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WO2018196975A1 - Sonde - Google Patents

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Publication number
WO2018196975A1
WO2018196975A1 PCT/EP2017/060043 EP2017060043W WO2018196975A1 WO 2018196975 A1 WO2018196975 A1 WO 2018196975A1 EP 2017060043 W EP2017060043 W EP 2017060043W WO 2018196975 A1 WO2018196975 A1 WO 2018196975A1
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WIPO (PCT)
Prior art keywords
probe
analyte
indicator
rna
sample
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Inventor
Sherif EL-KHAMISY
Sherif SHAWKY
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University of Sheffield
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University of Sheffield
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the invention relates to capture ligands and to probes including these.
  • 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.
  • existing detection technologies are expensive, labour intensive and time consuming, posing significant limitations to their wide-scale exploitation, particularly in economically deprived populations.
  • 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.
  • 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.
  • RT-PCR Real-Time PCR
  • b-DNA branched-DNA
  • TMA transcription-mediated Amplification
  • Gold nanoparticles, and other metal nanoparticles have unique optical properties, originating from their strong Surface Plasmon Resonance (SPR) phenomenon.
  • SPR Surface Plasmon Resonance
  • the SPR phenomenon is responsible for the intense colours of such nanoparticles.
  • metal nanoparticles, such as gold nanoparticles have been employed in many colorimetric assays for biological molecules.
  • 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).
  • a capture ligand for an analyte comprising SEQ ID NO: l ( 5 ' -TACCACAAGGCCTTTCGCG ACCCAACACTACT-3 ' ) .
  • 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.
  • 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.
  • 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.
  • the analyte comprises cell-free RNA, to improve sensitivity of detection.
  • 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.
  • 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.
  • a probe for an indicator of disease comprising a metal nanoparticle and one or more capture ligands specific for the indicator of hepatitis C or cancer carcinoma conjugated thereto.
  • 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.
  • 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.
  • the probe can operate across a wide pH range, resulting in an assay which is pH independent.
  • 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.
  • TOPI Topoisomerase 1
  • TOP2 Topoisomerase 2
  • TDP1 Tyrosyl- DNA phosphodiesterase 1
  • TDP2 Tyrosyl-DNA phosphodiesterase 2
  • 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.
  • 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.
  • 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.
  • citric acid offers a stable, readily available option for achieving these benefits.
  • the metal nanoparticle is a particle with a good zeta-potential, often in the range -40 to -60 mV, or -45 to -55 mV.
  • the particle will be a gold nanoparticle, although other metal particles, such as silver, platinum, palladium or combinations thereof may also be present.
  • 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.
  • 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.
  • 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.
  • the positively charged metal nanoparticles interact with negative charges on the analyte, preventing aggregation. In the absence of aggregation no colour change is observed.
  • the positively charged metal nanoparticle is a gold nanoparticle, although other metals may also be used, as described above for the probe.
  • 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.
  • 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.
  • CAB cetrimonium bromide
  • 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.
  • a method of detecting the presence of an analyte 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.
  • 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.
  • 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.
  • room temperature is intended to mean ambient temperature, typically in the temperature range 18 - 25°C.
  • the accuracy of the assay is significantly improved.
  • 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.
  • the positively charged nanoparticle 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.
  • 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.
  • red is intended to refer to light emitted at wavelengths in the range 590 - 700 nm
  • the term “blue” is intended to refer to light at wavelengths in the range 400 - 500 nm.
  • analyte concentration is 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.
  • 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.
  • the analyte comprises RNA.
  • the sample may be prepared using a membrane-based purification system
  • the purification system may include a DNase treatment step.
  • 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.
  • 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.
  • a probe for an analyte comprising a citrate functionalised gold nanoparticle and one or more of the capture ligands described above conjugated thereto.
  • 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.
  • 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.
  • the purification system includes a DNase treatment step.
  • 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);
  • RNase A Ribonuclease 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
  • 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
  • HCV RNA probe-analyte
  • 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/ ⁇ ), is cost effective and could readily be adopted for full automation.
  • Figure 1 shows the method described.
  • Citrate capped gold nanoparticies were functionalized with thiolated HCV RNA specific capture ligands forming probes.
  • the mixture incubated at room temperature.
  • Hydrogen tetrachloroa urate (III) trihydrate (HAuCI 4 -3H 2 0) >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.
  • HRTEM, JEM- 2100 High resolution transmission electron microscope
  • 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.
  • 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 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).
  • 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).
  • CTAB modified gold nanoparticles were synthesized using only the seed solution in a single-phase reaction without the growth solution.
  • 3.7 ml of 0.2 M aqueous solution of CTAB was mixed with 20 ⁇ 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 CTAB micelles are stabilised and the particles do not aggregate thereby allowing a shell like structure to form ( Figure 3a).
  • 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.
  • the high reaction temperature affects the final gold nanoparticulate shape and size (Scarabelli et al., 2015).
  • 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.
  • 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.
  • FIG 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.
  • 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 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.
  • 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.
  • the assay was performed by mixing 5 ⁇ of the probe with 10 ⁇ 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 ⁇ ) 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.
  • 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 ⁇ of 10 mg/ml Ribonuclease A to 35 ⁇ 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.
  • 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/ ⁇ ), 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 A 5 3o to the aggregated nanoparticles at A 6 so (A 5 3o/A 6 5o) was recorded and used to generate the standard curve, in which the A 5 3o/A 6 5o ratio was plotted against the viral RNA log concentration.
  • the viral load was determined by the assay using the generated standard curve, from their respective A 5 3o/A 6 5o ratios, using the equation generated by the standard curve. The results were expressed in IU/ ⁇ and converted to IU/ml by (IU ⁇ I*RNA elution volume in ⁇ /serum volume in ⁇ ). 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/ ⁇ . Each dilution was tested by the assay until aggregation occurred starting from about 4 IU/ ⁇ .
  • 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.
  • 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.
  • 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
  • probe concentration and type in this case potentially probe and cationic nanoparticle concentration
  • pH of the reaction mixture (2)
  • nucleic acid folding in this case RNA folding
  • the probe density of ⁇ 120 probes per gold nanoparticles 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 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.
  • Figures 8a and 8b TEM results for HCV positive and negative samples 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un ligand de capture pour analyte comprenant SEQ ID no : 1, ou comprenant une amorce antisens d'au moins 25 bases complémentaires de SEQ ID no : 2, où l'amorce initie l'hybridation à partir de n'importe lesquels des nucléotides 5 à 20, 43 à 118, 156 à 238, 253 à 279 ou 291 à 314 de SEQ ID no : 2. Une sonde pour un analyte comprenant ce ligand de capture et une sonde comprenant une nanoparticule métallique et un ou plusieurs ligands de capture spécifiquement pour l'indicateur de maladie conjugué à cette dernière. La présente invention concerne également des kits et des procédés d'essai de détection de la présence d'un analyte.
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