64 results found
Al Sulaiman D, Gatehouse A, Ivanov AP, et al., 2021, Length-Dependent, Single-Molecule Analysis of Short Double-Stranded DNA Fragments through Hydrogel-Filled Nanopores: A Potential Tool for Size Profiling Cell-Free DNA, ACS APPLIED MATERIALS & INTERFACES, Vol: 13, Pages: 26673-26681, ISSN: 1944-8244
Cai S, Pataillot-Meakin T, Shibakawa A, et al., 2021, Single-molecule amplification-free multiplexed detection of circulating microRNA cancer biomarkers from serum, Nature Communications, Vol: 12, ISSN: 2041-1723
MicroRNAs (miRNAs) play essential roles in post-transcriptional gene expression and are also found freely circulating in bodily fluids such as blood. Dysregulated miRNA signatures have been associated with many diseases including cancer, and miRNA profiling from liquid biopsies offers a promising strategy for cancer diagnosis, prognosis and monitoring. Here, we develop size-encoded molecular probes that can be used for simultaneous electro-optical nanopore sensing of miRNAs, allowing for ultrasensitive, sequence-specific and multiplexed detection directly in unprocessed human serum, in sample volumes as small as 0.1 μl. We show that this approach allows for femtomolar sensitivity and single-base mismatch selectivity. We demonstrate the ability to simultaneously monitor miRNAs (miR-141-3p and miR-375-3p) from prostate cancer patients with active disease and in remission. This technology can pave the way for next generation of minimally invasive diagnostic and companion diagnostic tests for cancer.
Gillespie P, Channon RB, Meng X, et al., 2021, Nucleic acid sensing via electrochemical oligonucleotide-templated reactions, BIOSENSORS & BIOELECTRONICS, Vol: 176, ISSN: 0956-5663
Channon RB, Gillespie P, Nazmul Islam M, et al., 2020, Electrochemical oligonucleotide templated reactions, Pages: 476-477
The biosensing of nucleic acids is an excellent approach for medical diagnosis, however established nucleic acid sensing technologies are typically limited by slow throughput, bulky equipment and the difficulty in achieving trace sensitivity with single nucleotide specificity. Here, we describe the first example of an electrochemical oligonucleotide templated reaction (EOTR). The target nucleic acid acts as a template for two probe-modified peptide nucleic acids. Reaction of the probe heads then generates an electrochemically active adduct. We couple EOTR with a lateral flow assay platform, towards developing a screening test for prostate cancer specific miRNA.
Delcassian D, Patel A, 2019, Nanotechnology and Drug Delivery, Bioengineering Innovative Solutions for Cancer, Editors: Ladame, Chang, Publisher: Academic Press, ISBN: 9780128138861
By covering the many different ways engineers can deliver innovative solutions to tackle cancer, this book is a valuable read for researchers who have an ambition to make an impact on people’s life in either an academic or industrial ...
Pavagada S, Channon RB, Chang JYH, et al., 2019, Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs (vol 55, pg 12451, 2019), CHEMICAL COMMUNICATIONS, Vol: 55, Pages: 13470-13470, ISSN: 1359-7345
Pavagada S, Channon RB, Chang JYH, et al., 2019, Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs, Chemical Communications, Vol: 55, Pages: 12451-12454, ISSN: 1359-7345
Herein we demonstrate the first example of oligonucleotide-templated reaction (OTR) performed on paper, using lateral flow to capture and concentrate specific nucleic acid biomarkers on a test line. Quantitative analysis, using a low-cost benchtop fluorescence reader showed very high specificity down to the single nucleotide level and proved sensitive enough for amplification-free, on-chip, detection of endogenous concentrations of miR-150-5p, a recently identified predictive blood biomarker for preterm birth.
Al Sulaiman D, Chang JYH, Bennett NR, et al., 2019, Hydrogel-coated microneedle arrays for minimally invasive sampling and sensing of specific circulating nucleic acids from skin interstitial fluid, ACS Nano, Vol: 13, Pages: 9620-9628, ISSN: 1936-0851
Minimally invasive technologies that can sample and detect cell-free nucleic acid biomarkers from liquid biopsies have recently emerged as clinically useful for early diagnosis of a broad range of pathologies, including cancer. Although blood has so far been the most commonly interrogated bodily fluid, skin interstitial fluid has been mostly overlooked despite containing the same broad variety of molecular biomarkers originating from cells and surrounding blood capillaries. Emerging technologies to sample this fluid in a pain-free and minimally-invasive manner often take the form of microneedle patches. Herein, we developed microneedles that are coated with an alginate–peptide nucleic acid hybrid material for sequence-specific sampling, isolation, and detection of nucleic acid biomarkers from skin interstitial fluid. Characterized by fast sampling kinetics and large sampling capacity (∼6.5 μL in 2 min), this platform technology also enables the detection of specific nucleic acid biomarkers either on the patch itself or in solution after light-triggered release from the hydrogel. Considering the emergence of cell-free nucleic acids in bodily fluids as clinically informative biomarkers, platform technologies that can detect them in an automated and minimally invasive fashion have great potential for personalized diagnosis and longitudinal monitoring of patient-specific disease progression.
Gillespie P, Ladame S, O'Hare D, 2019, Molecular methods in electrochemical microRNA detection, ANALYST, Vol: 144, Pages: 114-129, ISSN: 0003-2654
Channon RB, Pavagada S, Chang JYH, et al., 2019, Point-of-care nucleic Acid sensors via paper-based oligonucleotide-templated reactions, Pages: 815-816
This paper describes a new and inexpensive approach for specific sensing of endogenous concentrations of miRNAs extracted from blood, based on a fluorogenic oligonucleotide templated reaction (OTR) on a lateral flow assay (LFA). Our method is then applied to develop the first early screening test for Preterm birth.
Pavagada S, Ladame S, 2018, Platforms for bioorthogonal oligonucleotide-templated reactions: Translating Concepts into devices, Chimia (Aarau), Vol: 72, Pages: 809-814, ISSN: 0009-4293
The exponential improvements made in DNA sequencing technologies, together with the rapidly declining associated costs, has increasingly led to the expansion of the field of personalised genomic medicine. Changes in the sequence or copy number of specific deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules represent key signatures for the diagnosis, prognosis, classification and monitoring of a broad range of pathologies, most notably cancer. Technologies that can detect these changes require analytical tools that can detect DNA or RNA with high sensitivity and high specificity. Sensing based on bioorthogonal oligonucleotide-templated reactions (OTRs) has been recognised as an elegant strategy that satisfies these criteria and was successfully used for the quantitative detection of nucleic acids both in vitro and in vivo. Herein, we will focus on recent efforts to implement bioorthogonal OTRs into clinically useful biosensors using probes immobilised on or embedded in customised materials and platforms.
Al Sulaiman D, Cadinu P, Ivanov AP, et al., 2018, Chemically modified hydrogel-filled nanopores: a tunable platform for single-molecule sensing, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 18, Pages: 6084-6093, ISSN: 1530-6984
Label-free, single-molecule sensing is an ideal candidate for biomedical applications that rely on the detection of low copy numbers in small volumes and potentially complex biofluids. Among them, solid-state nanopores can be engineered to detect single molecules of charged analytes when they are electrically driven through the nanometer-sized aperture. When successfully applied to nucleic acid sensing, fast transport in the range of 10–100 nucleotides per nanosecond often precludes the use of standard nanopores for the detection of the smallest fragments. Herein, hydrogel-filled nanopores (HFN) are reported that combine quartz nanopipettes with biocompatible chemical poly(vinyl) alcohol hydrogels engineered in-house. Hydrogels were modified physically or chemically to finely tune, in a predictable manner, the transport of specific molecules. Controlling the hydrogel mesh size and chemical composition allowed us to slow DNA transport by 4 orders of magnitude and to detect fragments as small as 100 base pairs (bp) with nanopores larger than 20 nm at an ionic strength comparable to physiological conditions. Considering the emergence of cell-free nucleic acids as blood biomarkers for cancer diagnostics or prenatal testing, the successful sensing and size profiling of DNA fragments ranging from 100 bp to >1 kbp long under physiological conditions demonstrates the potential of HFNs as a new generation of powerful and easily tunable molecular diagnostics tools.
Cadinu P, Paulose Nadappuram B, Lee DJ, et al., 2017, Single molecule trapping and sensing using dual nanopores separated by a zeptoliter nanobridge, Nano Letters, Vol: 17, Pages: 6376-6384, ISSN: 1530-6984
There is a growing realization, especially within the diagnostic and therapeutic community, that the amount of information enclosed in a single molecule can not only enable a better understanding of biophysical pathways, but also offer exceptional value for early stage biomarker detection of disease onset. To this end, numerous single molecule strategies have been proposed, and in terms of label-free routes, nanopore sensing has emerged as one of the most promising methods. However, being able to finely control molecular transport in terms of transport rate, resolution, and signal-to-noise ratio (SNR) is essential to take full advantage of the technology benefits. Here we propose a novel solution to these challenges based on a method that allows biomolecules to be individually confined into a zeptoliter nanoscale droplet bridging two adjacent nanopores (nanobridge) with a 20 nm separation. Molecules that undergo confinement in the nanobridge are slowed down by up to 3 orders of magnitude compared to conventional nanopores. This leads to a dramatic improvement in the SNR, resolution, sensitivity, and limit of detection. The strategy implemented is universal and as highlighted in this manuscript can be used for the detection of dsDNA, RNA, ssDNA, and proteins.
Al Sulaiman D, Chang JYH, Ladame S, 2017, Subnanomolar detection of oligonucleotides via templated fluorogenic reaction in hydrogels: controlling diffusion to improve sensitivity, Angewandte Chemie - International Edition, Vol: 56, Pages: 5247-5251, ISSN: 1433-7851
Oligonucleotide-templated reactions are valuable tools for nucleic acid sensing both in vitro and in vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide-templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non-specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100 pm.
Al Sulaiman D, Chang JYH, Ladame S, 2017, Subnanomolar detection of oligonucleotides through templated fluorogenic reaction in hydrogels: Controlling diffusion to improve sensitivity, Angewandte Chemie, Vol: 129, Pages: 5331-5335, ISSN: 0044-8249
Oligonucleotide‐templated reactions are valuable tools for nucleic acid sensing both in vitro and in vivo. They are typically carried out under conditions that make any reaction in the absence of template highly unfavorable (most commonly by using a low concentration of reactants), which has a negative impact on the detection sensitivity. Herein, we report a novel platform for fluorogenic oligonucleotide‐templated reactions between peptide nucleic acid probes embedded within permeable agarose and alginate hydrogels. We demonstrate that under conditions of restricted mobility (that is, limited diffusion), non‐specific interactions between probes are prevented, thus leading to lower background signals. When applied to nucleic acid sensing, this accounts for a significant increase in sensitivity (that is, lower limit of detection). Optical nucleic acid sensors based on fluorogenic peptide nucleic acid probes embedded in permeable, physically crosslinked, alginate beads were also engineered and proved capable of detecting DNA concentrations as low as 100 pm.
Al Sulaiman D, Metcalf GAD, Ladame S, 2017, Engineering innovative solutions to screen for prostate cancer, CONVERGENT SCIENCE PHYSICAL ONCOLOGY, Vol: 3, ISSN: 2057-1739
Zuffo M, Ladame S, Doria F, et al., 2017, Tuneable coumarin-NDI dyads as G-quadruplex specific light-up probes, SENSORS AND ACTUATORS B-CHEMICAL, Vol: 245, Pages: 780-788, ISSN: 0925-4005
Panich S, Sleiman MH, Steer I, et al., 2016, Real-Time Monitoring of Ligand Binding to G-Quadruplex and Duplex DNA by Whispering Gallery Mode Sensing, ACS Sensors, Vol: 1, Pages: 1097-1102, ISSN: 2379-3694
The therapeutic potential of small molecules targeting G-quadruplexes has gained credibility since such structures were shown to form in human cells and to be highly prevalent in the human genome, most notably at telomere ends and in oncogene promoters. Herein, we perform whispering gallery mode (WGM) sensing for monitoring DNA–small molecule interactions. Unlike most existing technologies, WGM sensing offers numerous advantages including high sensitivity, real-time analysis, easy access to kinetic parameters, and much lower cost than current gold standards. In this work, interactions of five known DNA-binding ligands with either G-quadruplex or duplex DNA immobilized on a sphere microresonator have been assessed. The induced shift of the resonant mode from quadruplex (or duplex)–ligand binding was used to estimate kinetic parameters. Association and dissociation rate constants (kon and koff, respectively) as well as dissociation equilibrium constants (KD) were measured for these five ligands binding to both duplex and quadruplex DNA.
Metcalf GAD, Shibakawa A, Patel H, et al., 2016, Amplification-free detection of circulating microRNA biomarkers from body fluids based on fluorogenic oligonucleotide-templated reaction between engineered peptide nucleic acid probes: application to prostate cancer diagnosis, Analytical Chemistry, Vol: 88, Pages: 8091-8098, ISSN: 0003-2700
Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening.
Zuffo M, Doria F, Spalluto V, et al., 2015, Red/NIR G-Quadruplex Sensing, Harvesting Blue Light by a Coumarin-Naphthalene Diimide Dyad, CHEMISTRY-A EUROPEAN JOURNAL, Vol: 21, Pages: 17596-17600, ISSN: 0947-6539
Choi Y, Metcalf G, Haj Sleiman M, et al., 2014, Oligonucleotide-templated reactions based on Peptide Nucleic Acid (PNA) probes: Concept and biomedical applications., Bioorganic and Medicinal Chemistry
Sensing technologies based on Peptide Nucleic Acids (PNAs) and oligonucleotide-templated chemistry are perfectly suited for biomedical applications (e.g., diagnosis, prognosis and stratification of diseases) and could compete well with more traditional amplification technologies using expensive dual-labelled oligonucleotide probes. PNAs can be easily synthesised and functionalised, are more stable and are more responsive to point-mutations than their DNA counterpart. For these reasons, fluorogenic PNAs represent an interesting alternative to DNA-based molecular beacons for sensing applications in a cell-free environment, where cellular uptake is not required.
Fallah-Araghi A, Meguellati K, Baret J-C, et al., 2014, Enhanced Chemical Synthesis at Soft Interfaces: A Universal Reaction-Adsorption Mechanism in Microcompartments, PHYSICAL REVIEW LETTERS, Vol: 112, ISSN: 0031-9007
Sleiman MH, Ladame S, 2014, Synthesis of squaraine dyes under mild conditions: applications for labelling and sensing of biomolecules, CHEMICAL COMMUNICATIONS, Vol: 50, Pages: 5288-5290, ISSN: 1359-7345
Najah M, Mayot E, Mahendra-Wijaya IP, et al., 2013, New Glycosidase Substrates for Droplet-Based Microfluidic Screening, ANALYTICAL CHEMISTRY, Vol: 85, Pages: 9807-9814, ISSN: 0003-2700
Percivalle C, Mahmood T, Ladame S, 2013, Two-in-one: a pH-sensitive, acridine-based, fluorescent probe binds G-quadruplexes in oncogene promoters, MEDCHEMCOMM, Vol: 4, Pages: 211-215, ISSN: 2040-2503
Percivalle C, Bartolo J-F, Ladame S, 2013, Oligonucleotide-templated chemical reactions: pushing the boundaries of a nature-inspired process, ORGANIC & BIOMOLECULAR CHEMISTRY, Vol: 11, Pages: 16-26, ISSN: 1477-0520
Meguellati K, Fallah-Araghi A, Baret J-C, et al., 2013, Enhanced imine synthesis in water: from surfactant-mediated catalysis to host-guest mechanisms, CHEMICAL COMMUNICATIONS, Vol: 49, Pages: 11332-11334, ISSN: 1359-7345
Mahmood T, Wu Y, Loriot D, et al., 2012, Closing the ring to bring up the light: synthesis of a hexacyclic acridinium cyanine dye., Chemistry, Vol: 18, Pages: 12349-12356
The synthesis of a geometrically constrained and near-planar hexacyclic acridinium cyanine dye 9 is reported. When compared to its unlocked and non-fluorescent monomethine cyanine dye analogue 3, this photostable dye emits in the green area of the spectrum with a remarkable quantum yield close to unity in organic solvents and above 0.5 in water. A detailed steady-state and time-resolved spectroscopic study revealed that dye 9 forms emissive aggregates in water, which are responsible for a red-shifted and broadened emission band and longer emission lifetime, τ≈33 compared to 6.5-7.0 ns for the monomeric dye. Dye 9 also binds strongly to DNA (both duplex and quadruplex) in its monomeric form and is very efficiently taken up by cells, in which it accumulates primarily into the nucleus.
Meguellati K, Ladame S, 2012, Reversible Covalent Chemistries Compatible with the Principles of Constitutional Dynamic Chemistry: New Reactions to Create More Diversity, CONSTITUTIONAL DYNAMIC CHEMISTRY, Vol: 322, Pages: 291-314, ISSN: 0340-1022
Meguellati K, Ladame S, Spichty M, 2011, A conceptually improved TD-DFT approach for predicting the maximum absorption wavelength of cyanine dyes, DYES AND PIGMENTS, Vol: 90, Pages: 114-118, ISSN: 0143-7208
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