Modified primers for rapid and direct electrochemical analysis of coeliac disease associated HLA alleles

Joda, Hamdi; Beni, Valerio; Willems, Andreas; Frank, Rainer; Höth, Julian; Lind, Kristina; Strömbom, Linda; Katakis, Ioanis; O’Sullivan, Ciara K.

doi:10.1016/j.bios.2015.05.048

Cited by 14 publications

(7 citation statements)

References 37 publications

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“…Recombinase polymerase amplification was used as this method of isothermal amplification is remarkable for its simplicity, not requiring any initial melting or multiple primers. Furthermore, we exploited a methodology we previously reported for PCR amplification, where modified primers, referred to as tailed primers, are used to result in amplicons of double stranded DNA with two single stranded DNA tails on each end . This is facilitated by a “stopper”, which can be C3, C9, C18, which is located between the primer binding site and the single stranded tail and effectively acts to prevent the polymerase from further elongation.…”

Section: Resultsmentioning

confidence: 99%

Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers

et al. 2016

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In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which β-conglutin immobilized on the test line of a nitrocellulose membrane and β-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the β-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized β-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

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Section: Resultsmentioning

confidence: 99%

Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers

et al. 2016

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“…A 10 nM solution of horseradish peroxidase (HRP)‐labeled detecting probe (loaded in tank c Fig. ) was flowed over the electrodes and incubated for 2 min . The amperometric measurements were carried out by injection of TMB Liquid Mixture from Sigma (loaded in tank d , Fig.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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Celiac 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.

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“…Another application of a sandwich hybridization assay, using immobilized capture and soluble reporter probes, has been reported for the detection of PCR-amplified DNA sequences associated with celiac disease [81]. A 4 Â 4 planar array of 1 mm 2 gold working electrodes was used with shared, coplanar counter, and reference electrodes and was sealed into a microfluidic housing.…”

Section: Electrochemical Nucleic Acid Arraysmentioning

confidence: 99%

Electrochemical Arrays for Bioassay Applications

Cortón

Mikkelsen

2016

Trends in Bioelectroanalysis

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This chapter presents a survey of developments and bioassay applications of individually addressable electrochemical arrays covering the past two decades and including over 90 references. Many different array designs have been fabricated, from macroscopic devices that have two to four sensing elements to ultramicroelectrode arrays containing as many as 12,000 individually addressable micrometer-sized working electrodes. Some inexpensively fabricated arrays are intended for single sample analysis and are disposable, while others are encapsulated for incorporation into reusable microfluidic systems. Devices and assays are grouped according to the types of modifications done to the sensing elements, and sections are included to consider unmodified elements as well as modifications with enzymes, antibodies or antigens, nucleic acids, in addition to viable cells and tissues. Some trends have been identified, and possible future directions are suggested.

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Modified primers for rapid and direct electrochemical analysis of coeliac disease associated HLA alleles

Cited by 14 publications

References 37 publications

Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers

Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers

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

Electrochemical Arrays for Bioassay Applications

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