Electrochemical genosensor for the direct detection of tailed PCR amplicons incorporating ferrocene labelled dATP

An electrochemical genosensor for the detection and quantification of Karlodinium armiger is presented. The genosensor exploits tailed primers and ferrocene labelled dATP analogue to produce PCR products that can be directly hybridised on a gold electrode array and quantitatively measured using square wave voltammetry. Tailed primers consist of a sequence specific for the target, followed by a carbon spacer and a sequence specifically designed not to bind to genomic DNA, resulting in a duplex flanked by single stranded binding primers. The incorporation of the 7-(ferrocenylethynyl)-7-deaza-2′-deoxyadenosine triphosphate was optimised in terms of a compromise between maximum PCR efficiency and the limit of detection and sensitivity attainable using electrochemical detection via hybridisation of the tailed, ferrocene labelled PCR product. A limit of detection of 277aM with a linear range from 315aM to 10 fM starting DNA concentration and a sensitivity of 122 nA decade−1 was achieved. The system was successfully applied to the detection of genomic DNA in real seawater samples.info:eu-repo/semantics/acceptedVersio

Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides.

Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping

Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification

Aptamer-based assays and sensors are garnering increasing interest as alternatives to antibodies, particularly due to their increased flexibility for implementation in alternative assay formats, as they can be employed in assays designed for nucleic acids, such as molecular aptamer beacons or aptamer detection combined with amplification. In this work, we took advantage of the inherent nucleic acid nature of aptamers to enhance sensitivity in a rapid and facile assay format. An aptamer selected against the anaphylactic allergen β-conglutin was used to demonstrate the proof of concept. The aptamer was generated by using biotinylated dUTPs, and the affinity of the modified aptamer as compared to the unmodified aptamer was determined by using surface plasmon resonance to calculate the dissociation constant (KD), and no significant improvement in affinity due to the incorporation of the hydrophobic biotin was observed. The modified aptamer was then applied in a colorimetric competitive enzyme-linked oligonucleotide assay, where β-conglutin was immobilized on the wells of a microtiter plate, competing with β-conglutin free in solution for the binding to the aptamer. The limit of detection achieved was 68 pM, demonstrating an improvement in detection limit of three orders of magnitude as compared with the aptamer simply modified with a terminal biotin label. The concept was then exploited by using electrochemical detection and screen-printed electrodes where detection limits of 326 fM and 7.89 fM were obtained with carbon and gold electrodes, respectively. The assay format is generic in nature and can be applied to all aptamers, facilitating an easy and cost-effective means to achieve lower detection limits

Liposomal Nanoreactors for the Synthesis of Monodisperse Palladium Nanoparticles Using Glycerol

The synthesis of highly stable ultrasmall monodisperse populations of palladium nanoparticles in the range of 1-3 nm in size was achieved via polyol reduction within 1,2-dioleoyl-sn-glycero-3-phosphor-rac(1-glycerol) liposomal nanoreactors exploiting glycerol as both reducing and stabilizing agent. The liposome-based green method was compared with synthesis in solution, and the reducing agent concentration and the lipidic composition of the liposomal nanoreactors were demonstrated to have a strong effect on the final size and homogeneity of the palladium nanoparticles. Glycerol molecules acting as capping agent demonstrated the ability to stabilize the palladium nanoparticles over a long period of time, maintaining their homogeneity in size and shape. The obtained palladium nanoparticles were characterized using transmission electron microscopy, selected area electron diffraction, Fourier transform infrared and Raman spectroscopies, X-ray diffraction, and dynamic light scattering to determine their morphology, size, charge, surface chemistry, and crystal structure. The catalytic activity of the palladium nanoparticles was also tested for a reduction reaction

Postfunctionalization of Keggin silicotungstates by general coupling procedures

International audienceno abstrac

Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products

International audienceno abstrac

Bleed-to-read disposable microsystems for the genetic and serological analysis of celiac disease markers with amperometric detection

Coeliac disease is an auto-immune disorder induced by ingestion of gluten in genetically predisposed individuals. Its diagnostics is more accurate using a combination of immunologic and genetic tests to detect of high levels of certain auto-antibodies and the presence human leukocyte antigen HLA-DQ2 or HLA-DQ8 genetic markers. In this work, we report the design and testing of automated microsystems combining sample treatment, storage, fluidic transport and detection in a single platform able to carry out genetic or serologic analysis for detection of celiac disease markers. These microsystems share a common footprint and many fluidic features and are thus able to perform a complete assay. The microsystem for the genetic assay extracts and amplifies the DNA prior to detection, while the serology microsystem contains a filter and chamber for the generation and subsequent dilution of plasma. The performance of both platforms is demonstrated and compared with reference methods with an excellent correlation, which makes the developed platform amenable for clinical studies