WO2018196975A1

WO2018196975A1 - Probe

Info

Publication number: WO2018196975A1
Application number: PCT/EP2017/060043
Authority: WO
Inventors: Sherif EL-KHAMISY; Sherif SHAWKY
Original assignee: University of Sheffield
Current assignee: University of Sheffield
Priority date: 2017-04-27
Filing date: 2017-04-27
Publication date: 2018-11-01
Anticipated expiration: 2019-10-27

Abstract

A capture ligand for an analyte comprising SEQ ID NO:1, or comprising an antisense primer of at least 25 bases complementary to SEQ ID NO:2, wherein the primer initiates hybridisation from any of nucleotides 5 to 20, 43 to 118, 156 to 238, 253 to 279 or 291 to 314 of SEQ ID NO:2. A probe for an analyte comprising this capture ligand and a probe comprising a metal nanoparticle and one or more capture ligand specifically for the indicator of disease conjugated thereto. Also assay kits and methods of detecting the presence of an analyte.

Description

Probe

The invention relates to capture ligands and to probes including these. In particular the invention relates to capture ligands and probes for analytes, to assay kits containing the probes, to a method of detecting an analyte. The early, rapid and inexpensive detection of disease is paramount to society. Key diseases in this spectrum include hepatitis C, a disease affecting approximately 3% of the global population, and cancer which affects around 40% of the global population at some point in their life. However, existing detection technologies are expensive, labour intensive and time consuming, posing significant limitations to their wide-scale exploitation, particularly in economically deprived populations.

Hepatitis C viral (HCV) infection usually progresses to fatty liver and hepatocellular carcinoma, posing significant health and economic challenges to the society. Since an approved vaccination against HCV is yet to be established, the prime combating strategies rely on newly developed medications coupled to robust and affordable means of early viral detection and quantification.

Currently, HCV diagnosis is achieved by serologic and Nucleic Acid Testings (NATs). A major drawback of the serological approach is the inability to detect acute infections and the associated complications of immunosuppression patients. NATs are based mainly on Real-Time PCR (RT-PCR), branched-DNA (b-DNA), and transcription-mediated Amplification (TMA). NATs are relatively expensive, labour intensive, and require adequately equipped labs, posing significant limitations to their point of care testing. Thus, alternative approaches for HCV RNA detection and quantification are urgently needed.

Gold nanoparticles, and other metal nanoparticles, have unique optical properties, originating from their strong Surface Plasmon Resonance (SPR) phenomenon. The SPR phenomenon is responsible for the intense colours of such nanoparticles. Thus, metal nanoparticles, such as gold nanoparticles, have been employed in many colorimetric assays for biological molecules. For example, Mirkin and co-workers were the first to develop a modified gold nanoparticle crosslinking method for the direct detection of nucleic acids (Elghanian et al., 1997; Larguinho et al., 2015). Despite the high sensitivity and specificity of this method, it requires firm temperature control for precise target detection. Li and co-workers developed a method for the direct detection of nucleic acids using unmodified gold nanoparticles (Li and Rothberg 2004a, 2005). The technique is based on the adsorption of single and double stranded nucleic acids onto the surface of gold nanoparticles. Despite its sensitivity and specificity, it requires precise control of the probe, salt, and gold nanoparticle concentrations.

A non-crosslinking method was introduced by Sato and co-workers (Sato et al., 2003), which is based on functionalizing gold nanoparticles with a single stranded thiol-modified probe. Depending on the ionic strength of the medium, the gold nanoparticles aggregate. However, the desired results are only achieved if the target is of the same length as the probe, limiting applicability. Baptista and co-workers improved this technique allowing the detection of long nucleic acids; however the Baptista technique remains dependent on either high ionic strength, or the pH, to induce gold nanoparticle aggregation (Baptista et al., 2008; Larguinho, Canto et al., 2015).

It would therefore be desirable to provide a detection method which is at least as accurate as current NATs techniques, whilst being quantitative, sensitive, rapid and inexpensive, with high specificity that is not dependent on solution environmental factors, such as pH. The invention is intended to overcome or ameliorate at least some aspects of this problem.

Accordingly, in a first aspect of the invention there is provided a capture ligand for an analyte comprising SEQ ID NO: l ( 5 ' -TACCACAAGGCCTTTCGCG ACCCAACACTACT-3 ' ) . Often the capture ligand will be thiolated, often with an alkanethiol, often at the 5'- terminus. The presence of the thiol allows for conjugation of the capture ligand to a substrate, improving the robustness of the capture ligand. The selection of an alkanethiol offers a chain flexibility which provides for folding of the capture ligand, and movement relative to any substrate to which it is conjugated. This improves "capture" and binding/hybridisation.

Typically, the analyte comprises an indicator of disease, often hepatitis C or cancer, often the indicator is an RNA sequence that codes for hepatitis C, such that the capture ligand binds to the RNA sequence that codes for hepatitis C. This provides for an extremely specific detection of hepatitis C.

In a second aspect of the invention there is provided a capture ligand comprising an antisense primer of at least 25 bases complementary to SEQ ID NO: 2 (5'- GCCAGCCCCCTGATGGGGGCGACACTCCACCATGAATCACTCCCCTGTGAGGAACTACTGTCTT CACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTC CCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGT CCTTTCTTGGATAAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCAAGACTGCTAGCC GAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCG GGAGGTCTCGTAGACCGTGCACC-3'), wherein the primer initiates hybridisation from any of nucleotides 5 to 20, 43 to 118, 156 to 238, 253 to 279 or 291 to 314 of SEQ ID NO: 2, preferably wherein the primer initiates hybridisation from any of nucleotides 172 to 229 or 253 to 279 of SEQ ID NO : 2. The primer therefore initiates hybridisation in one of the 5' UTR loops. Any of the loops may be suitable, however, often the primer may initiate hybridisation in loop Hid or Illb, as these loops are highly conserved within the 5' UTR sequence. Often the primer will be in the range 25 - 45 bases, often in the range 30 - 40. Often, the capture ligand will have at least 80% homology with the RNA sequence that codes for hepatitis C. However, the homology may be in the range 80, 85, 90, 95, 97 % - 100% homology to the sequence. Often the capture ligand comprises an antisense primer of 32 bases complementary to the highly conserved region in the HCV RNA 5'-untranslated region (5'UTR) in almost all genotypes and subtypes.

Often the analyte comprises cell-free RNA, to improve sensitivity of detection. Often the cell-free RNA is isolated from blood, urine, cerebro-spinal fluid, serum, saliva or combinations thereof. Therefore, the sample containing the analyte will be one of these substances, most often the sample containing the analyte will be blood.

In a third aspect of the invention there is provided a probe for an analyte, comprising a metal nanoparticle and one or more of the capture ligand of the first or second aspects of the invention conjugated thereto.

In a fourth aspect of the invention there is provided a probe for an indicator of disease (often hepatitis C or cancer), the probe comprising a metal nanoparticle and one or more capture ligands specific for the indicator of hepatitis C or cancer carcinoma conjugated thereto. Often the indicator will be for hepatitis C or rectal cancer carcinoma, often the indicator will be for hepatitis C. The use of the probes of the third and fourth aspects of the invention allow the minimisation of the factors that may have a deleterious effect on the output results, whilst enhancing the specificity, sensitivity and detection limit. Most importantly, this approach allows quantitative detection of the analyte to be achieved in clinical samples, in short time and with reasonable cost, and it could be easily adapted for full automation. Furthermore, the probe can be used with other RNA transcripts, such as those which are indicators of cancer, illustrating its broad utility as a detection technology in particular for nucleic acids. In addition, the probe can operate across a wide pH range, resulting in an assay which is pH independent.

Where the indicator is for a cancer carcinoma, the capture ligand is often is selected from a transcript of TOPI (Topoisomerase 1), TOP2 (Topoisomerase 2), TDP1 (Tyrosyl- DNA phosphodiesterase 1) and TDP2 (Tyrosyl-DNA phosphodiesterase 2), as these are strong indicators for, in particular, rectal cancer carcinoma. As with capture ligands for HCV RNA, the transcript for cancer carcinoma is often thiolated, and thiolation is often with an alkanethiol.

Often, the metal nanoparticle of the probe is surface functionalised, generally to induce a negatively charged surface to the metal nanoparticle for interaction with oppositely charged nanoparticles during use of the probe. Often, the surface functionalisation comprises functionalisation with a carboxylic acid, and often the carboxylic acid is selected from citric acid, adipic acid, malonic acid, succinic acid, and combinations thereof. In many cases the carboxylic acid will be selected from citric acid. The use of carboxylic acids creates a negative charge on the surface of the nanoparticle, without preventing conjugation with the capture ligands. This is because, where the capture ligand is thiolated, the thiol group has a greater affinity for the surface of the metal than the carboxylic acid, allowing strong covalent bonds to be formed between the capture ligand and the surface of the metal nanoparticle. This provides a probe with high stability against high salt, and with reduced aggregation. The use of citric acid offers a stable, readily available option for achieving these benefits.

It may be the case that the metal nanoparticle is a particle with a good zeta-potential, often in the range -40 to -60 mV, or -45 to -55 mV. Often the particle will be a gold nanoparticle, although other metal particles, such as silver, platinum, palladium or combinations thereof may also be present. It may be the case that the surface functionalised metal nanoparticle has size in the range 15 - 30 nm, 15 - 25 nm, often around 20±2 nm . At these sizes, the nanoparticle can conjugate to in the range 80 - 160 capture ligands per metal nanoparticle. As such, the nanoparticle may be conjugated to in the range 80 - 160, 100 - 140, or often around 120 capture ligands. At these levels, there are enough capture ligands to provide good sensitivity and a strong response, without introducing steric hindrance that would prevent binding to the analyte.

In a fifth aspect of the invention there is provided an assay kit for an analyte comprising a probe according to the fourth aspect of the invention and a positively charged metal nanoparticle. The positively charged metal nanoparticle induces aggregation of the probe in the absence of the analyte, causing a colour change. In the presence of the analyte, the positively charged metal nanoparticles interact with negative charges on the analyte, preventing aggregation. In the absence of aggregation no colour change is observed.

Often, the positively charged metal nanoparticle is a gold nanoparticle, although other metals may also be used, as described above for the probe. Often the positively charged metal nanoparticle will be selected from metals with good zeta-potentials, often in the ranges described above for the probe nanoparticles. It will also often be the case that the positively charged metal nanoparticles are of roughly uniform size.

Often the positively charged metal nanoparticle is surface modified. Where surface modification is present, this may include cysteamine and/or cetrimonium bromide (CTAB). Often, surface modification includes cysteamine because this compound offers exceptional reliability of results and clear signals providing easy quantification of results. Without being bound by theory, this is believed to be because cysteamine has a short chain length, allowing good distribution of the positively charged metal nanoparticles along the bound analyte backbone.

It may be the case that a ratio of probe to positively charged metal nanoparticle in the kit is in the range 1 : 1 - 1 : 3, 1 : 1.1 - 1.25 or often around 1 : 2, such that there is more positively charged metal nanoparticle present than probe.

In a sixth aspect of the invention there is provided a method of detecting the presence of an analyte, the method comprising the steps of: a. providing a probe for an indicator of disease, the probe comprising a metal nanoparticle and one or more capture ligands specific for the indicator of disease conjugated thereto; b. contacting the probe with a sample to allow binding of the analyte with the capture ligands; c. providing a positively charged metal nanoparticle; and d. observing a colour of the sample.

The indicator of disease will often be hepatitis C, or cancer, most often hepatitis C.

The method may include the additional step of heating the probe and sample, prior to provision of the positively charged metal nanoparticle. The heating will often denature the capture ligand and/or analyte. In the absence of this heating step, the risk of false negatives is increased as aggregation on addition of the positively charged metal nanoparticle can occasionally be observed even in the presence of the analyte. This is believed to be as a result of interaction between any surface modification on the probe and the capture ligands, particularly between acidic groups on the surface modification and nitrogenous groups on the capture ligand. Heating the sample prevents this aggregation. In view of this, the probe and sample may be heated to a temperature in the range 90 - 100°C, often in the range 95 - 98°C. Often heating will be for a time in the range 1 - 5 minutes, or 1 - 10 minutes.

Often, after heating, the probe and sample are incubated at room temperature, this allows for the analyte to unwind such that the probe may bind to it more effectively. As used herein, the term "room temperature" is intended to mean ambient temperature, typically in the temperature range 18 - 25°C. When an incubation period is provided, the accuracy of the assay is significantly improved. Often the incubation period is for a time in the range 1 - 15 minutes, often 5 - 10 minutes. Incubations of this time scale are sufficient to maximise binding interactions, without undue delay in obtaining the test results.

After heating and incubation, where used, the positively charged nanoparticle is provided. This either stabilises the probe-analyte combination, and no colour change is observed, or it causes aggregation of the probe, causing a colour change. The colour change is clearly visible to the naked eye, and is usually a change in the colour of the solution from an initial red colour to a blue colour. Therefore, there is no requirement for colour charts to determine the presence/absence of analyte. As a result, the presence of the analyte can indicated by a red colour after the addition of the positively charged metal nanoparticles and/or the absence of the analyte by a blue colour. As used herein, the term "red" is intended to refer to light emitted at wavelengths in the range 590 - 700 nm, and the term "blue" is intended to refer to light at wavelengths in the range 400 - 500 nm.

If a quantitative determination of analyte concentration is required, this will typically be achieved using spectroscopic methods, based upon a calibration of the intensity of the Amax absorption signal; typically, for gold nanoparticles, this will be in the range 510 - 540 nm, often in the range 520 - 530 nm.

Typically, if colour change is to occur, this will be within 30 minutes of adding the positively charged metal nanoparticle. Often, this will be after a time in the range 10 - 25 minutes, or 15 - 20 minutes, most often within 25 minutes.

Often the analyte comprises RNA. Where this is the case, the sample may be prepared using a membrane-based purification system, the purification system may include a DNase treatment step. In some cases the purification system will be the SV Total RNA Isolation System (Promega). This system is a membrane-based purification system that provides a fast and simple technique for preparation of intact total RNA from tissues, cultured cells and white blood cells. This purification system substantially reduces genomic DNA contamination. The method may be completed using traditional laboratory techniques, such as centrifuges, and spectrophotometers (often hand held), or it may be carried out using "lab-on-a-chip" techniques.

There is therefore provided a capture ligand for a RNA sequence that codes for hepatitis C comprising at least one of:

SEQ ID NO: l, wherein SEQ ID NO: l is thiolated; and an antisense primer of at least 25 bases complementary to SEQ ID NO: 2, wherein the primer initiates hybridisation from any of nucleotides 5 to 20, 43 to 118, 156 to 238, 253 to 279 or 291 to 314 of SEQ ID NO: 2, preferably nucleotides 172 to 229 or 253 to 279 of SEQ ID NO: 2.

The capture ligand having at least 80% homology with the RNA sequence that codes for hepatitis C, wherein the RNA is cell-free and is isolated from serum.

There is additionally provided a probe for an analyte, comprising a citrate functionalised gold nanoparticle and one or more of the capture ligands described above conjugated thereto. Alternatively, the probe may comprise a citrate functionalised gold nanoparticle and one or more capture ligands specific for the RNA sequence that codes for hepatitis C conjugated thereto. The probe may be used in an assay kit, the kit further comprising a cysteamine surface modified gold nanoparticle. The kit, probe and capture ligands being used in method of detecting the presence of RNA that codes for hepatitis C, the method comprising the steps of: a. providing a probe for RNA that codes for hepatitis C, the probe comprising a citrate modified gold nanoparticle and one or more capture ligands specific for the RNA that codes for hepatitis C conjugated thereto; b. contacting the probe with a sample to allow binding of the HCV RNA with the capture ligands; c. heating the probe and sample to a temperature in the range 90 - 100°C for a time in the range 1 - 5 minutes to denature the HCV RNA and the capture ligand; d. incubating the probe and sample at a temperature in the range 18 - 25°C for a time period in the range 1 - 15 minutes; e. providing a cysteamine surface modified gold nanoparticle; and f. observing a colour of the sample wherein the presence of the analyte is indicated by a red colour and/or the absence of the analyte by a blue colour.

The sample may be prepared using a membrane-based purification system wherein the purification system includes a DNase treatment step. Unless otherwise stated, each of the integers described may be used in combination with any other integer as would be understood by the person skilled in the art. Further, although all aspects of the invention preferably "comprise" the features described in relation to that aspect, it is specifically envisaged that they may "consist" or "consist essentially" of those features outlined in the claims. In addition, all terms, unless specifically defined herein, are intended to be given their commonly understood meaning in the art.

Further, in the discussion of the invention, unless stated to the contrary, the disclosure of alternative values for the upper or lower limit of the permitted range of a parameter, is to be construed as an implied statement that each intermediate value of said parameter, lying between the smaller and greater of the alternatives, is itself also disclosed as a possible value for the parameter.

In addition, unless otherwise stated, all numerical values appearing in this application are to be understood as being modified by the term "about".

In order that the invention may be more readily understood, it will be described further with reference to the figures and to the specific examples hereinafter.

Figure 1 is a schematic illustration depicting the assay principle and procedures;

Figure 2 shows the characterization of citrate-modified gold nanoparticies where (a) shows a TEM image of the citrate-modified gold nanoparticies, (b) and (c) are spectra of the citrate-modified gold nanoparticies showing SPR Ama_X at approximately 520 nm; Figure 3 shows the characterization of CTAB and cysteamine surface modified gold nanoparticies where (a) shows a TEM image of CTAB gold nanoparticies showing the nanoparticies appearing as clusters surrounded or internalized within a shell, (b) is a TEM image showing cysteamine gold nanoparticies, and (c) provides the extinction spectra of the cysteamine nanoparticies and CTAB nanoparticies, with Amax of 528 and 526 nm respectively;

Figure 4 shows (a) the size of the CTAB surface modified gold nanoparticies and (b) and their charge; Figure 5 shows the analysis of HCV clinical samples using cysteamine and CTAB surface modified gold nanoparticles where (a) is an extinction spectra of positive and negative HCV samples detected by the cysteamine and CTAB surface modified gold nanoparticles, (b) a photograph showing the change in colour using CTAB surface modified gold nanoparticles, (c) a photograph showing the change in colour using cysteamine surface modified gold nanoparticles, and (d) assay results when performed on HCV positive sample with and without the addition of Ribonuclease A (RNase A);

Figure 6 show UV-Vis absorption spectra of the gold nanoparticles in the presence of different messenger RNAs (transcripts) extracted from human colon cell lines of different concentrations for each transcript, after performing the assay. The concentrations are expressed in nanogram as determined by their absorbance at 260 nm. TOPOl: Topoisomerase 1, TOP02: Topoisomerase 2alfa, TDP1: Tyrosyl- DNA phosphodiesterases 1, and TDP2: Tyrosyl- DNA phosphodiesterases 2;

Figure 7 summarises the size and zeta potential of the probe and positively charged nanoparticles of the assay, and a comparison between the Real Time PCR and the assay where (a) shows charges of all gold nanoparticles prepared, (b) size of the different gold nanoparticles and the HCV positive & negative samples, (c) the mean viral loads (IU/ml) and standard deviations for the samples tested using the assay ( 15203 ± 1898) and the RT-PCR (15203 ± 1898) respectively, (d) sensitivity and specificity of the assay, (e) time taken to complete the assay, and (f) cost per sample for the assay compared to RT-PCR, cost including all materials, chemicals and plastics used for the assays, and the RNA extraction cost; and

Figure 8 shows the characterization of the probe-analyte (HCV RNA) solution where (a) is TEM images for HCV positive samples after performing the assay showing the distribution of the nanoparticles (black spheres) along and within the RNA molecules and in solution, (b) is TEM images showing the aggregation of the nanoparticles in absence of HCV RNA, (c) is TEM images for another HCV positive sample after performing the assay, which is consistent with images in (a) and (d) shows the sample of (c) after being subjected to the developed assay after treatment with Ribonuclease A.

Assay

As discussed, a novel quantitative colorimetric assay has been prepared, and tested for the direct detection of unamplified HCV RNA in clinical samples and DNA repair transcripts from cell lines. Stability or aggregation of the probes is aided by the use of cationic gold nanoparticies, making the assay less sensitive to ionic strength or pH, which limits false positives. The assay is simple with a turnover time of ~30 min including RNA extraction, sensitive (93.3%), has high specificity, with a low detection limit (4.57 IU/μΙ ), is cost effective and could readily be adopted for full automation. Figure 1 shows the method described.

(1) Citrate capped gold nanoparticies, were functionalized with thiolated HCV RNA specific capture ligands forming probes.

(2) The probe was then mixed with the RNA sample and heated at 95 °C for 3 minutes.

(3) Right panel; the HCV viral RNA was hybridized (bound) to the probe by sequence complimentarily. Left panel; no HCV RNA is present, thus no hybridization takes place.

The mixture incubated at room temperature.

(4) The positively charged cysteamine modified gold nanoparticies were added.

(5) Right panel; in the presence of HCV RNA, the mixture solution remains red, reflecting the dispersion of the gold nanoparticies onto the folded HCV RNA, thereby protecting the probes from aggregation by the cysteamine modified gold nanoparticies. Left panel; in the absence of a complementary target, the cysteamine modified gold nanoparticies bind to the capture ligands phosphate backbone electrostatically, thereby reducing the inter- particle distance between the probes and the cysteamine modified gold nanoparticies. This results in aggregation and colour change from red to blue.

Materials and methods Chemicals and equipment

Hydrogen tetrachloroa urate (III) trihydrate (HAuCI₄-3H₂0) >99%, Dithiothrietol, Dibasic and mono basic phosphate, Sodium dodecyl sulfate (SDS), Sodium Chloride, Sodium borohydride, Hexadecyltrimethyl ammonium bromide (CTAB), Tri-sodium citrate dehydrate, and Ribonuclease A were purchased from Sigma-Aldrich. Cysteamine (2- Mercaptoethylemine HCI) >98% was purchased from Acros Organics. SV-Total RNA isolation system was purchased from Promega. Artus HCV RG RT-PCR Kit was purchased from Qiagen. RPMI-1640 medium, L-glutamine, Penicillin/ streptomycin was purchased from Lonza, while fetal bovine serum from Gibco. NAP-5 columns (illustra NAP-5) were from GE Healthcare. UV-vis spectra were recorded with Eppendorf Bio-spectrophotometer basic. A zetasizer ZC system (Malvern Instrument Ltd., Zeta sizer Nano series, UK) was used for size and potential measurements. High resolution transmission electron microscope (HRTEM, JEM- 2100) was used for imaging Serum sample collection

Samples were collected following ethical approval and informed consent of all subjects. HCV infected serum samples were collected (n=28), which included 23 samples with chronic HCV infection and 5 samples with acute HCV infection. Healthy volunteers provided 17 serum samples. Rapid HCV and Hepatitis B virus (HBV) antibody tests were performed for all samples. Only one sample was HBV positive. Real-time PCR was conducted for all the samples using Artus HCV RG RT-PCR Kit according to the manufacturers' instructions.

Synthesis, functionalization and characterization of citrate

Citrate modified gold nanoparticles were prepared by the traditional sodium citrate reduction method of Gold (III) chloride (Hill and Mirkin, 2006; Turkevich, 1985a, 1985b). Transmission Electron Microscope (TEM), UV-Vis. spectroscopy and Dynamic light scattering (DLS) were used for characterisation. The citrate modified gold nanoparticles were functionalized each with an alkanethiol modified RNA target specific capture ligand using well known salt aging process (Hurst et al., 2006). Functionalization of the alkanethiol capture ligands followed the Hill and Mirkin method (Hill and Mirkin, 2006). The capture ligand is an antisense primer of 32 bases complementary to the highly conserved region in the HCV RNA 5'untranslated region (5'UTR) in almost all genotypes and subtypes. The probe (5'-TACCACAAGGCCTTTCGCGACCCAACACTACT'-3) was alkane-thiol modified at its 5'-terminus. In parallel reactions, functionalization was performed for Topoisomerase 1 & 2 (TOPI and & TOP2), Tyrosyl- DNA phosphodiesterases 1 & 2 (TDP1 and TDP2) transcripts, by alkanethiol specific probes for each transcript extracted from Rectal Cancer carcinoma (RKOs) cell lines. Each transcript specific probe was functionalized to the gold nanoparticle solution separately.

The morphology of these particles was identified by TEM analysis. As-synthesized gold nanoparticles were spherical and uniformly distributed with average diameter of ~20 nm (Figure 2a) and average charge of -48 mv. The HCV probe was synthesized by functionalization of the citrate gold nanoparticles with the alkanethiol modified capture ligand. TEM analysis of the probe revealed an increase in the size of the citrate gold nanoparticles from ~20 nm to ~38 nm (Figure 2b), which was confirmed by DLS with a corresponding reduction of surface charge from -48 mv to -27 mV. A spectrum of the as-synthesized gold nanoparticles revealed a Amax at 520 nm, while the probe showed a slight red shift to 530 nm (Figure 2c). The red shift was accompanied by a noticeable absorption broadening upon cysteamine gold nanoparticle induced aggregation of the probe (Figure 2c). The molar concentration of gold nanoparticles was calculated to be 4 nM with ~2.436x l0¹² nanoparticles per ml (Liu et al., 2007). This data demonstrates conjugation of probes to the particles and confirms the ability of cysteamine gold nanoparticles to induce aggregation of the probe.

The probe amount and density conjugated to citrate gold nanoparticles were measured. At high amounts, steric hindrance at the nanoparticle surface takes place, which results in electrostatic repulsion between the analyte and the probe, and thus no hybridization/binding takes place (Doria et al., 2010). It was estimated that ~ 1.44 nmol capture ligands were conjugated to the gold nanoparticles. Accordingly, the amount of capture ligands per one nanoparticle was ~ 120, which is in agreement with previously reported functionalization methods (Hurst et al., 2006).

Synthesis and characterization of Cysteamine and CTAB modified gold nanoparticles Cysteamine modified gold nanoparticles were synthesized using the sodium borohydride reduction method as described by Kim (Kim et al., 2009). The cysteamine gold nanoparticles were characterized using TEM, DLS, and spectrophotometrically.

CTAB modified gold nanoparticles were synthesised following the established seed- mediated growth using two solutions (El-Sayed et al., 2006; Huang et al., 2007b; Li et al., 2013; Scarabelli et al., 2015). Herein, CTAB modified gold nanoparticles were synthesized using only the seed solution in a single-phase reaction without the growth solution. In a typical experiment, 3.7 ml of 0.2 M aqueous solution of CTAB was mixed with 20 μΙ of 1 mM gold chloride under vigorous stirring at 50 °C. Then, 1 ml of ice-cold 10 mM sodium borohydride was added in three portions at 20 min intervals. The solution colour changed from yellow to light brown after the first addition of the borohydride and colour intensity increased after each addition. Stirring was continued for 3 hours at 50 °C and then stored at room temperature, in the dark, for 2 days. A clear pink-coloured solution was obtained after two days, which was characterised using the methods described above for the other gold nanoparticles. CTAB and cysteamine gold nanoparticle morphology were characterized using TEM (Figure 3a and 3b, respectively). Cysteamine gold nanoparticles were spherical and uniformly distributed with an approximate size of 40 nm and a Amax of ~528 nm (Figure 3c), with an average positive charge of +39. The Molar concentration was calculated as described (Liu et al., 2007) and found to be ~0.5 nM with ~3.04x lOⁿ nanoparticles per ml. The charge and size of CTAB gold nanoparticles were + 100 mv and 30 nm, respectively (Figures 4a and 4b). CTAB gold nanoparticle extinction spectrum revealed one sharp peak in the visible region with Amax at 526 nm, suggesting spherical nanoparticles with uniform size (Figure 3c). However, their TEM analysis (Figure 3a) showed a core/shell like structure of many gold nanoparticles coated within a shell. This may be due to nanoparticulate seed formation followed by internalization within the CTAB micelle. If the seeds are formed in the presence of the high concentration of CTAB and at a high temperature of reaction, the CTAB micelles are stabilised and the particles do not aggregate thereby allowing a shell like structure to form (Figure 3a). Moreover, the addition rate of the reductant (3 times at 1 h intervals) and its high concentration led to complete reduction of all the gold ion states to gold metal. Indeed, the high reaction temperature affects the final gold nanoparticulate shape and size (Scarabelli et al., 2015). Thus, the high temperature employed here and long growth time (2 days) led to further growth of the traditional seed particles into bigger nanoparticles and prevented the CTAB molecules from crystallization.

Selection of cysteamine gold nanoparticles for the assay

It was found that cysteamine surface modified nanoparticles gave excellent results regardless of the cysteamine nanoparticle or probe concentration. The best results were obtained when the concentration of cysteamine nanoparticles was twice the probe concentration, although reliable results were obtained over a range of relative concentrations. Moreover, the spectrum of samples following cysteamine surface modified nanoparticles had sharp peaks and showed a clear difference between the positive and negative samples. This allowed a linear relation to be devised and thus enabled quantitative detection of RNA. CTAB surface modified particles were also tested, a comparison between the two cationic gold nanoparticles is shown in Figures 5a - 5d which display the absorption peaks and colour intensity of the assay using each of the two cationic surface modified nanoparticles. In figure 5a, the extinction spectra of positive and negative HCV samples detected by the cysteamine and CTAB nanoparticles are shown. There is a difference in the peak intensity between the two cationic nanoparticles, specifically, cysteamine nanoparticles showed better SPR and well defined peak than the CTAB nanoparticles. Figure 5b is a photograph showing the change in colour using CTAB nanoparticles in the assay. Whilst they may be observed, the colours of both the positive and negative samples are faint relative to the results of figure 5c, where cysteamine nanoparticles were used. These results indicate that the use of cysteamine surface-modified gold nanoparticles may provide an assay where the results are easier to interpret. This may be because cysteamine is a smaller molecule than CTAB, and this may have allowed better distribution on the folded HCV RNA backbone.

Figure 5d shows the results of the assay when performed on a HCV positive sample with and without the addition of Ribonuclease A (RNase A). The tube on the right shows faint red colour indicating the presence of HCV RNA (the original colour being more intense, but sample dilution to allow a more direct comparison between tubes reduced this intensity). The RNase A treated tube on the left was nearly colourless indicating the absence of RNA. The difference in colour confirms that the analyte has an influence on the stability of the probe and cationic nanoparticle solution. HCV RNA extraction evaluation

HCV RNA extraction was performed using Promega SV-total RNA isolation system according to the modified HCV extraction manufacturer's protocol (Otto et al., 1998).

Total RNA Promega Kit was chosen for RNA extraction because it includes a DNase treatment step, which eliminates DNA from the final product. As DNA can lead to false positive results, the provision of pure RNA improves reproducibility.

Cell lines and total RNA extraction

RKOs cancer cell lines (Homo sapiens, Tissue: colon, Carcinoma) were cultured at 37 °C and 5% CO2 in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 1% L- glutamine and 1% penicillin/streptomycin. Total RNA extraction was conducted using Promega SV-total RNA isolation system following manufacture instructions and each transcript was further purified by homemade magnetic nanoparticles (Eissa et al., 2014; Shawky et al., 2014), to enhance the purity of the extracted transcript, and exclude the interference of other RNA molecules from the cell lines.

It was determined whether the assay could be applied to cell lines. Four RNA transcripts implicated in the repair of protein-linked DNA breaks were tested using probes with capture ligands specific for each transcript (Ashour et al., 2015, Elsayed et al., 2016, Meisenberg et al., 2016). In agreement with HCV detection, the results of the additional four transcripts confirm the broad utility of the assay technology in RNA detection (Figure 6). A summary of the size and charge of nanoparticles. As shown in Figure 6, the spectral behaviour is quite similar to that of the HCV RNA. The Amax decreases and shifts to longer wavelength as the concentration decreases. According to the shown spectral behaviour, the developed assay could be used in the detection and quantification of any nucleic acid. Colorimetric/spectrophotometric gold nanoparticulate assay for RNA detection

The assay was performed by mixing 5 μΙ of the probe with 10 μΙ of RNA containing analyte, heating at 95°C for 3 minutes, and incubating at room temperature for 5-10 minutes. Positively charged gold nanoparticles (10 μΙ) were then added to the solution and mixed well. Solution colour was developed immediately and observed by naked eye while mixing. The colour was stable for at least 30 minutes. Solutions were scanned from 400 nm to 750 nm using spectrophotometer.

To confirm that the colour change is due to RNA solely, the assay was performed on two HCV positive samples before and after digestion with Ribonuclease A enzyme. Each sample was divided into two portions, of which one portion was treated with the enzyme by adding 10 μΙ of 10 mg/ml Ribonuclease A to 35 μΙ of HCV RNA and then incubated for 20 minutes at room temperature. After completion of the digestion process, the assay was performed on both samples. The colour of the two samples was then observed and samples were also analysed using TEM.

Quantification of HCV RNA using real-time PCR and the developed assay As the concentration of the RNA increased the probe Amax height at wavelength 530 nm proportionally increased, representing the non-aggregated combination of the probes, analyte and cationic gold nanoparticles. The opposite occurred as the RNA concentration decreased, the probe Amax height declined with concomitant shift and broadness in the peak to a higher wavelength, indicating an increase in aggregated gold nanoparticles population. Thus, at high RNA concentration, the ratio of the non-aggregated/aggregated is high, indicating the predominance of the dispersed probes and their stability and vice versa. Results were compared to RT-PCR and showed a concordance of ~96.3%, indicating excellent reliability.

HCV RNA viral load was determined using Artus Qiagen HCV Realtime RT-PCR Kit, used in accordance with the manufacturer's instructions, for all sera samples. Quantification of HCV RNA using the assay was performed by preparing serial dilutions of HCV RNA concentration (10-1200 IU/μΙ), and application of the assay. The spectral absorbance for each concentration was scanned spectrophotometrically in duplicate, and the ratio of the non-aggregated nanoparticles at A₅3o to the aggregated nanoparticles at A₆so (A₅3o/A₆5o) was recorded and used to generate the standard curve, in which the A₅3o/A₆5o ratio was plotted against the viral RNA log concentration. In all HCV RNA samples the viral load was determined by the assay using the generated standard curve, from their respective A₅3o/A₆5o ratios, using the equation generated by the standard curve. The results were expressed in IU/μΙ and converted to IU/ml by (IU^I*RNA elution volume in μΙ/serum volume in μΙ). Comparison between the developed assay and the Real-Time PCR viral load is shown in Figures 7c-f. The receiver operating characteristic curve (ROC curve) for the assay was generated to determine the specificity, sensitivity, and the detection limit, using SPSS software (IBM, SPSS Statistics, version 20 package). The detection limit was further confirmed by performing serial dilutions of HCV sample down to 1 IU/μΙ. Each dilution was tested by the assay until aggregation occurred starting from about 4 IU/μΙ.

Figure 7(a) shows that HCV negative samples have positive charge while HCV positive samples have negative charge. Figure 7(b) shows a significant size increase for the negative samples, this size increase confirms the aggregation of the nanoparticles. Figure 7(c) shows the mean viral loads (IU/ml) and standard deviations for the samples tested using the assay as 15203 ± 1898 and RT-PCR 15203 ± 1898 respectively. The complete identity of these figures shows that they have the ability to detect the same viral loads with the same level of accuracy. Figure 7(d) shows a comparison between the sensitivity and specificity of the assay and PCR techniques. The assay showed a sensitivity and specificity of 93.3% and 100% respectively, while PCR showed a 96.8% sensitivity and 100% specificity. The similarity of these percentages again shows that the assay is of comparable accuracy to existing PCT techniques. Figure 7(e) compares the overall time of the assay for one sample relative to the use of PCR. The assay can be completed in ~30 minutes compared to 230 minutes for the PCR. Figure 7(f) compares the cost per sample for the assay relative to existing PCT techniques. The cost differential at the time of calculation was $4.5 for the assay relative to $33 for a PCR test.

Charge and size of different gold nanoparticles and comparison between realtime PCR and the assay For HCV positive samples the charge was negative indicating distribution and spreading of cationic gold nanoparticles along the RNA molecules, thereby preventing the aggregation of the probe (Figure 7a). In contrast, HCV negative samples (aggregated nanoparticles) were positively charged indicating the alignment of the cationic gold nanoparticles onto the probe backbone, and reduction of the inter-particle distance between the probes and the cationic nanoparticles (Figure 7a). This was associated with a reduction in inter-particle distance, leading to an increase in the size to ~900 nm (Figure 7b). However, the HCV positive sample size increased to only ~ 190 nm (Figure 7b), which is probably due to differences in the amount of the dispersed to non- dispersed nanoparticles, which depends mainly on the RNA concentration of the tested sample. Assay TEM analysis for HCV positive and HCV negative samples, and Ribonuclease A treated sample before and after digestion

TEM analysis was conducted immediately after performing the assay for both HCV positive and negative samples. Moreover, it was performed on the Ribonuclease treated and mock-treated sample to verify that RNA folding contributes to probe stability. Factors affecting assay performance

The assay reproducibility, sensitivity and specificity for nucleic acid analytes are often affected by:

(1) probe concentration and type (in this case potentially probe and cationic nanoparticle concentration), (2) pH of the reaction mixture,

(3) temperature, and

(4) nucleic acid folding (in this case RNA folding).

The probe density of ~ 120 probes per gold nanoparticles (nanoparticle size of roughly 20 nm) improves the probes stability against aggregation and provides sufficient levels of capture ligand for proper probe/analyte binding.

Solution pH had no impact on the assay. The non-pH dependency of the assay is advantageous as the pH dependency of existing tests can lead to false-negative results.

The last two parameters which often affect assay performance are reaction temperature and nucleic acid folding. It was found that with this assay improved results could be obtained where the probe-analyte mixture was heated. This reduced aggregation of the probe-analyte solution on addition of the cationic nanoparticles, because the electrostatic interaction within the probe (particularly between the capture ligand and the surface modification of the probe) is reduced. By reducing the electrostatic interaction, stronger bonds could be formed with the analyte. A brief denaturation step was therefore conducted for the probe-analyte (RNA) mixture and prior to the addition of the cationic gold nanoparticles. This allowed for the unfolding of the RNA tertiary structure and subsequent hybridization with its complementary probes.

The mixture was then incubated at room temperature prior to the addition of the cationic nanoparticles. This incubation period improved reliability of the assay as it allowed for RNA hybridization to the probe and subsequent RNA re-folding to adopt its most thermodynamically stable form before addition of the cationic nanoparticles.

TEM results for HCV positive and negative samples (Figures 8a and 8b) were compared and they confirmed that where the analyte is RNA, RNA folding adaptation occurs, coating the probe after binding (hybridization), resulting in multiple layers of RNA stabilizing the probe. The shape adopted by the re-folded RNA is predicted to offer a high surface area for the cationic gold nanoparticles to be distributed along the RNA molecules, preventing capture ligand interaction with the probe and preserving their distribution. Figures 8a and 8c show representative TEM images for HCV positive samples after performing the assay showing the distribution of the nanoparticles (black spheres) along and within the RNA molecules and in solution. The RNA folding protects the probe from aggregation and provides a large surface area for cationic nanoparticle distribution. Figure 8b shows that in the absence of RNA, the inter-particle distance between the nanoparticles decreases and thus induces aggregation.

To further validate the assay, HCV samples were re-analysed with and without prior digestion with RNase A. In RNase A treated samples, aggregation of the gold nanoparticles occurred, as was the case for negative samples, indicating absence of RNA. In contrast, in mock treated samples, the stability of gold nanoparticles was noticeable with a detectable red colour (Figure 5d), which was further confirmed by TEM (Figures 8c and 8d) where digestion of RNA molecules caused aggregation of the cationic nanoparticles with some residues of RNA.

It would be appreciated that the capture ligands, probes, assays and methods of the invention are capable of being implemented in a variety of ways, only a few of which have been illustrated and described above.

Claims

1. A capture ligand for an analyte comprising SEQ ID NO: 1.

2. A capture ligand according to claim 1, wherein SEQ ID NO: l is thiolated.

3. A capture ligand according to claim 1 or claim 2, wherein the analyte comprises an indicator of hepatitis C.

4. A capture ligand according to claim 3, wherein the analyte comprises an RNA sequence that codes for hepatitis C.

5. A capture ligand comprising an antisense primer of at least 25 bases complementary to SEQ ID NO: 2, wherein the primer initiates hybridisation from any of nucleotides 5 to 20, 43 to 118, 156 to 238, 253 to 279 or 291 to 314 of SEQ ID NO: 2.

6. A capture ligand according to claim 5, wherein the primer initiates hybridisation from any of nucleotides 172 to 229 or 253 to 279 of SEQ ID NO: 2.

7. A capture ligand according to any of claims 4 to 6, having at least 80% homology with the RNA sequence that codes for hepatitis C.

8. A capture ligand according to any preceding claim, wherein the analyte comprises cell-free RNA.

9. A capture ligand according to claim 8, wherein the cell-free RNA is isolated from blood, urine, cerebro-spinal fluid, serum, saliva or combinations thereof.

10. A probe for an analyte, comprising a metal nanoparticle and one or more of the capture ligand of any of claims 1 to 9 conjugated thereto.

11. A probe for an indicator of disease, the probe comprising a metal nanoparticle and one or more capture ligands specific for the indicator of disease conjugated thereto.

12. A probe according to claim 11, wherein the indicator of disease is selected from an indicator of hepatitis C or an indicator of rectal cancer carcinoma.

13. A probe according to claim 12, wherein the indicator of rectal cancer carcinoma is selected from a transcript of TOPI, TOP2, TDP1 and TDP2.

14. A probe according to any of claims 10 to 13, wherein the metal nanoparticle is surface functionalised.

15. A probe according to claim 14, where in the surface functionalisation comprises functionalisation with a carboxylic acid.

16. A probe according to claim 15, wherein the carboxylic acid is selected from citric acid, adipic acid, malonic acid, succinic acid, and combinations thereof.

17. A probe according to any of claims 10 to 16, wherein the metal nanoparticle is a gold nanoparticle.

18. An assay kit for an analyte comprising a probe according to any of claims 10 to

17. and a positively charged metal nanoparticle.

19. An assay kit according to claim 18, wherein the positively charged metal nanoparticle is a surface modified gold nanoparticle.

20. An assay kit according to claim 18 or claim 19, wherein the positively charged metal nanoparticle is surface modified with cysteamine or cetrimonium bromide.

21. A method of detecting the presence of an analyte, the method comprising the steps of: a. providing a probe for an indicator of disease, the probe comprising a metal nanoparticle and one or more capture ligands specific for the indicator of disease conjugated thereto; b. contacting the probe with a sample to allow binding of the analyte with the capture ligands; c. providing a positively charged metal nanoparticle; and d. observing a colour of the sample.

22. A method according to claim 21, wherein the indicator of disease is selected from an indicator of hepatitis C or an indicator of rectal cancer carcinoma.

23. A method according to claim 21 or claim 22, comprising the additional step of heating the probe and sample.

24. A method according to claim 23, wherein heating the probe and the sample denatures the capture ligand and/or the analyte.

25. A method according to claim 23 or claim 24, wherein the probe and sample are heated to a temperature in the range 90 - 100°C, and/or for a time in the range 1 - 5 minutes.

26. A method according to any of claims 21 to 25, wherein the probe and sample are incubated at room temperature.

27. A method according to any of claims 23 to 26, wherein the probe and sample are incubated at room temperature after heating.

28. A method according to claim 26 or claim 27, wherein incubation is for a time period in the range 1 - 15 minutes.

29. A method according to any of claims 21 to 28, wherein the presence of the analyte is indicated by a red colour and/or the absence of the analyte by a blue colour.

30. A method according to any of claims 21 to 29, wherein the analyte comprises RNA, and the sample is prepared using a membrane-based purification system.

31. A method according to claim 30, wherein the purification system includes a DNase treatment step.

32. A method according to any of claims 21 to 31, completed on a chip.