Patent Application
20160265064
Cell-free nucleic acids (cfNAs) released from tumors are present in the blood of patients with cancer as they have the potential to act as markers for cancer diagnosis and management. Cancer patients often have higher levels of cfNAs than do healthy individuals. Examples of tumor-related mutated sequences, include the Kirsten rat sarcoma-2 virus (KRAS) gene that is associated with lung cancer, colorectal cancer, and ovarian cancer, and the BRAF gene associated with melanoma. The ability to detect mutated sequences (e.g., KRAS and BRAF) linked to cancer could allow specific monitoring of tumor-related sequences. Furthermore, detection of cfNAs in plasma or serum could serve as a liquid biopsy, replacing tumor tissue biopsies in certain diagnostic applications.
The existing approaches that are able to monitor cfNAs rely on the polymeric chain reaction (PCR) or DNA sequencing. The implementation of DNA sequencing is usually too expensive for routine clinical use, and the slow turnaround time (2-3 weeks) is not ideal for optimal treatment outcomes. PCR is not typically effective for the detection of point mutations, but the introduction of peptide nucleic acid (PNA) clamps boosts the accuracy of this approach. The PNAs serve as sequence-selective clamps that prevent amplification of wild-type DNA during clamp PCR, and the mutated sequence is then selectively amplified. PCR is prone to interference from the components of biological samples, and as such clamp PCR is not able to detect cfNA mutations in blood or serum samples directly. Instead the samples require pre-processing from large-volume samples (e.g. >5 ml) and then purification of the cfNAs. Additionally, clamp PCR can introduce bias based on the amplification efficiency of different sequences.
Chip-based methods leveraging electronic or electrochemical readout represent attractive alternatives for clinical sample analysis because they are amenable to automation, are low cost and have the potential for high levels of multiplexing and sensitivity. This type of testing approach has been applied successfully to the analysis of cancer as well as a variety of infectious pathogens, but the feasibility of analyzing cfNAs for cancer-related mutations in clinical samples has not been established. Previous efforts to achieve point mutation detection based on electrochemical methods have relied on stringent control of assay conditions or mismatch-sensitive enzymes, but these types of approaches do not yield significant selectivity in heterogeneous patient samples where a mutated sequence may be outnumbered by a high level of the wild-type sequence A method that is more sensitive, specific and efficient than the existing methods, and one which is also able to detect cfNA mutations directly in serum or blood, is therefore desirable.
Disclosed herein are systems, devices, and methods for the electrochemical detection of a first variant of a target sequence in a sample. Electrochemical sensors are functionalized with molecules that render them specific for a nucleic acids sequence of interest, and a series of probes (“clamp”) molecules are used to eliminate cross-reactivity with wild-type DNA and with deliberately-selected mutants. The electrochemical clamp assay is an advantageous technique over PCT as it can successfully be applied to unpurified serum samples. This is of particular benefit because it can reduce bias in the pool of sequences that are isolated. In addition, very small volumes (e.g., less than about 5 mls, less than about 4 mls, less than about 3 mls, less than about 2 mls, less than about 1 ml, less than about 500 μl and less than about 50 μL) of serum can be analyzed with this approach, whereas 5 ml of serum is required to yield enough purified cfNA for analysis via clamp PCR. Moreover, the analysis time can be shorter (e.g., about 5 minutes, less than about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes or about 1 minute) than the 2-3 hours required by PCR, and the several days required by DNA sequencing techniques. The electrochemical clamp assay is therefore suitable for a point-of-care device.
According to one aspect, there is provided a detection system for electrochemically detecting a variant of a target sequence in a sample, the target sequence being present as a plurality of variants within the sample, the system comprising: an electrode comprising a first probe on its surface, said first probe being capable of binding a first variant of the target sequence and; a second probe capable of binding a second variant of the target sequence, wherein the second probe is added to the sample, thereby preventing binding of the second variant to the first probe.
According to another aspect, there is provided a method for electrochemical detection of a variant of a target sequence in a sample, the target sequence being present as a plurality of variants within the sample, the method comprising: contacting an electrode comprising a first probe (e.g., an electrochemical probe) on its surface with the sample, said first probe, preferably deployed on the surface of the electrode and being capable of binding a first variant of the target sequence, adding a second probe to the sample, said second probe being capable of binding a second variant of the target sequence, thereby preventing binding of the second variant to the first probe; and measuring an electrochemical signal generated by the binding of the first variant of the target sequence to the first probe, wherein the electrochemical signal is indicative of the presence of the first variant within the sample.
According to a further aspect there is provided a point-of care diagnostic device configured to perform the method described herein.
According to another aspect there is provided a kit comprising: a biosensor comprising an electrode; a first probe affixed to surface of the electrode, said first probe being capable of binding a first variant of a target sequence in a sample, said sample containing a plurality of variants of the target sequence; a second probe, capable of binding a second variant of a target sequence in a sample containing a plurality of variants of the target sequence, thereby preventing binding of the second variant to the first probe.
According to a still further aspect there is provided a method of detecting a variant of a cancer-related sequence mutation in sample from a patient, the method comprising: contacting an electrode comprising a first probe on its surface with the sample, said first probe being capable of binding a first variant of the cancer-related sequence mutation, adding at least a second probe to the sample, said second probe being capable of binding a second variant of the cancer-related sequence mutation, thereby preventing binding of the second variant to the first probe; and measuring an electrochemical signal generated by the binding of the first variant of the cancer-related sequence mutation to the first probe, wherein the electrochemical signal is indicative of the presence of the first variant within the sample.
In some embodiments of the electrochemical detection systems and methods provided herein, the electrode is a microelectrode. In other embodiments, the microelectrode is a nanostructured microelectrode (“NME”). NMEs are microelectrodes that feature nanostructured surfaces. Surface nanotexturing or nanostructures provide the electrode with an increased surface area, allowing greater sensitivity, particularly in biosensing applications. Manufacturing of NMEs can be performed by electrodeposition. By varying the parameters such as deposition time, deposition potential, supporting electrolyte types and metal ion sources, NMEs of a variety of sizes, morphologies and compositions may be generated. In certain instances, NMEs have a dendritic or fractal structure. Exemplary NMEs for use in the systems and methods described herein are described in International Patent Publication WO2010/025547, which is hereby incorporated by reference in its entirety. In additional embodiments, the electrode is on a microfabricated chip.
Other electrode structures can also be used in the detection systems and methods described herein, including planar surfaces, wires, tubes, cones and particles. Commercially available macro- and micro-electrodes are also suitable for the embodiments described herein.
In some embodiments of the electrochemical detection systems and methods provided herein the first and the second probe is an oligonucleotide. In some embodiments the oligonucleotide is a peptide nucleic acid (PNA).
In some embodiments of the systems and methods provided herein the target sequence to be detected is a cell-free nucleic acid (cfNA). Most DNA and RNA in the body are located within cells, but a small amount can be found circulating freely in the blood. A substantial proportion of such DNA and RNA molecules (cfNAs) are thought to come from cells undergoing apoptosis or necrosis, which release their contents into the blood stream. The analysis of cfNAs offers a non-invasive approach for the diagnosis of a variety of diseases/disorders that are capable of being diagnosed using genetic analysis. In some embodiments of the systems and methods described herein, cfNAs (present at significant levels in the blood of cancer patients) are analyzed to reveal the mutational spectrum of a tumor without the need for invasive sampling of tissue. Non-limiting examples of cfNAs released from tumors that can be detected using the systems and methods as herein described include the Kirsten rat sarcoma-2 virus (KRAS) gene that is associated with lung cancer, colorectal cancer, and ovarian cancer, and the BRAF gene associated with melanoma. In other embodiments of the systems and methods described herein, analysis of fetal-derived cfNAs found within the maternal blood is useful for detecting and monitoring fetal diseases and pregnancy-associated complications. In some other embodiments of the systems and methods described herein, the quantitation of elevated levels of cfNAs provides an indication of the level of clinical severity of acute medical emergencies, including trauma and stroke.
The patient sample can be a blood sample, for example a whole blood sample, a plasma sample or a serum sample. In specific embodiments the electrochemical detection system enables direct analysis of cancer-related mutations in cfNAs from unprocessed patient serum samples.
The foregoing and other objects and advantages will be apparent upon consideration of the following detailed description, taken in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout, and in which:
To provide an overall understanding of the systems, devices, and methods described herein, certain illustrative implementations will be described.
A given patient sample may contain one of the 7 mutant alleles and a large amount of wild-type nucleic acids (NAs), as illustrated in
Using photolithographic patterning, an array of forty sensors is defined to form a bioelectronic integrated circuit (IC) (
As shown in
The clamps hybridize to the six non-target mutants and the wild-type sequence, sequestering them in the sample, and leaving only the 134A mutation unhybridized. Only the mutant 134A can hybridize to the immobilized probe; all other mutant alleles and the wild-type allele are blocked by their clamps and simply remain in solution and are washed away.
As shown in
The sequences of the seven mutant KRAS alleles, and of the oligonucleotides used as probes and clamps, are shown below. DNA sequences are shown 5′ to 3′, and PNA sequences are shown N to C terminus. Underlined portions denote point mutations.
A sample including complementary mutant target, non-complementary mutants, wild-type sequence, total human RNA, and a clamp cocktail was used to measure the positive signal at electrochemical sensors functionalized with probes (P135 A, P135 C, P135 T, P134 A, P134 C, P134 T, and P138 A) corresponding to each of the mutant alleles. Sensors were challenged with mixtures of nucleic acids with (positive control) and without (negative control) mutant target of interest. The positive control contained all of the seven mutant oligonucleotides with 1 nM concentration of each, 100 nM of wild-type (WT) synthetic oligonucleotides, 50 pg/μL cfNAs from healthy donors, and seven clamps except one that is complementary for target of interest. The negative control contained all of the above except target of interest and its clamp. As shown in
In order to investigate whether the clamps were necessary for accurate point mutation detection, a sensor was challenged with purified nucleic acids from a wild-type patient sample, a mutant-positive patient sample, and a healthy donor in presence and absence of the clamp for the wild-type sequence. Although hybridization and washing were performed at an elevated temperature, a signal increase for all three samples was observed if the clamp for the wild-type sequence was not present in solution (
To evaluate the sensitivity of the electrochemical clamp assay, the dependence of the electrochemical signal on RNA concentration when an electrode on which a 134A mutant probe is hybridized was challenged with exosomal RNA isolated from cells derived from a lung cancer cell line (A549 cells) carrying the 134A mutation, was investigated. Concentrations of RNA ranged from 1 fg/μL to 100 pg/μL. Seven clamps except the clamp for the 134A mutation were added to the sample. Controls included (i) a blank, (ii) RNA containing the wild-type sequence isolated from cells derived from a glioblastoma cell line (exosomal RNA from U733v3 cells) (NCT in
The use of the electrochemical detection system to detect other mutations in the sequence of other genes, is illustrated in
The electrochemical clamp assay was used to analyze cfNA in processed and non-processes serum samples from lung cancer patients (KRAS) and melanoma cancer patients (BRAF) (Table 1 and 2).
Table 1 shows the results of the analysis of KRAS mutations in cfNAs isolated and purified from lung cancer patients, and also in unprocessed lung cancer patient serum. As a control, serum from a healthy donor (HD) was processed and analyzed in the same way. A universal probe mixture (Cys-Gly-CTA CGX CAXXAG CTC CAA C (where, X=mixture of A, T, and G with unimolar ratio), allowed all possible known KRAS mutant sequences of interest to be screened in cfNAs in a single experiment. For each sample analyzed using the electrochemical clamp assay, a mean signal of −1.0±0.3 nA (plus three standard deviations) measured in the healthy donor's sample was used as a cutoff value for determining the presence or absence of the KRAS mutation. A sample with a current level higher than the cutoff value is positive for the KRAS mutation, whereas a sample with a current level lower than the cutoff value is negative for the KRAS mutation. A previously-validated clamp PCR method was used to confirm the presence or absence of the KRAS mutation. In this clamp PCR method, when ΔCt-1≧2, the sample is positive for the KRAS mutation, and when ΔCt-1<0, the sample is negative for the KRAS mutation. When 0<ΔCt-1<2, another parameter (ΔCt-2) is taken into consideration. When ΔCt-2>6, the sample is negative for the KRAS mutation. The results of the electrochemical clamp assay and clamp PCR are comparable. The electrochemical clamp assay was also used to detect KRAS mutations in unprocessed lung cancer patient serum. As demonstrated in all three assays, three (3) of the fourteen (14) lung cancer patient samples were positive for KRAS mutation. The signal changes observed in electrochemical assay for the undiluted serum is lower than in the processed samples, which is expected due to much lower levels in the purified sample. The electrochemical clamp assay is able to detect mutated KRAS in unprocessed serum, in comparison to clamp PCR method. The inability of clamp PCR to produce detectable amplification in patient sample is demonstrated in
Table 2 shows the results of the analysis of BRAF mutations in cfNAs isolated and purified from melanoma patients, and also in unprocessed melanoma patient serum. As a control, serum from a healthy donor (HD) was processed and analyzed in the same way.
The sequences of the mutant BRAF allele, and of the oligonucleotides used as probes and clamps are shown below. DNA sequences are shown 5′ to 3′, and PNA sequences are shown N to C terminus. Underlined portions denote point mutations.
For each sample analyzed using the electrochemical clamp assay, a mean signal of −1.0±0.3 nA (plus three standard deviations) measured in the healthy donor's sample was used as a cutoff value for determining the presence or absence of the BRAF mutation. A sample with a current level higher than the cutoff value is positive for the BRAF mutation, whereas a sample with a current level lower than the cutoff value is negative for the BRAF mutation. A previously-validated clamp PCR method was used to confirm the presence or absence of the BRAF mutation. In this clamp PCR method, when ΔCt-1≧2, the sample is positive for the BRAF mutation, and when ΔCt-1<0, the sample is negative for the BRAF mutation. The results of the electrochemical clamp assay and clamp PCR are comparable. The electrochemical clamp assay was also used to detect BRAF mutations in unprocessed melanoma patient serum. As demonstrated in all three assays, three (3) of the seven (7) melanoma patient samples were positive for BRAF mutation. The signal changes observed in electrochemical assay for the undiluted serum is lower than in the processed samples, which is expected due to much lower levels in the purified sample. Unlike the clamp PCR method, the electrochemical clamp assay is able to detect mutated BRAF in unprocessed serum.
III. Distinguishing Between DNA or RNA cfNAs
To identify whether the electrochemical clamp assay detects genomic DNA or transcribed RNA analytes the assay was challenged with cfNAs, cfNAs digested with DNase I, and cfNAs digested with RNase A. As illustrated in
Materials
HAuCl4, potassium ferricyanide (K3[Fe(CN)6), and hexaamine ruthenium (III) chloride (Ru(NH3)6Cl3) were obtained from Sigma-Aldrich. ACS-grade acetone, isopropyl alcohol (IPA), and perchloric acid were obtained from EMD; hydrochloric acid was purchased from VWR.
Phosphate-buffered saline (PBS, pH 7.4, 1X) was obtained from Invitrogen. All of the PNA probes and PNA clamps were obtained from PNA Bio, USA. PCR primers, synthetic DNA targets, and DNA clamps were obtained from ACGT, Canada. The A549 lung cancer cell line (catalog number CCL-185) and cultured medium (F-12K medium, catalog number 30-2004 supplemented with 10% (v/v) FBS and 5% CO2 atmosphere) were obtained from ATCC, Canada. Lung cancer and melanoma patient serums were obtained from Bioreclamation Inc., USA.
Chip Fabrication
Six-inch silicon wafers were passivated using a thick layer of thermally grown silicon dioxide. A layer of about 25 nm Ti was deposited. A gold layer of about 350 nm was deposited on the chip using electron-beam-assisted gold evaporation. The gold film was patterned using standard photolithography and a lift-off process. A Ti layer of about 5 μm was deposited. A layer of insulating Si3N4 of about 500 nm was deposited using chemical vapor deposition; apertures of about were imprinted on the electrodes using standard photolithography, and bond pads of about 0.4 mm×2 mm were exposed using standard photolithography.
Fabrication of NMEs
Chips were cleaned by sonication in acetone for about 5 min, rinsed with isopropyl alcohol and DI water, and dried using a flow of nitrogen. Electrodeposition was performed at room temperature; 5 pm apertures on the fabricated electrodes were used as the working electrode and were contacted using the exposed bond pads. Au sensors were generated using a deposition solution containing a solution of about 50 mM HAuCl4 and about 0.5 M HCl using DC potential amperometry at about 0 mV for about 100 s. After washing with DI water and drying, the Au sensors were coated with Pd to form nanostructures by replating in a solution of about 5 mM H2PdCl4 and about 0.5 M HClO4 at about −250 mV for about 10 s. The control of sensor surface area has been characterized extensively and in this study, the average surface area was 4.75±0.3×10−4 cm2 as determined by electrochemical Pd oxide stripping.
Clamp Chip Protocol
A 2 μM probe solution in water was prepared from a 20% acetonitrile solution containing about 100 pM PNA probe. Probe solutions were then heated to about 65° C. for about 5 min and chilled on ice for about 5 min before deposition. About 50 μL of the probe solution was dropped onto the chips and incubated for overnight in a dark humidity chamber at room temperature for immobilization of probe. The deposition used lead to a surface coverage of about 2×1013 molecules/cm2. The chip was washed for about 10 min with PBS at about 60° C. followed by washing for about 10 min at room temperature. After initial electrochemical scanning the chips were then treated with different targets at about 60° C. Optimal hybridization time was determined to be about 15 min. After washing for about 10 min with PBS at about 55° C., followed by washing for about 10 min at room temperature of the chip, a final electrochemical scan was performed.
cfNAs Isolation from Exosomes
MW9 mutant BRAF 1799A melanoma exosomes and U373v3 glioblastoma exosomes (wild type BRAF and wild type KRAS control) were obtained from the laboratory of Prof. Janusz Rak's (Montreal Children's Hospital Research Institute, McGill University). MW9 and U373v3 exosomes were isolated by ultracentrifugation method and RNA was extracted by Trizol (Invitrogen). A549 exosomal RNA (mutant KRAS 134A) and exosomal RNA from patient serums was extracted, using Norgen biotek kit catalog number 51000. Isolated RNA had a A260/A280 ratio>2, indicating a high level of purity.
cDNA Synthesis and Clamp PCR
A volume of 2 purified cfNA (30-754 ng) was used for cDNA synthesis, in 20 μL reaction, with random hexamer primers and Superscript III reverse transcriptase, Invitrogen kit. A volume of about 2 μL cDNA was used in 50 μL not-competitive clamp PCR reaction with about 2 μM final concentration of gene specific primers, or in a 201.1 of real-time clamp PCR reaction, Panagene kit.
Mutant BRAF and Mutant KRAS Clamp Optimization
To validate that 60° C. was an appropriate specific temperature for the sensor assay, clamp PNA was tested in a qualitative PCR assay. The PCR program was as follows: template denaturing at about 95° C. for about 3 minutes followed by about 35 cycles of template denaturing at about 95° C. for about 30 seconds, primer annealing and DNA chain extension at about 60° C. for about one minute. The PCR products were visualized using agarose gel electrophoresis. PCR primers for BRAF (95 bp PCR product): Forward primer: FPBRAF3 (5′-CCT-CAC-AGT-AAA-AAT-AGG-TGA-TTT-TGG-3′), Reverse primer: RPBRAF3 (5′-CAC-AAA-ATG-GAT-CCA-GAC-AAC-TGT-TC-3′). PCR primers for KRAS (80 bp PCR product): Forward primer: FPKRAS (5′-GCC-TGC-TGA-AAA-TGA-CTG-AAT-ATA-3′), Reverse primer: RPKRAS (5′-TTA-GCT-GTA-TCG-TCA-AGG-CAC-TC-3′).
Real-Time Competitive Clamp PCR
Mutant BRAF and mutant KRAS real-time competitive clamp PCR were performed using a Panagene kit (mutant BRAF product number PNAC-2001 and mutant KRAS product number PNAC-1002). The real time clamp PCR was performed on ABI 7500 thermocycler and the SYBR Green reading was set at about 72° C. The PCR program was: template denaturing at about 94° C. for about 5 min followed by about 40 cycles of template denaturing at about 94° C. for about 30 sec, PNA clamp at about 70° C. for about 20 sec, primer annealing at about 63° C. for about 30 sec and DNA chain extension at about 72° C. for about 30 sec.
KRAS and BRAF Detection in Whole Serum
For mutation detection in whole serum we co-deposited 6-mercaptohexanol (MCH) with the probe to minimize nonspecific binding. For KRAS mutation detection, we used a universal probe for KRAS point mutations that was a combination of all of the possible mutant probes. An aqueous solution containing about 2 μM of PNA probes was heated to about 65° C. for about 5 min and, after annealing, about 18 μM of MCH was mixed with this probe solution. The solution was dropped onto chip and left overnight, followed by washing as described above. Serum samples were prepared by adding about 12.5 μL of lysis buffer (1×PBS containing about 10% NP40 and about 10% Triton X100), about 1 μL of 10 μM clamps for wild-type, and about 3 μL of RNAase inhibitor (Ambion, Am 2694) to about 50 μL of patients' serum. After initial electrochemical scanning, the above serum sample was dropped onto chip and incubated at about 60° C. for about 15 min. After washing, a final electrochemical scan was performed.
Electrochemical Analysis and Scanning Electron Microscopy (SEM)
All electrochemical experiments were carried out using a Bioanalytical Systems Epsilon potentiostat with a three-electrode system featuring a Ag/AgCl reference electrode and a platinum wire auxiliary electrode. Electrochemical signals were measured in a 0.1×PBS containing about 10 μM [Ru(NH3)6]Cl3, and about 4 mM K3[Fe(CN)6]. Differential pulse voltammetry (DPV) signals were obtained with a potential step of about 5 mV, pulse amplitude of about 50 mV, pulse width of about 50 ms, and a pulse period of about 100 ms. Signal changes corresponding to specific target were calculated with background-subtracted currents: change in currents=(Iafter−Ibefore) (where Iafter=current after target binding, Ibefore=current before target binding). SEM images were obtained using an Aspex 3025 SEM.
Variations and modifications will occur to those of skill in the art after reviewing this disclosure. The disclosed features may be implemented, in any combination and subcombination (including multiple dependent combinations and subcombinations), with one or more other features described herein. The various features described or illustrated above, including any components thereof, may be combined or integrated in other systems. Moreover, certain features may be omitted or not implemented. All references cited are hereby incorporated by reference herein in their entireties and made part of this application.
Examples of changes, substitutions, and alterations are ascertainable by one skilled in the art and could be made without departing from the scope of the information disclosed herein. All references cited herein are incorporated by reference in their entirety and made part of this application.
This application claims the benefit of U.S. Provisional Application No. 62/131,434, filed on Mar. 11, 2015, which is hereby incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62131434 | Mar 2015 | US |
