Accurate and early detection of biomarkers provides the molecular evidence for disease management... more Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction. Biomarkers, defined as measurable indicators of disease, are valuable tools for patient screening, prognosis, informing treatment and treatment monitoring. The detection of biomarkers typically relies on functional nanoprobes (for example, labelled antibody or peptide) due to their high sensitivity and specificity 1. Ideal functional nanoprobes should have high binding affinity to the target, cost-effective production, scalability and flexible storage to benefit population-based patient screening, which is particularly important for the rapid detection of emerging diseases like COVID-19 (refs. 2,3). The controlled and precise attachment of the target binder (for example, antibody) to material surfaces in optimal densities and orientations to maximize the binding affinity, multivalency and target-specific recognition are also crucial for improved diagnostic outcomes 4-6. Although various strategies are available for attaching biomolecules to materials, these methods are challenging because of problems with conjugation efficiency, site specificity and inhibited target binding. As such, it is important to
“Lab-in-a-drop”: a miniaturised fabrication-free diagnostics platform by which traditional bioass... more “Lab-in-a-drop”: a miniaturised fabrication-free diagnostics platform by which traditional bioassays are downsized into a droplet and programmed for convenient detection.
The intrinsic affinity of DNA molecules toward metallic ions can drive the specific formation of ... more The intrinsic affinity of DNA molecules toward metallic ions can drive the specific formation of copper nanostructures within the nucleic acid helix structure in a sequence-dependent manner. The resultant nanostructures have interesting fluorescent and electrochemical properties, which are attractive for novel biosensing applications. However, the potential of using DNA-templated nanostructures for precision disease diagnosis remains unexplored. Particularly, DNAtemplated nanostructures show high potential for the universal amplification-free detection of different RNA biomarker species. Because of their low cellular levels and differing species-dependent length and sequence features, simultaneous detection of different messenger RNAs, microRNAs, and long non-coding RNAs species with a single technique is challenging. Here, we report a contemporary technique for facile in situ assembly of DNA-templated copper nanoblocks (CuNBs) on various RNA species targets after hybridization-based magnetic isolation. Our approach circumvents the typical limitations associated with amplification and labeling procedures of current RNA assays. The synthesized CuNBs enabled amplification-free fM-level RNA detection with flexible fluorescence or electrochemical readouts. Furthermore, our nanosensing technique displays potential for clinical application, as demonstrated by non-invasive analysis of three diagnostic RNA biomarkers from a cohort of 10 prostate cancer patient urinary samples with 100%-concordance (quantitative reverse transcriptionpolymerase chain reaction (PCR) validation). The good analytical performance and versatility of our method may be useful in both diagnostics and research fields.
The TMPRSS2:ERG gene fusion is one of a series of highly promising prostate cancer (PCa) biomarke... more The TMPRSS2:ERG gene fusion is one of a series of highly promising prostate cancer (PCa) biomarker alternatives to the controversial serum PSA. Current methods for detecting TMPRSS2:ERG are limited in terms of long processing time, high cost and the need for specialized equipment. Thus, there is an unmet need for less complex, faster, and cheaper methods to enable gene fusion detection in the clinic. We describe herein a simple, rapid and inexpensive assay which combines robust isothermal amplification technique with a novel visualization method for evaluating urinary TMPRSS2:ERG status at less than USD 5 and with minimal equipment. The assay is sensitive, and rapidly detects as low as 10 5 copies of TMPRSS2:ERG transcripts while maintaining high levels of specificity.
TMPRSS2 (Exon 1)-ERG (Exon 4) is the most frequent gene fusion event in prostate cancer (PC), and... more TMPRSS2 (Exon 1)-ERG (Exon 4) is the most frequent gene fusion event in prostate cancer (PC), and is highly PC-specific unlike the current serum prostate specific antigen (PSA) biomarker. However, TMPRSS2-ERG levels are currently measured with quantitative reverse-transcription PCR (RT-qPCR) which is time-consuming and requires costly equipment, thus limiting its use in clinical diagnostics. Herein, we report a novel rapid, cost-efficient and minimal-equipment assay named "FusBLU" for detecting TMPRSS2-ERG gene fusions from urine. TMPRSS2-ERG mRNA was amplified by isothermal reverse transcription-recombinase polymerase amplification (RT-RPA), magnetically-isolated, and detected through horseradish peroxidase (HRP)-catalyzed colorimetric reaction. FusBLU was specific for TMPRSS2-ERG mRNA with a low visual detection limit of 10 5 copies. We also demonstrated assay readout versatility on 3 potentially useful platforms. The colorimetric readout was detectable by naked eye for a quick yes/no evaluation of gene fusion presence. On the other hand, a more quantitative TMPRSS2-ERG detection was achievable by absorbance/electrochemical measurements. FusBLU was successfully applied to 12 urinary samples and results were validated by gold-standard RT-qPCR. We also showed that sediment RNA was likely the main source of TMPRSS2-ERG mRNA in urinary samples. We believe that our assay is a potential clinical screening tool for PC and could also have wide applications for other disease-related fusion genes.
Point mutations in DNA are useful biomarkers that can provide critical classification of disease ... more Point mutations in DNA are useful biomarkers that can provide critical classification of disease for accurate diagnosis and to inform clinical decisions. Conventional approaches to detect point mutations are usually based on technologies such as real-time polymerase chain reaction (PCR) or DNA sequencing, which are typically slow and require expensive lab-based equipment. While rapid isothermal strategies such as recombinase polymerase amplification (RPA) have been proposed, they tend to suffer from poor specificity in discriminating point mutations. Herein, we describe a novel strategy that enabled exquisite point mutation discrimination with isothermal DNA amplification, using mismatched primers in conjunction with a two-round enrichment process. As a proof of concept, the method was applied to the rapid and specific identification of drug-resistant Mycobacterium tuberculosis using RPA under specific conditions. The assay requires just picogram levels of genomic DNA input, is sensitive and specific enough to detect 10% point mutation loading, and can discriminate between closely related mutant variants within 30 min. The assay was subsequently adapted onto a low-cost 3D-printed isothermal device with real-time analysis capabilities to demonstrate a potential point-of-care application. Finally, the generic applicability of the strategy was shown by detecting three other clinically important cancer-associated point mutations. We believe that our assay shows potential in a broad range of healthcare screening processes for detecting and categorizing disease phenotypes at the point of care, thus reducing unnecessary therapy and cost in these contexts.
We report a new multiplexed strategy for the electrochemical detection of regional DNA methylatio... more We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl(-1) of DNA with no sequencing requirement.
The long non-coding RNA PARTICLE (Gene PARTICL- 'Promoter of MAT2A-Antisense RadiaTion Induce... more The long non-coding RNA PARTICLE (Gene PARTICL- 'Promoter of MAT2A-Antisense RadiaTion Induced Circulating LncRNA) partakes in triple helix (triplex) formation, is transiently elevated following low dose irradiation and regulates transcription of its neighbouring gene - Methionine adenosyltransferase 2A. It now emerges that PARTICLE triplex sites are predicted in many different genes across all human chromosomes. In silico analysis identified additional regions for PARTICLE triplexes at >1600 genomic locations. Multiple PARTICLE triplexes are clustered predominantly within the human and mouse tumor suppressor WW Domain Containing Oxidoreductase (WWOX) gene. Surface plasmon resonance diffraction and electrophoretic mobility shift assays were consistent with PARTICLE triplex formation within human WWOX with high resolution imaging demonstrating its enrichment at this locus on chromosome 16. PARTICLE knockdown and over-expression resulted in inverse changes in WWOX transcripts l...
Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative deter... more Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG's contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed-or deoxyinosinebase modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates. DNA methylation in higher eukaryotes occurs as a result of the addition of a methyl group to the carbon-5 position of cytosine-guanine (CpG) dinucleotides to form 5-methylcytosine. Due to the critical role of DNA methylation in a number of biological functions (e.g. X-chromosome inactivation, genomic imprinting and regulation of gene expression) 1-4 , a number of technologies have been developed to study and characterise the methylation patterns at a locus-specific level (e.g. DNA melting curve analysis, PCR-based methods). A common feature of these applications is the use of bisulfite modification 5-7 , wherein unmethylated cytosines are selectively deaminated to uracils and participate in subsequent PCR amplifications as thymines but 5-methylcytosines are left unaltered, which allows the visualisation of this epigenetic change using a variety of applications 8,9. As such, the bisulfite-conversion process results in a pool of templates which has cytosine/thymine degeneracy at the cytosine positions of CpG dinucleotides, and PCR primers designed against these regions must therefore be able to bind to both cytosines (methylated) and thymines (unmethylated) when unbiased amplification is sought. However, under non-optimized conditions a PCR primer that binds to a mixed-template CpG site may differentially amplify the methylated or unmethylated DNA strand, thereby inducing bias in the data and an inaccurate representation of the methylation state of the amplified products 10,11. Dealing with this potential bias represents a major challenge in bisulfite PCR assay design. The use of 'CpG-free' primers (i.e. primers that do not bind to a region with CpG dinucleotides) has been considered to
Simple nucleic acid detection methods could facilitate the progress of disease diagnostics for cl... more Simple nucleic acid detection methods could facilitate the progress of disease diagnostics for clinical uses. An attractive strategy is label-free surface-enhanced Raman scattering (SERS) due to its capability of providing structural fingerprinting of analytes that are close to or on nanomaterial surfaces. However, current label-free SERS approaches for DNA/RNA biomarker detection are limited to short and synthetic nucleic acid targets and have not been fully realized in clinical samples due to two possible reasons: (i) low target copies in limited patient samples and (ii) poor capability in identifying specific biomarkers from complex samples. To resolve these limitations and enable label-free SERS for clinical applications, we herein present a novel strategy based on multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) to enrich multiple RNA biomarkers, followed by label-free SERS with multivariate statistical analysis to directly detect, identify and dist...
The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its... more The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously (PrimerSuite), (ii) screen for primer-primer dimerizing artefacts (PrimerDimer), and (iii) support multiplex PCR assays (PrimerPlex). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).
Small (Weinheim an der Bergstrasse, Germany), Mar 22, 2016
Cancer diagnosis and patient monitoring require sensitive and simultaneous measurement of multipl... more Cancer diagnosis and patient monitoring require sensitive and simultaneous measurement of multiple cancer biomarkers considering that single biomarker analysis present inadequate information on the underlying biological transformations. Thus, development of sensitive and selective assays for multiple biomarker detection might improve clinical diagnosis and expedite the treatment process. Herein, a microfluidic platform for the rapid, sensitive, and parallel detection of multiple cancer-specific protein biomarkers from complex biological samples is presented. This approach utilizes alternating current electrohydrodynamic-induced surface shear forces that provide exquisite control over fluid flow thereby enhancing target-sensor interactions and minimizing non-specific binding. Further, the use of surface-enhanced Raman scattering-based spectral encoding with individual barcodes for different targets enables specific and simultaneous detection of captured protein biomarkers. Using this...
The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplificati... more The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community. Isothermal exponential amplification reaction (EXPAR) is an emerging amplification technique that is used to amplify short oligonucleotides for molecular diagnostics 1. In contrast with the conventional PCR, EXPAR operates at a constant temperature and provides high amplification efficiency, allowing 10 6-10 9 fold amplification of short oligonucleotides within minutes 2-4. Owing to its advantages, many studies have reported the use of EXPAR for molecular diagnostics 5-7. Briefly, EXPAR amplifies DNA by four major steps (Fig. 1). First, EXPAR is initiated when the target primes to the trigger sequence of the template, forming a partial double-stranded duplex. This is followed by the extension by DNA polymerase, forming an extended double-stranded DNA containing a nicking enzyme recognition site. A nicking enzyme then cleaves the upper strand and DNA polymerase displaces the cleaved trigger by strand displacement to generate additional trigger sequences. This occurs repeatedly and exponentially 1. Despite its advantages, target-independent amplification is a major limitation of EXPAR, which in turn, leads to lowered specificity and sensitivity 1,5,8. Non-specific amplification of EXPAR was suggested to arise from the interaction between the single-stranded template and the DNA polymerase independent of the target 3. Due to the issue of high levels of non-specific amplification with EXPAR, several studies have proposed ways to resolve this problem. Since the non-specific amplification is sequence dependent, Qian et al. demonstrated that the variability in template performance is linked to sequence motifs 9. Therefore, they developed computational methods to predict EXPAR template performance based on the template sequence. Wang et al. described the use of graphene oxide (GO) to minimize the level of non-specific amplification by preventing the binding of the DNA polymerase to the template 10. In the presence of a target, the template sequestered by GO can then be released, initiating EXPAR. This thus led us to hypothesize that other small molecules could also be used to enhance EXPAR performance.
Sensitive and accurate identification of specific DNA mutations can influence clinical decisions.... more Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samp...
au. Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterize... more au. Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells and an increased risk of late transformation to acute myeloid leukemia or primary myelofibrosis. Approximately 15% of MPN cases do not carry mutations in JAK2, CALR, or MPL and are thus often referred to as triple-negative cases. These are caused by a diverse set of rare mutations in cytokine receptors, JAK-STAT signaling pathway components, or epigenetic modifiers. In addition, some cases diagnosed as MPN are reactive rather than clonal disorders, so a negative result from a genetic screen can be informative. To obtain a comprehensive rapid molecular diagnosis for most MPNs, we developed an assay to detect genetic mutations (single nucleotide variants and/or small insertions/deletions) in 86 genes using targeted exon resequencing (AmpliSeq) and a bench-top semiconductor machine (Ion Torrent Personal Genome Machine). Our assay reliably detects well characterized mutations in JAK2, CALR, and MPL, but also rarer mutations in ASXL1, TET2, SH2B3, and other genes. Some of these mutations are novel. We find multiple mutations in advanced cases, suggesting cooperation between Janus kinase-STAT pathway mutations and epigenetic mutations in disease progression. This assay can be used to follow molecular progression, clonal heterogeneity, and drug resistance in MPNs.
A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers... more A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers for accurately distinguishing between benign and malignant cancer forms. This is causing high degree of overdiagnosis and overtreatment of otherwise clinically insignificant cases. As around half of all malignant PCa cases display a gene fusion mutation between the TMPRSS2 promoter sequence and the ERG coding sequence (TMPRSS2:ERG) detectable in urine; non-invasive screening of TMPRSS2:ERG mRNA in patient urine samples could improve specificity of current PCa diagnosis. However, current gene fusion detection methodologies are largely dependent on RNA enzymatic amplification, which requires extensive sample manipulation, costly labels for detection, and it is prone to bias/artifacts. Herein we introduce the first successful amplification-free electrochemical assay for direct detection of TMPRSS2:ERG mRNA in PCa urinary samples by selectively isolating and adsorbing TMPRSS2:ERG mRNA onto bare gold electrodes without requiring any surface modification. We demonstrated excellent limit-of-detection (10 cells) and specificity using PCa cell line models; and showcased clinical utility by accurately detecting TMPRSS2:ERG in a collection of 17 urinary samples obtained from PCa patients. Furthermore, these results were validated with current gold standard reverse transcription (RT)-PCR approach with 100% concordance.
Effective disease management strategies to prevent catastrophic crop losses require rapid, sensit... more Effective disease management strategies to prevent catastrophic crop losses require rapid, sensitive and multiplexed detection methods for timely decision making. To address this need, a rapid, highly specific and sensitive point-of-care method for multiplex detection of plant pathogens was developed by taking advantages of surface-enhanced Raman scattering (SERS) labelled nanotags and recombinase polymerase amplification (RPA), which is a rapid isothermal amplification method with high specificity. In this study, three agriculturally important plant pathogens (Botrytis cinerea, Pseudomonas syringae, and Fusarium oxysporum) were used to demonstrate potential translation into the field. The RPA-SERS method was faster, more sensitive than polymerase chain reaction and could detect as little as 2 copies of B. cinerea DNA. Furthermore, multiplex detection of the three pathogens was demonstrated for complex systems such as the Arabidopsis thaliana plant and commercial tomato crops. To demonstrate the potential for on-site field applications, a rapid single-tube RPA/SERS assay was further developed and successfully performed for a specific target outside of a laboratory setting.
Accurate and early detection of biomarkers provides the molecular evidence for disease management... more Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction. Biomarkers, defined as measurable indicators of disease, are valuable tools for patient screening, prognosis, informing treatment and treatment monitoring. The detection of biomarkers typically relies on functional nanoprobes (for example, labelled antibody or peptide) due to their high sensitivity and specificity 1. Ideal functional nanoprobes should have high binding affinity to the target, cost-effective production, scalability and flexible storage to benefit population-based patient screening, which is particularly important for the rapid detection of emerging diseases like COVID-19 (refs. 2,3). The controlled and precise attachment of the target binder (for example, antibody) to material surfaces in optimal densities and orientations to maximize the binding affinity, multivalency and target-specific recognition are also crucial for improved diagnostic outcomes 4-6. Although various strategies are available for attaching biomolecules to materials, these methods are challenging because of problems with conjugation efficiency, site specificity and inhibited target binding. As such, it is important to
“Lab-in-a-drop”: a miniaturised fabrication-free diagnostics platform by which traditional bioass... more “Lab-in-a-drop”: a miniaturised fabrication-free diagnostics platform by which traditional bioassays are downsized into a droplet and programmed for convenient detection.
The intrinsic affinity of DNA molecules toward metallic ions can drive the specific formation of ... more The intrinsic affinity of DNA molecules toward metallic ions can drive the specific formation of copper nanostructures within the nucleic acid helix structure in a sequence-dependent manner. The resultant nanostructures have interesting fluorescent and electrochemical properties, which are attractive for novel biosensing applications. However, the potential of using DNA-templated nanostructures for precision disease diagnosis remains unexplored. Particularly, DNAtemplated nanostructures show high potential for the universal amplification-free detection of different RNA biomarker species. Because of their low cellular levels and differing species-dependent length and sequence features, simultaneous detection of different messenger RNAs, microRNAs, and long non-coding RNAs species with a single technique is challenging. Here, we report a contemporary technique for facile in situ assembly of DNA-templated copper nanoblocks (CuNBs) on various RNA species targets after hybridization-based magnetic isolation. Our approach circumvents the typical limitations associated with amplification and labeling procedures of current RNA assays. The synthesized CuNBs enabled amplification-free fM-level RNA detection with flexible fluorescence or electrochemical readouts. Furthermore, our nanosensing technique displays potential for clinical application, as demonstrated by non-invasive analysis of three diagnostic RNA biomarkers from a cohort of 10 prostate cancer patient urinary samples with 100%-concordance (quantitative reverse transcriptionpolymerase chain reaction (PCR) validation). The good analytical performance and versatility of our method may be useful in both diagnostics and research fields.
The TMPRSS2:ERG gene fusion is one of a series of highly promising prostate cancer (PCa) biomarke... more The TMPRSS2:ERG gene fusion is one of a series of highly promising prostate cancer (PCa) biomarker alternatives to the controversial serum PSA. Current methods for detecting TMPRSS2:ERG are limited in terms of long processing time, high cost and the need for specialized equipment. Thus, there is an unmet need for less complex, faster, and cheaper methods to enable gene fusion detection in the clinic. We describe herein a simple, rapid and inexpensive assay which combines robust isothermal amplification technique with a novel visualization method for evaluating urinary TMPRSS2:ERG status at less than USD 5 and with minimal equipment. The assay is sensitive, and rapidly detects as low as 10 5 copies of TMPRSS2:ERG transcripts while maintaining high levels of specificity.
TMPRSS2 (Exon 1)-ERG (Exon 4) is the most frequent gene fusion event in prostate cancer (PC), and... more TMPRSS2 (Exon 1)-ERG (Exon 4) is the most frequent gene fusion event in prostate cancer (PC), and is highly PC-specific unlike the current serum prostate specific antigen (PSA) biomarker. However, TMPRSS2-ERG levels are currently measured with quantitative reverse-transcription PCR (RT-qPCR) which is time-consuming and requires costly equipment, thus limiting its use in clinical diagnostics. Herein, we report a novel rapid, cost-efficient and minimal-equipment assay named "FusBLU" for detecting TMPRSS2-ERG gene fusions from urine. TMPRSS2-ERG mRNA was amplified by isothermal reverse transcription-recombinase polymerase amplification (RT-RPA), magnetically-isolated, and detected through horseradish peroxidase (HRP)-catalyzed colorimetric reaction. FusBLU was specific for TMPRSS2-ERG mRNA with a low visual detection limit of 10 5 copies. We also demonstrated assay readout versatility on 3 potentially useful platforms. The colorimetric readout was detectable by naked eye for a quick yes/no evaluation of gene fusion presence. On the other hand, a more quantitative TMPRSS2-ERG detection was achievable by absorbance/electrochemical measurements. FusBLU was successfully applied to 12 urinary samples and results were validated by gold-standard RT-qPCR. We also showed that sediment RNA was likely the main source of TMPRSS2-ERG mRNA in urinary samples. We believe that our assay is a potential clinical screening tool for PC and could also have wide applications for other disease-related fusion genes.
Point mutations in DNA are useful biomarkers that can provide critical classification of disease ... more Point mutations in DNA are useful biomarkers that can provide critical classification of disease for accurate diagnosis and to inform clinical decisions. Conventional approaches to detect point mutations are usually based on technologies such as real-time polymerase chain reaction (PCR) or DNA sequencing, which are typically slow and require expensive lab-based equipment. While rapid isothermal strategies such as recombinase polymerase amplification (RPA) have been proposed, they tend to suffer from poor specificity in discriminating point mutations. Herein, we describe a novel strategy that enabled exquisite point mutation discrimination with isothermal DNA amplification, using mismatched primers in conjunction with a two-round enrichment process. As a proof of concept, the method was applied to the rapid and specific identification of drug-resistant Mycobacterium tuberculosis using RPA under specific conditions. The assay requires just picogram levels of genomic DNA input, is sensitive and specific enough to detect 10% point mutation loading, and can discriminate between closely related mutant variants within 30 min. The assay was subsequently adapted onto a low-cost 3D-printed isothermal device with real-time analysis capabilities to demonstrate a potential point-of-care application. Finally, the generic applicability of the strategy was shown by detecting three other clinically important cancer-associated point mutations. We believe that our assay shows potential in a broad range of healthcare screening processes for detecting and categorizing disease phenotypes at the point of care, thus reducing unnecessary therapy and cost in these contexts.
We report a new multiplexed strategy for the electrochemical detection of regional DNA methylatio... more We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl(-1) of DNA with no sequencing requirement.
The long non-coding RNA PARTICLE (Gene PARTICL- 'Promoter of MAT2A-Antisense RadiaTion Induce... more The long non-coding RNA PARTICLE (Gene PARTICL- 'Promoter of MAT2A-Antisense RadiaTion Induced Circulating LncRNA) partakes in triple helix (triplex) formation, is transiently elevated following low dose irradiation and regulates transcription of its neighbouring gene - Methionine adenosyltransferase 2A. It now emerges that PARTICLE triplex sites are predicted in many different genes across all human chromosomes. In silico analysis identified additional regions for PARTICLE triplexes at >1600 genomic locations. Multiple PARTICLE triplexes are clustered predominantly within the human and mouse tumor suppressor WW Domain Containing Oxidoreductase (WWOX) gene. Surface plasmon resonance diffraction and electrophoretic mobility shift assays were consistent with PARTICLE triplex formation within human WWOX with high resolution imaging demonstrating its enrichment at this locus on chromosome 16. PARTICLE knockdown and over-expression resulted in inverse changes in WWOX transcripts l...
Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative deter... more Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG's contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed-or deoxyinosinebase modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates. DNA methylation in higher eukaryotes occurs as a result of the addition of a methyl group to the carbon-5 position of cytosine-guanine (CpG) dinucleotides to form 5-methylcytosine. Due to the critical role of DNA methylation in a number of biological functions (e.g. X-chromosome inactivation, genomic imprinting and regulation of gene expression) 1-4 , a number of technologies have been developed to study and characterise the methylation patterns at a locus-specific level (e.g. DNA melting curve analysis, PCR-based methods). A common feature of these applications is the use of bisulfite modification 5-7 , wherein unmethylated cytosines are selectively deaminated to uracils and participate in subsequent PCR amplifications as thymines but 5-methylcytosines are left unaltered, which allows the visualisation of this epigenetic change using a variety of applications 8,9. As such, the bisulfite-conversion process results in a pool of templates which has cytosine/thymine degeneracy at the cytosine positions of CpG dinucleotides, and PCR primers designed against these regions must therefore be able to bind to both cytosines (methylated) and thymines (unmethylated) when unbiased amplification is sought. However, under non-optimized conditions a PCR primer that binds to a mixed-template CpG site may differentially amplify the methylated or unmethylated DNA strand, thereby inducing bias in the data and an inaccurate representation of the methylation state of the amplified products 10,11. Dealing with this potential bias represents a major challenge in bisulfite PCR assay design. The use of 'CpG-free' primers (i.e. primers that do not bind to a region with CpG dinucleotides) has been considered to
Simple nucleic acid detection methods could facilitate the progress of disease diagnostics for cl... more Simple nucleic acid detection methods could facilitate the progress of disease diagnostics for clinical uses. An attractive strategy is label-free surface-enhanced Raman scattering (SERS) due to its capability of providing structural fingerprinting of analytes that are close to or on nanomaterial surfaces. However, current label-free SERS approaches for DNA/RNA biomarker detection are limited to short and synthetic nucleic acid targets and have not been fully realized in clinical samples due to two possible reasons: (i) low target copies in limited patient samples and (ii) poor capability in identifying specific biomarkers from complex samples. To resolve these limitations and enable label-free SERS for clinical applications, we herein present a novel strategy based on multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) to enrich multiple RNA biomarkers, followed by label-free SERS with multivariate statistical analysis to directly detect, identify and dist...
The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its... more The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously (PrimerSuite), (ii) screen for primer-primer dimerizing artefacts (PrimerDimer), and (iii) support multiplex PCR assays (PrimerPlex). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).
Small (Weinheim an der Bergstrasse, Germany), Mar 22, 2016
Cancer diagnosis and patient monitoring require sensitive and simultaneous measurement of multipl... more Cancer diagnosis and patient monitoring require sensitive and simultaneous measurement of multiple cancer biomarkers considering that single biomarker analysis present inadequate information on the underlying biological transformations. Thus, development of sensitive and selective assays for multiple biomarker detection might improve clinical diagnosis and expedite the treatment process. Herein, a microfluidic platform for the rapid, sensitive, and parallel detection of multiple cancer-specific protein biomarkers from complex biological samples is presented. This approach utilizes alternating current electrohydrodynamic-induced surface shear forces that provide exquisite control over fluid flow thereby enhancing target-sensor interactions and minimizing non-specific binding. Further, the use of surface-enhanced Raman scattering-based spectral encoding with individual barcodes for different targets enables specific and simultaneous detection of captured protein biomarkers. Using this...
The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplificati... more The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community. Isothermal exponential amplification reaction (EXPAR) is an emerging amplification technique that is used to amplify short oligonucleotides for molecular diagnostics 1. In contrast with the conventional PCR, EXPAR operates at a constant temperature and provides high amplification efficiency, allowing 10 6-10 9 fold amplification of short oligonucleotides within minutes 2-4. Owing to its advantages, many studies have reported the use of EXPAR for molecular diagnostics 5-7. Briefly, EXPAR amplifies DNA by four major steps (Fig. 1). First, EXPAR is initiated when the target primes to the trigger sequence of the template, forming a partial double-stranded duplex. This is followed by the extension by DNA polymerase, forming an extended double-stranded DNA containing a nicking enzyme recognition site. A nicking enzyme then cleaves the upper strand and DNA polymerase displaces the cleaved trigger by strand displacement to generate additional trigger sequences. This occurs repeatedly and exponentially 1. Despite its advantages, target-independent amplification is a major limitation of EXPAR, which in turn, leads to lowered specificity and sensitivity 1,5,8. Non-specific amplification of EXPAR was suggested to arise from the interaction between the single-stranded template and the DNA polymerase independent of the target 3. Due to the issue of high levels of non-specific amplification with EXPAR, several studies have proposed ways to resolve this problem. Since the non-specific amplification is sequence dependent, Qian et al. demonstrated that the variability in template performance is linked to sequence motifs 9. Therefore, they developed computational methods to predict EXPAR template performance based on the template sequence. Wang et al. described the use of graphene oxide (GO) to minimize the level of non-specific amplification by preventing the binding of the DNA polymerase to the template 10. In the presence of a target, the template sequestered by GO can then be released, initiating EXPAR. This thus led us to hypothesize that other small molecules could also be used to enhance EXPAR performance.
Sensitive and accurate identification of specific DNA mutations can influence clinical decisions.... more Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samp...
au. Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterize... more au. Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells and an increased risk of late transformation to acute myeloid leukemia or primary myelofibrosis. Approximately 15% of MPN cases do not carry mutations in JAK2, CALR, or MPL and are thus often referred to as triple-negative cases. These are caused by a diverse set of rare mutations in cytokine receptors, JAK-STAT signaling pathway components, or epigenetic modifiers. In addition, some cases diagnosed as MPN are reactive rather than clonal disorders, so a negative result from a genetic screen can be informative. To obtain a comprehensive rapid molecular diagnosis for most MPNs, we developed an assay to detect genetic mutations (single nucleotide variants and/or small insertions/deletions) in 86 genes using targeted exon resequencing (AmpliSeq) and a bench-top semiconductor machine (Ion Torrent Personal Genome Machine). Our assay reliably detects well characterized mutations in JAK2, CALR, and MPL, but also rarer mutations in ASXL1, TET2, SH2B3, and other genes. Some of these mutations are novel. We find multiple mutations in advanced cases, suggesting cooperation between Janus kinase-STAT pathway mutations and epigenetic mutations in disease progression. This assay can be used to follow molecular progression, clonal heterogeneity, and drug resistance in MPNs.
A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers... more A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers for accurately distinguishing between benign and malignant cancer forms. This is causing high degree of overdiagnosis and overtreatment of otherwise clinically insignificant cases. As around half of all malignant PCa cases display a gene fusion mutation between the TMPRSS2 promoter sequence and the ERG coding sequence (TMPRSS2:ERG) detectable in urine; non-invasive screening of TMPRSS2:ERG mRNA in patient urine samples could improve specificity of current PCa diagnosis. However, current gene fusion detection methodologies are largely dependent on RNA enzymatic amplification, which requires extensive sample manipulation, costly labels for detection, and it is prone to bias/artifacts. Herein we introduce the first successful amplification-free electrochemical assay for direct detection of TMPRSS2:ERG mRNA in PCa urinary samples by selectively isolating and adsorbing TMPRSS2:ERG mRNA onto bare gold electrodes without requiring any surface modification. We demonstrated excellent limit-of-detection (10 cells) and specificity using PCa cell line models; and showcased clinical utility by accurately detecting TMPRSS2:ERG in a collection of 17 urinary samples obtained from PCa patients. Furthermore, these results were validated with current gold standard reverse transcription (RT)-PCR approach with 100% concordance.
Effective disease management strategies to prevent catastrophic crop losses require rapid, sensit... more Effective disease management strategies to prevent catastrophic crop losses require rapid, sensitive and multiplexed detection methods for timely decision making. To address this need, a rapid, highly specific and sensitive point-of-care method for multiplex detection of plant pathogens was developed by taking advantages of surface-enhanced Raman scattering (SERS) labelled nanotags and recombinase polymerase amplification (RPA), which is a rapid isothermal amplification method with high specificity. In this study, three agriculturally important plant pathogens (Botrytis cinerea, Pseudomonas syringae, and Fusarium oxysporum) were used to demonstrate potential translation into the field. The RPA-SERS method was faster, more sensitive than polymerase chain reaction and could detect as little as 2 copies of B. cinerea DNA. Furthermore, multiplex detection of the three pathogens was demonstrated for complex systems such as the Arabidopsis thaliana plant and commercial tomato crops. To demonstrate the potential for on-site field applications, a rapid single-tube RPA/SERS assay was further developed and successfully performed for a specific target outside of a laboratory setting.
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