar range of optimal frequencies for EIS measurement, spanning around a decade of frequency. For both types of electrode, the imaginary component gives optimal signals at slightly higher frequencies than that for real and modulus data, again spanning a decade of frequencies. The very large changes in the electrode dimensions from macro to IME have had little effect on the optimum frequency range for measurement, consistent with the response being largely independent of electrode area, which simplifies EIS measurement. Differential analysis of complementary versus mock hybridisation using fold-change gave a similar optimal frequency range to that of complementary hybridisation vs. immobilised probe signals (Table 2), confirming that the same measurement range can be used.

(58) TABLE-US-00002 TABLE 2 7-point optimal frequency range in Hz for Macro Gold Electrode based on complementary versus mock hybridisation comparison. Signal Type No. of points Optimal range Modulus 7 [4, 44] Real component 7 [3, 30] Imaginary Component 7 [20, 255]

(59) To enable these data to be obtained rapidly, multisine techniques have been employed to apply the required multiple frequencies simultaneously, with FFT to analyse the results and extract these data. FIG. 3 shows a comparison of the EIS Nyquist plot for the previously used method of sequential application of single sines to the measured responses for 5 multisine (over one decade of frequency) and 15 multisine (over two decades of frequency) EIS measurements for a protein macroelectrode experimental system. Experimental data collection, analysis and display was achieved on a PC in several minutes for sequential application, around 7 seconds for 5 sines and around 23 seconds for 15 sines. The component frequencies for this multisine experiment have been selected to span the frequency range determined by statistical analysis, which spans the semicircular charge transfer feature in the EIS Nyquist plot shown. The extremely close correspondence of all data (typically to within 0.05%) indicates that the multisine EIS approach leads to more rapid EIS parameter extraction compatible with EIS measurement and analysis (and hence a TTR) of seconds, without compromising the accuracy of measurement.

Example 2—Investigating Real Time Kinetics Measurement Using EIS

(60) In this Example, the kinetics of probe-target hybridisation on commercial gold IDEs from Abtech were studied. An electrochemical cleaning cycle was utilised, applying to both electrodes in the IDE pair a linear potential sweep between −0.6 V and +1.65 V versus Ag/AgCl in 50 mM aqueous H.sub.2SO.sub.4 solution at a sweep rate of 50 mVs for 30-40 complete cycles, until a stable cyclic voltammogram (CV) characteristic of clean gold electrodes was seen. Before preparing the DNA (69 mer ITI 021) solution, the DNA probes were purified by passing them through a MicroSpin™ G-25 column (Amersham Biosciences, Buckinghamshire, UK) after cleavage of the disulfide protected nucleotides with 5 mM of TCEP solution.

(61) Immediately after cleaning, thiol-DNA probe layers were immersed in a 10 μM DNA solution in 2×SSC buffer and 10 mM of each of [Fe(CN).sub.6].sup.3− and [Fe(CN).sub.6].sup.4− (10 mM [Fe(CN).sub.6].sup.3−/4−) at room temperature. The EIS measurement was started as soon as the electrode was immersed in the DNA solution and was left to run for 3-4 h. As previously, a 10 mV RMS amplitude sinusoidal voltage was applied between the electrodes in the IDE pair at a DC voltage of 0 V throughout in these experiments, as the presence of equal concentrations of [Fe(CN).sub.6].sup.3− and [Fe(CN).sub.6].sup.4− ensured that the DC potential of each electrode was pinned at the reduction potential of [Fe(CN).sub.6].sup.3−/4−. Then, the modified surface was washed with 2×SSC for a few minutes and blocked with MCH 1 mM in water at room temperature for 30 minutes. After washing for 10-20 minutes in 2×SSC buffer, the electrode EIS signal was measured again in 10 mM [Fe(CN).sub.6].sup.3−/4− 2×SSC buffer to check for changes after the blocking step. The electrodes were then immersed in the target (complementary or not) DNA dissolved in 2×SSC and containing 10 mM [Fe(CN).sub.6].sup.3−/4− to allow EIS measurements, again at 0 V DC.

(62) FIG. 4 shows typical impedance plots of these 69-mer thiol-DNA modified probe electrodes, before and after hybridisation with 1 μM of complementary target (ITI 025). The high frequency semicircle is the common feature for both macro and IDE electrodes, and gives information on the charge transfer through the probe film layer at the electrode surface. After addition of 1 μM of complementary target the diameter of this high frequency semicircle increases, as expected, due to complementary target-probe binding in the probe layer, whilst the lower frequency diffusion feature remains essentially unchanged, indicating (as expected) little effect on diffusion between the electrodes.

(63) FIG. 5 shows another example of IDEs prepared in the same way. In this case, after the blocking, a negative control was carried out: for a few hours the EIS was monitored in a solution containing 1 μM non complementary (ITI 012) target and 10 mM [Fe(CN).sub.6].sup.3−/4− in 2×SSC. As expected, no changes were observed in the impedance signal, indicating no non complementary target probe binding. After this the electrode was rinsed in 2×SSC buffer and the response measured in a solution of 1 nM complementary target DNA and 10 mM [Fe(CN).sub.6].sup.3−/4− in 2×SSC. After 1 h, when the response was stable, the electrode was immersed in 50 nM target solution and measured overnight. The difference between probe and 1 nM target is small but significant, whilst it is easily seen for 50 nM. Thus EIS is probing complementary target binding using the established method of waiting for equilibration.

(64) FIG. 6 now shows typical EIS measurements made in real time: the parameter sensitive to probe film formation and probe-target hybridisation is the electron transfer resistance, Ret, for [Fe(CN).sub.6].sup.3−/4−, which has been calculated from finding the width of the semicircular feature in the Nyquist plot of each of the EIS spectra. This has been plotted (as Ret for electron transfer) as function of time in this Figure.

(65) These data are rich in information, and show the establishment of a probe film (diamonds), blocking and washing (squares) and the kinetics of probe-target hybridisation (triangles). When the gold electrode is exposed to probe film solution (diamonds) the value of Ret rises over the first hour or so due to probe film formation, then falls to a steady-state value after 3 4 hours, indicating a stable surface film. This is confirmed by removing the probe solution and washing, as there is little change in the observed value. Adding mercaptohexanol (MCH) to block any remaining gold surface also causes little change in resistance, as does measuring the resistance over time in buffer with [Fe(CN).sub.6].sup.3−/4− (squares), which again indicates a stable probe film. Having established a stable probe film, the kinetic technique is then used to monitor probe-target binding in the solution containing complementary target and ferri/ferrocyanide. On exposing the probe film to this solution (triangles), an immediate increase in Ret is seen due to complementary target probe binding. The initial response is immediate, with the first point showing an increase in Ret and with the value more than doubling within the first hour. This method enables the measurement of EIS response kinetically every few seconds (see multisine IDF). The rate of increase in probe-target binding would typically be expected to be first order in (and certainly dependent on) target concentration; therefore analysis of the rate of rise of EIS is then possible on the seconds to minutes timescale to give target concentration. It is satisfactory that the impedance increases more slowly over several hours after this, showing the long-time approach to an equilibrium response which limits the TTR of equilibrium measurement. On removing the target solution, washing and then measuring the response in buffer with [Fe(CN).sub.6].sup.3−/4− (circles), after a transient change in Ret the value returns initially to that observed previously, showing that the response is indicative of probe-target binding.

(66) In order to confirm that probe layer formation and hybridisation had occurred on the gold electrode, biotin-labelled target was used and then incubated (for 1 h at room temperature) with streptavidin-labelled Qdots (20 nM in QD buffer).

(67) It is clear from the resulting fluorescence image (FIG. 7) that as expected the regions of highest fluorescence intensity are on the gold fingers of the IDE. This confirms the enhancement of Ret observed after hybridisation is due to probe-target hybridisation in a film on the gold IDE surfaces.

Example 3—Comparison of DNA and PNA Probes

(68) EIS Protocol for DNA Probes (FIG. 9)

(69) After cleaning, the gold macrodisk electrodes were incubated with a solution of 200 nM thiol-modified oligonucleotide solution+800 mM mercaptohexanol in 1 M NaCl+5 mM MgCl.sub.2+1 mM EDTA for 16 h at 30° C. Thiol-modified oligonucleotides were provided from the manufacturer as disulfides with a mercaptoethyl protection group. This mercaptoethyl protection group was removed prior immobilisation by incubation with 5 mM TCEP for 30 min followed by gel extraction clean-up with Illustra spin G-25 micro columns (GE Healthcare). The electrodes with immobilised probe were blocked with an aqueous solution of 1 mM mercaptohexanol for 1 h at 30° C. Then the electrodes were washed with the immobilisation buffer (1 M NaCl+5 mM MgCl.sub.2+1 mM EDTA), 1×PBS and 1×PBS+10 mM EDTA for 10 min each.

(70) EIS measurements were performed with a three electrode system with an Ag/AgCl reference electrode and a platinum wire counter electrode (both from Metrohm (Runcorn, UK) connected to an Autolab potentiostat (Metrohm, Runcorn, UK) before and after hybridisation. EIS measurements were performed at 0.24 V with in amplitude of 10 mV at a frequency range between 100,000 Hz-0.1 Hz (15 frequencies) in 1 mM K.sub.3[Fe(CN).sub.6]+60 mM KCl. Electrodes were hybridised with 1 μM complementary artificial target (20 mer oligonucleotide) in 2×SSC for 2 h at 50° C. and with the hybridisation buffer alone without target (negative control), respectively. After hybridisation electrodes were washed with 2×SSC, 0.2×SSC solution and the EIS measurement buffer for 10 min each.

(71) EIS Protocol for PNA Probes (FIG. 10)

(72) After cleaning the gold macrodisk electrodes were incubated with a solution of 1.5 μM thiol-modified PNA solution+30 μM mercaptohexanol in 50% (v/v) DMSO for 16 h at 30° C. after incubation at 30° C. for 10 min. Electrodes were rinsed in 50% (v/v) DMSO and incubated in 1 mM mercaptohexanol in 50% (v/v) DMSO for 1 h at 30° C. Then the electrodes were washed with 50% (v/v) DMSO and the EIS measurement buffer (0.1 mM K.sub.3[Fe(CN).sub.6]+10 phosphate buffer for 10 min each.

(73) EIS measurements were performed with a three electrode system with an Ag/AgCl reference electrode and a platinum wire counter electrode (both from Metrohm (Runcorn, UK) connected to an Autolab potentiostat (Metrohm, Runcorn, UK) before and after hybridisation. EIS measurements were performed at 0.24 V with in amplitude of 10 mV at a frequency range between 100,000 Hz-0.1 Hz (15 frequencies) in 0.1 mM K.sub.3[Fe(CN).sub.6]+10 phosphate buffer.

(74) Electrodes were hybridised with 1 μM complementary artificial target (20 mer oligonucleotide) and 1 μM non-complementary artificial target (20 mer oligonucleotide) in 2×SSC for 2 h at 50° C. and with the hybridisation buffer alone without target (negative control), respectively. After hybridisation electrodes were washed with 2×SSC, 0.2×SSC solution and the EIS measurement buffer for 10 min each.

Example 4—Detection of E. coli rRNA (FIG. 11)

(75) EIS Protocol for RNA Detection with PNA Probes

(76) After cleaning the gold macrodisk electrodes were incubated with a solution of 1.5 μM thiol-modified PNA solution+30 μM mercaptohexanol in 50% (v/v) DMSO for 16 h at 30° C. after incubation at 30° C. for 10 min. Electrodes were rinsed in 50% (v/v) DMSO and incubated in 1 mM mercaptohexanol in 50% (v/v) DMSO for 1 h at 30° C. Then the electrodes were washed with 50% (v/v) DMSO and the EIS measurement buffer (0.1 mM K.sub.3[Fe(CN).sub.6]+10 phosphate buffer for 10 min each.

(77) EIS measurements were performed with a three electrode system with an Ag/AgCl reference electrode and a platinum wire counter electrode (both from Metrohm (Runcorn, UK) connected to an Autolab potentiostat (Metrohm, Runcorn, UK) before and after hybridisation. EIS measurements were performed at 0.24 V with in amplitude of 10 mV at a frequency range between 100,000 Hz-0.1 Hz (15 frequencies) in 0.1 mM K.sub.3[Fe(CN).sub.6]+10 phosphate buffer.

(78) Electrodes were hybridised with nucleic acid target solution in 2×SSC for 2 h at 50° C. Ribosomal 16S RNA extracted from E. coli was applied as full length rRNA. After hybridisation electrodes were washed with 2×SSC, 0.2×SSC solution and the EIS measurement buffer for 10 min each.

(79) RNA Extraction Protocol

(80) Inoculate 2.5 mL Luria-Bertani (LB) medium (10 g/L Bacto-tryptone, 5 g/L yeast extract, 10 g/L NaCl) with an E. coli DH10β colony from a LB agar plate and incubate for 16 h at 37° C. in a shaking incubator. Prepare TE buffer containing 15 mg/ml lysozyme. Add 2 volumes of RNA protect Bacteria Reagent into 1 volume of bacterial culture and vortex immediately, and then incubate for 5 min at room temperature Centrifuge for 10 min at 13,500 rpm and remove supernatant Add 10-20 μl QIAGEN Proteinase K to 200 μL TE buffer containing lysozyme and re-suspend the pellet by pipetting Incubate on roller mixer at room temperature for 30 min Add 700 μL buffer RLT and vortex vigorously. If there is a white precipitate centrifuge at 13,500 rpm and use supernatant in following steps Add 500 μL 100% ethanol and shake vigorously Transfer 700 μl lysate to an RNeasy Mini spin column in a 2 ml collection tube and centrifuge for 30 s at 13,500 rpm. Discard flow through and add remainder of sample and centrifuge again. Discard flow through Add 350 μL of RW1 to the spin column and centrifuge for 30 s at 13,500 rpm and discard flow through Mix 10 μl DNase I stock solution 70 μl Buffer RDD, invert and spin down. Add the 80 μl of the solution directly to the column and incubate at room temperature 15 min Add 350 μl Buffer RW1 to the RNeasy spin column and incubate for a further 5 min Centrifuge for 30 s@13,500 rpm, discard the flow-through Transfer the column to a 2 ml tube and add 500 uL RPE buffer to the column. Centrifuge 30 s@13,500 rpm Repeat this step with 2 min centrifugation@13,500 rpm Elute rRNA in 30 μL deionised water and quantify using the nanodrop.

Example 5—Detection of MRSA gDNA Using Method of Invention (FIG. 12)

(81) Gold electrodes were used in a three electrode system with a platinum counter and silver/silver chloride reference electrode. The gold electrode surface was cleaned by cyclic voltammetry in 0.1 M H.sub.2SO.sub.4, scanning the potential between 0 and 1.6 V 10 times and between 0 and 1.3 V 10 times at a scan rate of 0.1 V/s. In the case of gold macro electrodes additional cleaning steps were included in the protocol which preceded cyclic voltammetry and these were 1) electrode polishing with alumina slurry and 2) submersion of electrodes in piranha solution for 10 mins.

(82) Once clean the gold electrode was incubated for 16 h with a solution containing 1.5 μM thiol-modified PNA solution+30 μM mercaptohexanol in 50% (v/v) DMSO. Blocking was then carried out by incubating the electrode in 1 mM mercaptohexanol for 1 h. Upon completion of blocking the electrode was washed in 50% DMSO for 2 h and in EIS measurement buffer (0.1 mM K.sub.3[Fe(CN).sub.6]+0.1 mM K.sub.4[Fe(CN).sub.6]+pH 7.0 10 mM phosphate buffer).

(83) To obtain methicillin resistance S. aureus (MRSA) and methicillin susceptible S. aureus (MSSA) gDNA bacteria were sub cultured onto Columbia blood agar and incubated overnight at 37° C. in a CO.sub.2 atmosphere. Cells were inoculated into saline and the optical density measured using a Densicheck (bioMerieux). This gave values in McFarland units, proportional to the cellular concentration of bacteria in the suspension. A bacterial cell suspension in saline solution or mock wound fluid (MWF) of approximately 10.sup.8 cells/mL was produced in this way and ten-fold dilutions ranging down to 10.sup.2 cells/mL prepared from this suspension.

(84) The bacterial cells were pelleted by centrifuging 1 mL of the suspension at 5000×g for 10 mins. The supernatant was discarded and the bacterial pellet re-suspended in 200 μL of enzymatic lysis buffer (2×TE Buffer, 1.2% Triton X, 50 μg/mL Lysostaphin), before incubating for 30 mins at 37° C. 200 μL of bacterial lysate was added to 20 μL Proteinase K and DNA extracted using the bioMerieux NucliSens easyMAG automated platform. Guanidine Thiocyanate was the active chaotropic agent in the lysis buffer, acting as a protein denaturant in the purification and extraction of nucleic acids from cellular material. The purified nucleic acid solution was then removed from the vessel without dislodging the magnetic silica pellet—DNA was eluted in 100 μL of water.

(85) EIS measurements were performed at a DC potential of 0.24 V with an amplitude of 10 mV rms using a frequency range between 100,000 Hz-0.1 Hz (15 frequencies) in 0.1 mM K.sub.3[Fe(CN).sub.6]+0.1 mM K.sub.4[Fe(CN).sub.6]+10 mM phosphate buffer. The MRSA gDNA sample was prepared by mixing 45 μL of sample with 5 μL of 20×SSC and then heating at 95° C. for 5 mins, storing on ice for 2 mins and heating at 25° C. for 5 mins. The electrode was incubated with the sample for 2 h at 55° C. with shaking (650 rpm). Following incubation with sample, electrodes were washed with 2×SSC, 0.2×SSC and EIS measurement buffer for 10 mins in each. EIS measurements were performed pre and post hybridisation.

(86) Preparation of Mock Wound Fluid (MWF)

(87) Ringer's (Krebs) Solution: 118.4 mM NaCl (mwt 58.44˜6.91 g/l) 4.7 mM KCl (mwt 74.56˜0.350 g/l) 2.52 mM CaCl.sub.2 (mwt 147.02˜0.370 g/l) 1.18 mM MgSO.sub.4 (mwt 246.5˜0.290 g/l) 1.18 mM KH.sub.2PO.sub.4 (mwt 136.09˜0.160 g/l) 25 mM NaHCO.sub.3 (mwt 84.01˜2.10 g/l) pH 7.4

(88) All components are dissolved in 900 ml of deionised water and solution pH is adjusted to 7.4. Adjust volume to 1 liter with deionised water and confirm the pH prior to use. Ringer's solution is mixed 1:1 with Foetal Bovine serum (Gibco ref 16000-036) to produce mock wound fluid.

Example 6—Online EIS Experiments

(89) Electrode Preparation and Measurement Information

(90) For online detection screen printed gold electrodes (Working electrode diameter 1.6 mm) were purchased from DropSens (Oviedo, Spain). Each electrode was pre-cleaned by cyclic voltammetry in 0.1 M H.sub.2SO.sub.4. Electrode potential was scanned between 0 and 1.6 V for 20 cycles with care being taken to remove any bubbles forming on the surface with a pipette. A second round of cleaning in 0.1 M H.sub.2SO.sub.4 was then carried out where cyclic voltammetry was again performed, this time electrodes were scanned between potentials of 0 and 1.3 V for 20 cycles. Finally, the electrodes were thoroughly rinsed with deionised water and dried under a stream of nitrogen. After cleaning, screen printed electrodes were incubated with a solution of 1.5 μM thiol-modified PNA solution+30 μM mercaptohexanol in 50% (v/v) DMSO for 16 h at room temperature in a humidity chamber. In order to block the surface, electrodes were rinsed in 50% (v/v) DMSO and incubated in 1 mM mercaptohexanol in 50% (v/v) DMSO for 1 h at room temperature in a humidity chamber. Finally, the electrodes were washed with 50% (v/v) DMSO and the EIS measurement buffer (0.1 mM K.sub.4[Fe(CN).sub.6]+0.1 mM K.sub.3[Fe(CN).sub.6]+pH 7.0 10 mM phosphate buffer. Online EIS measurements were performed with a screen printed electrode (WE-Au, CE-Pt, RE-Ag) connected to an Autolab potentiostat. EIS measurements were performed at a DC potential of 0.03 V with an amplitude of 10 mV rms using a frequency range between 100,000 Hz-0.1 Hz (15 frequencies) in 0.1 mM K.sub.4[Fe(CN).sub.6]+0.1 mM K.sub.3[Fe(CN).sub.6]+pH 7.0 10 mM phosphate buffer.

(91) DNA Fragmentation Experiments

(92) The behaviour of the DNA sample following heat pre-treatment was analysed. This was done to see if sample fragmentation influenced the EIS result. These experiments were performed on an Agilent 2100 Expert Bioanalyzer (Agilent Technologies; Palo Alto, Calif., USA). Samples of isolated bacterial gDNA were prepared by heating at 95° C. for 0, 1 or 5 mins in a variety of solutions including pure water and 2×SSC. Following treatment 1 μL of sample was loaded into individual wells on a DNA 500 Labchip kit (Agilent Technologies; Palo Alto, Calif., USA). Each chip contained 12 wells and was loaded as required prior to electrophoresis. Upon completion of the automated electrophoresis program the results were analysed using the proprietary software. This enabled the resolution and positioning of individual peaks and also allowed quantification of DNA by integration to find peak area.

(93) Chemical Structures of Spacer Molecules

(94) Spacer molecules were incorporated into the PNA probe in order to improve hybridisation efficiency at the electrode surface. The chemical structures of the AEEA (Probe 01) and AEEEA (Probe 02) ethylene glycol linkers were as follows:

(95) ##STR00006##
Results
qPCR and Quantification of gDNA Samples

(96) Samples of genomic DNA were prepared in two ways:

(97) 1) gDNA was extracted from a sample of MRSA at 108 cells/mL (1 McFarland standard) spiked into wound fluid. The obtained DNA was then serially 1:10 diluted to give a range of concentrations equivalent to 108 to 102 cells/mL.

(98) 2) MRSA was cultivated at 108 cells/mL in wound fluid and then 1:10 serially diluted in wound fluid to give a range of preparations ranging from 108 to 102 cells/mL. The DNA extraction process was performed on each concentration of MRSA.

(99) qPCR was then performed on the samples prepared using the two methods and it was found that the cycle threshold was lower and showed a greater degree of linearity from samples prepared using method 1. This meant that dilution of gDNA extracted from a culture of 108 cells/mL produced more reliable dilution series than by diluting cultures of MRSA and then performing a DNA extraction. results are shown in FIG. 13.

(100) To better understand any variation observed in EIS data, extracts of genomic DNA were quantified using a NanoDrop spectrophotometer (see Table 3). It can be seen that yields of MRSA genomic DNA showed considerable variation along with levels of recovered DNA from human wound fluid alone. The heterogeneous nature of the DNA extraction process is likely to contribute to variation observed in the EIS data. Efforts were made to ensure good reproducibility of data. As shown in FIG. 14 (FIG. 14 shows qPCR data demonstrating variation in gDNA yield upon extraction from 108 cells/mL MRSA in wound fluid) reproducibility was good from a single batch of wound fluid. Batch to batch variation was higher and this can be attributed to the variable nature of human wound fluid and other experimental factors such as aggregation of MRSA and variability inherent in the process of enzymatically digesting the MRSA cell wall with lysotaphin.

(101) TABLE-US-00003 TABLE 3 MRSA total DNA quantification using the NanoDrop 1. 06/01 2. 06/01 3. 06/01 4. 06/01 1. 18/01 2. 18/01 3. 18/01 4. 18/01 MRSA gDNA Quantification (Nanodrop) [ng/μL] 10.sup.8 1164.5 434.2 350.0 1164.5 340.8 263.3 701.4 529.0 10.sup.7 47.5 22.1 35.1 47.5 63.1 69.5 19.0 179.7 WF neat 79.0 47.2 81.2 79.0 188.4 378.6 99.3 — WF 10.sup.−1 6.5 4.3 71.3 6.5 16.3 16.7 26.3 — MRSA qPCR [Ct] 10.sup.8 10.93 12.10 11.63 — 10.7 9.7 10.09 9.96 10.sup.7 13.23 13.81 15.26 — 12.53 13.53 13.29 13.16 WF neat — — — — — — — — WF 10.sup.−1 — — — — — — — —
Prototype Potentiostat for Point of Care Testing

(102) With point of care testing in mind a prototype potentiostat was designed and assembled. The potentiostat was assembled with parts totalling less than US$200 and was able to measure phase and magnitude changes over a frequency range of 100,000 to 0.1 Hz. The system was initially evaluated using a fully complementary short artificial target and it was found that increases in charge transfer resistance following target addition were observable. FIG. 15 shows (A) an image of the prototype potentiostat, and (B) a Nyquist plot pre and 10 minutes post introduction of a 23 bp fully complementary oligonucleotide (1 μM).

(103) DNA Fragmentation

(104) FIGS. 16 A, B & C show bioanalyser data of MRSA gDNA following heat treatment at 95° C. for 0, 1 and 5 mins respectively. It can be seen that heat denaturation time coincided with the production of smaller fragments of DNA. Similar samples were also analysed by gel electrophoresis (FIG. 17) and whilst DNA fragmentation was observed from heat treated fragments sizing was not possible due to smearing of the sample.

Example 7—Further EIS Experiments

(105) Materials and Methods

(106) DNA oligonucleotides were purchased from Metabion (Martinsried, Germany). PNA oligonucleotides were ordered via Cambridge Research Biochemicals (Cleveland, UK) from Panagene (Daejeon, South Korea). PCR kit and DNeasy blood and tissue kit were purchased from Qiagen (Crawley, UK). Potassium ferricyanide, potassium ferrocyanide, sodium saline citrate (SSC), monosodium phosphate, disodium phosphate and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (Poole, UK). Lambda exonuclease (Epicentre Biotechnologies, Madison, Wis., USA) Deionised water was used throughout the study (>18 MΩ).

(107) TABLE-US-00004 TABLE 4 Sequences and structures of oligonucleotides used during the study. Oligo 3′ 5′ Sequence name Type Modif. Modif. 5′-3′ 1 P48 DNA — Thiol-C6 ACTAGGTGTTGG mecA TGAAGATATACC (SEQ ID NO: 1) 2 mecA DNA — — AAAATCGATGGT primer 1 AAAGGTTGGC (SEQ ID NO: 2) 3 mecA DNA — — AGTTCTGCAGTA primer 2 CCGGATTTGC (SEQ ID NO: 3) 4 PNA48 PNA — 0.6 nm ACTAGGTGTTGG Thiol-C6 TGAAGATATAC (SEQ ID NO: 4) 5 PNA PNA — 1.6 nm- ACTAGGTGTTGG 48_01 Thiol-C6- TGAAGATATAC AEEA (SEQ ID NO: 5) 6 PNA PNA — 3.8 nm- ACTAGGTGTTGG 48_02 Thiol-C11- TGAAGATATAC AEEEA (SEQ ID NO: 6) 7 PNA PNA — 2 nm- ACTAGGTGTTGG 48_02 Thiol- TGAAGATATAC (His).sub.6 (SEQ ID NO: 7)
DNA Extraction from S. Aureus

(108) Bacteria were sub cultured onto Columbia blood agar and incubated overnight at 37° C. in a 5% CO.sub.2 atmosphere. Cells were inoculated into saline and the optical density measured using a Densicheck (bioMerieux). This gave values in McFarland units, proportional to the cellular concentration of bacteria in the suspension. A bacterial cell suspension of approximately 108 cells/mL was produced in this way and ten-fold dilutions ranging down to 102 cells/mL prepared from this suspension. Real time PCR was performed to characterise the DNA yields from the dilution series.

(109) The bacterial cells were pelleted by centrifuging 1 mL of the suspension at 5000×g for 10 mins. The supernatant was discarded and the bacterial pellet re-suspended in 200 μL of enzymatic lysis buffer (2×TE buffer, 1.2% Triton X, 50 μg/mL Lysostaphin), before incubating for 30 mins at 37° C. 200 μL of bacterial lysate was added to 20 μL Proteinase K and DNA extracted using the bioMerieux NucliSens easyMAG automated platform. Guanidine thiocyanate was the active chaotropic agent in the lysis buffer, acting as a protein denaturant in the purification and extraction of nucleic acids from cellular material. The purified nucleic acid solution was then removed from the vessel without dislodging the magnetic silica pellet—DNA was eluted in 100 μL of water.

(110) Electrochemical Impedance Spectroscopy (EIS)

(111) Gold disk electrodes (2 mm diameter) were purchased from IJ Cambria Scientific (Carms, UK). Each solid gold working electrode was thoroughly pre-cleaned by mechanical polishing with 0.05 μm alumina powder (IJ Cambria Scientific (Carms, UK) for 1 min, rinsing with water and immersing in an ultrasonic water bath for 1 min (to eliminate any residual alumina) and finally cleaning for 10 min in piranha solution (6 mL concentrated H.sub.2SO.sub.4+2 mL 30% (v/v) H.sub.2O.sub.2 solution). Then the electrodes were thoroughly washed with water and dried under a stream of nitrogen. After cleaning, the gold disk electrodes were incubated with a solution of 1.5 μM thiol-modified PNA solution+30 μM mercaptohexanol in 50% (v/v) DMSO for 16 h at 30° C. Electrodes were rinsed in 50% (v/v) DMSO and incubated in 1 mM mercaptohexanol in 50% (v/v) DMSO for 1 h at 30° C. Then the electrodes were washed with 50% (v/v) DMSO and the EIS measurement buffer (0.1 mM K.sub.3[Fe(CN).sub.6]+0.1 mM K.sub.4[Fe(CN).sub.6]+10 mM phosphate buffer) for 2 h and 1 h respectively.

(112) EIS measurements in batch end point assays were performed using a three electrode system with an Ag/AgCl reference electrode and a platinum wire counter electrode (both from Metrohm (Runcorn, UK) connected to an Autolab potentiostat running FRA software (Metrohm, Runcorn, UK). EIS measurements were performed at a DC potential of 0.24 V with an amplitude of 10 mV rms using a frequency range between 100,000 Hz-0.1 Hz (15 frequencies) in 0.1 mM K.sub.3[Fe(CN).sub.6]+0.1 mM K.sub.4[Fe(CN).sub.6]+10 mM phosphate buffer. The DNA sample was prepared by mixing 45 μL of sample with 5 μL of 20×SSC and then heating at 95° C. for 5 mins, storing on ice for 2 mins and heating at 30° C. for 5 mins. The electrode was incubated with the sample for 2 h at 55° C. with shaking (650 rpm). Following incubation with sample, electrodes were washed with 2×SSC, 0.2×SSC and EIS measurement buffer for 10 mins in each. EIS measurements were performed pre and post hybridisation.

(113) The online assay was performed by recording continuous EIS measurements with a screen printed electrode. A single well from a Schott Nexterion 16-well self-adhesive superstructure (Stafford, UK) was cut out and fitted around the electrode in which 50 μL of EIS measurement buffer was present. The well was sealed with an adhesive lid from the Schott Nexterion 16-well self-adhesive superstructure kit (Stafford, UK). 45 μL of sample was mixed with 5 μL of 10×EIS measurement buffer and pre-treated by heating at 95° C. for 5 mins, storing on ice for 2 mins and heating at 30° C. for 5 mins. Once the sample was prepared the EIS measurement buffer was removed from the electrode surface and replaced with the 50 μL sample+measurement buffer solution. The adhesive lid was resealed and EIS measurements continued.

(114) Results and Discussion

(115) In a previous study a probe sequence for the mecA gene was identified and optimised for the impedimetric detection of a 550 base pair mecA PCR product. The probe (P48) was found to be most effective at binding the mecA sequence in a 5′ configuration in DNA form on a microarray system and in PNA form for EIS measurements.

(116) Direct Detection of Genomic DNA from MRSA in Batch End Point Assay Format

(117) EIS measurements were carried out pre and post hybridisation and recorded in the form of a Nyquist Plot (see FIG. 18) in order to obtain values for the charge transfer resistance (RCT). Following incubation with MRSA gDNA extracted from a suspension of 107 cells/mL it was found that significant increases in RCT were apparent (see FIG. 18). To obtain values for RCT, data was fitted using a Randles circuit (at the top of the Figure) and the fitting function within the FRA Autolab software. The uncertainty associated with fitting RCT was in the range of 6-12% for the reported experiments. For a particular concentration of gDNA the RCT value obtained post hybridisation was divided by the value obtained pre hybridisation. This approach towards expressing the data provided a measure of the signal increase which corrected for variation in the RCT starting values. Plots showing such data have y-axes denoted as “Signal Increase Ratio”. Similar approaches to representing hybridisation induced impedimetric increases have been employed in journal publications. Where used, standard deviation (S.D) is defined as the square root of the variance.

(118) Hybridisation Efficiency—Effect of Incorporating Different Spacers into the Probe Sequence

(119) In microarrays and other surface based DNA detection technologies, the kinetics of DNA hybridisation can be improved by enhancing accessibility of the surface probe to species in solution. The hybridisation kinetics are likely to be hindered with the probe sequence in close proximity to the electrode surface and therefore, three probes (01, 02 & 03) with identical sequence to the original (P48) but containing additional spacer schemes were tested for their response after hybridisation with MRSA gDNA at a concentration of 107 cells/mL. The details of the probe spacers are presented in Table 4. The AEEA abbreviation is used to represent a linker containing two ethylene glycol units and the AEEEA abbreviation to represent a linker containing three ethylene glycol units. The chemical structures of the spacer molecules are those already described above.

(120) FIG. 19 shows that of the four probes tested, probe P48_02 showed the greatest signal increase ratio following sample incubation. C11 spacers (spacers with 11 C atoms in their backbone) proved the most effective in this study when attempting to improve DNA binding kinetics at the solid-liquid interface. Enhancement of DNA binding kinetics with the C11 spacer is attributed to the formation of a more densely packed and better ordered mixed film and the likely explanation is thought to be due to increased van der Waals' forces from the C11 spacer. Also the use of a long PEG molecule in combination with C11 spacer allows good flexibility of the probe molecules and ensures the probe sequence protrudes out above the alkanethiol film which coats the electrode surface. For the rest of the study, probe P48_02 was employed as the recognition element on the electrode surface.

(121) Hybridisation Efficiency—Role of DNA Fragmentation in Assay Performance

(122) In these experiments, samples from bacteria cultured at 107 cells/mL were incubated with the electrode for 2 hours prior to washing and measurement. Accompanying gel electrophoresis experiments were performed where the degree of DNA fragmentation during sample denaturation was assessed. This was carried out by heating samples of MRSA gDNA (107 cells/mL) for time periods of 0, 1 and 5 minutes, at temperatures of either 75 or 95° C. in pure water or 2×SSC. It was found that observable fragmentation took place when the sample was heated in 2×SSC for 5 minutes at 95° C. (See FIGS. 20A & 20B). The MRSA genome is approximately 2.8 Mb. The untreated gDNA extracted from MRSA contained large fragments ranging from 1000 to 15000 bp and the fragments observed after heat denaturation for 5 mins were found to be average around 120 bp.

(123) From FIG. 20 it can be seen that denaturation of the genomic DNA for 5 mins at 95° C. in 2×SSC coincided with an increase in DNA fragmentation and an increase in the impedimetric signal. The role of DNA fragmentation has been assessed for glass microarrays with long hybridisation times but has hitherto not been investigated for the binding of genomic DNA for impedimetric detection. It is known that incubation of DNA at a high temperature such at 95° C. causes fragmentation of long strands of DNA and reduces PCR efficiency. Thermal DNA fragmentation has been shown to produce strands of less than 800 bp. The DNA obtained post heat denaturation in this test may be single stranded therefore making it appear shorter when sized post electrophoresis. It is believed that the 95° C. incubation fragmented the high molecular weight MRSA gDNA and this resulted in improved impedance signals. Having obtained an understanding of the roles of spacer choice and DNA fragmentation and with a capillary gel electrophoresis measurement which provided an approximate size for the DNA targets, an assay for MRSA gDNA was devised and evaluated.

(124) Development of a Batch End Point Assay for MRSA gDNA

(125) An MRSA batch end point assay was developed. A DNA denaturation time of 5 minutes was employed in order to achieve fragmentation and non-specific sequences of gDNA from E. coli and methicillin susceptible Staphylococcus aureus (MSSA) were also tested so that assay specificity could be evaluated. The concentrations of MRSA tested ranged from 103-108 cells/mL.

(126) FIG. 21A shows that it was possible to detect MRSA gDNA hybridisation having extracted the DNA from MRSA cells spiked into saline. Using a definition of the signal increases ratio from incubations of 0 MRSA cells/mL plus three standard deviations the L.O.D was 106 cells/mL. The significance of this result lies in the fact that it was possible to detect hybridisation of the mecA gene without performing PCR on the extracted DNA. The maximum concentration of the mecA gene from samples spiked at 106 cells/mL and extracted was ˜500 fM.

(127) To confirm the specificity of the assay for MRSA gDNA, incubation of probe modified electrodes was carried out in the presence of E. coli gDNA and MSSA gDNA at comparable concentrations to the MRSA tests. FIG. 21(B) presents signal increases arising from such incubations and it can be seen that these signal increases were not observed in the presence of E. coli and MSSA gDNA. Concentrations of 3-6 pM were equivalent to yields of DNA extracted from bacterial suspensions of 107 cells/mL.

(128) Online MRSA gDNA Detection from Samples Spiked into Human Wound Fluid and Specificity Tests.

(129) The ability to detect binding of gDNA in real time and in the presence of an interfering matrix would amount to a tremendous advantage in terms of a point-of-care test. With this in mind the assay was transferred from gold macrodisk electrodes to screen printed electrodes onto which sample introduction could take place at room temperature and in small volumes. A further advantage of screen printed electrodes is their price (<$3 each) which makes them an attractive component for a point of care test. In these experiments, EIS measurements were performed in a continuous fashion with a full Nyquist plot being produced approximately every two minutes. 100 μL of EIS measurement buffer was incubated on the electrode and after a series of baseline measurements was replaced with 100 μL of previously heat denatured MRSA gDNA (95° C. for 5 mins) or similarly treated human DNA extracted from wound fluid preparations. Charge transfer resistance was plotted versus time and it was possible to measure signal increase ratio at various time points following sample addition (FIG. 22) and thereby obtain binding isotherms associated with the process of DNA hybridisation (FIG. 23).

(130) From FIG. 22 it can be seen that MRSA gDNA spiked into and recovered from human wound fluid caused a much larger increase in the impedimetric signal than DNA samples extracted from a dilution series of human wound fluid. Therefore in this format it was possible to measure MRSA gDNA hybridisation above a background signal caused by extracted human DNA. FIG. 23 shows that when equal amounts of MRSA and E. coli gDNA were added to the sensor, it was possible to discriminate between specific and non-specific binding in this case. The significance of these results lies in the fact that MRSA detection was shown possible in an online test where detection times were much shorter. For example, the data in FIG. 22 show signal increases 10 minutes after sample addition while FIG. 23 shows that a divergence between the binding curves following addition of MRSA and E. coli 107 cells/mL was apparent only approximately 5 minutes after sample addition.

(131) In a wider context these results show that detection of MRSA gDNA was possible without electrode modification through the use of nanostructures or polymeric layers. Additionally, signal enhancement strategies such as gold nanoparticles and protein G have been employed as possible methods for detecting PCR product hybridisation by EIS. The relative simplicity of the electrode preparation process detailed here and the lack of protein or nanoparticle based signal amplification steps are an advantage over many other electrochemical detection schemes.

(132) The data on spacer choice and target fragmentation time show that a relationship between probe length, fragment length and EIS signal exists. The fragmentation data suggests target strand lengths of approximately 120 bp in length are responsible for the EIS signal increases. Much of the literature on EIS based nucleic acid detection reports results obtained with short (˜20 bp) artificial oligonucleotides.

(133) The relative simplicity of the current detection scheme (and the fact that a prototype portable potentiostat with good equivalence to a bench top potentiostat and is compatible with screen printed electrodes and which costs less than US $200) has already been produced, means that the assay is well placed for implementation in point-of-care scenarios.

CONCLUSIONS

(134) A sensitive and specific biosensor for the label free detection of MRSA has been demonstrated. The use of a PNA probe sequence allows sensitive detection of MRSA genomic DNA and the assay does not require a PCR amplification step. DNA fragmentation and distance of the probe sequence from the electrode surface are shown to be important factors in assay performance. Fragments were found to be around 120 bp in length, which are longer than the DNA sequences typically reported in EIS studies. Detection of MRSA gDNA was shown to be possible in a batch assay on macro gold electrodes and in an online format on screen printed electrodes. Also it was possible to make detections when DNA was extracted from an interfering matrix such as human wound fluid.