Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing

Billah, Morsaline; Hodges, Chris; Hays, Henry C.W.; Millner, Paul A.

doi:10.1016/j.bioelechem.2010.08.005

Cited by 46 publications

(24 citation statements)

References 35 publications

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“…This irregular texture may prevent the analyte from binding to the receptor when is confined at the surface. The effect of steric hindrance has been previously identified as an issue in biosensor fabrication [23,30,44].…”

Section: Resultsmentioning

confidence: 99%

“…The polymer modified electrodes were incubated with 5 mM sulfosuccinimidyl 4-[Nmaleimidomethyl] cyclohexane-1-carboxylate (Sulfo-SMCC, Sigma) solution in 10 mM PBS-EDTA (10mM) pH 7.2 for one hour, as described in previous papers [30].Freshly reduced nanobodies (from 2.2) were then added to the sulfo-SMCC modified electrode and incubated for 1 hr. These electrodes were incubated for 30 min in PBS pH 7.4 to remove any non-specifically bound receptor.…”

Section: Oriented Conjugation Of Spacer Modified Nanobodiesmentioning

confidence: 99%

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The development and optimisation of nanobody based electrochemical immunosensors for IgG

Goode

Dillon

Millner

2016

Sensors and Actuators B: Chemical

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Article:Goode, J orcid.org/0000-0002-3642-1291, Dillon, G and Millner, PA orcid.org/0000-0002-4813-4302 (2016 ReuseUnless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. This has led to the development of alternative immuno-reagents such as non-antibody binding proteins (NABPs). These are low molecular weight proteins which largely avoid the aforementioned advantages of antibodies. They are commonly produced by bacteria enabling the use of DNA technology to manipulate bioreceptors at the molecular level.Single chain VHHs (commonly known as nanobodies) are an antibody derived NABP adapted from camelid heavy chain antibodies which are the isolated binding domain. Whilst nanobodies have been used for diagnostic and therapeutic applications, they have limited demonstration in biosensors.In this study, both antibodies and nanobodies were used to construct a biosensor. In addition nanobody performance was optimised by introducing a novel peptide spacer. The role of nanobody orientation and spacing was thus investigated and spacer length was optimised, leading to an increase in the sensitivity of the biosensor. Highlights: Nanobodies have been used on impedimetric immunosensors to detect rabbit IgG  Unmodified nanobody sensors displayed a decrease in resistance upon analyte recognition.  Nanobodies were modified with a peptide spacer, effectively reversing this phenomena.  The use of a spacer also increased the sensitivity of the immunosensor.

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Section: Resultsmentioning

confidence: 99%

Section: Oriented Conjugation Of Spacer Modified Nanobodiesmentioning

confidence: 99%

The development and optimisation of nanobody based electrochemical immunosensors for IgG

Goode

Dillon

Millner

2016

Sensors and Actuators B: Chemical

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“…This can be measured by monitoring the changes in the electron transfer capability of well-known redox systems, such as [Fe(CN)6] 4− /[Fe(CN)6] 3− . Indirect ways of measuring such alterations include EIS and CV [33]. EIS studies were used to follow the Au-SPE modification after each chemical change.…”

Section: Control Of the Surface Modificationmentioning

confidence: 99%

Novel biosensing device for point-of-care applications with plastic antibodies grown on Au-screen printed electrodes

Moreira

Dutra

Noronha

et al. 2013

Sensors and Actuators B: Chemical

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A gold screen printed electrode (Au-SPE) was modified by merging Molecular Imprinting and Self-Assembly Monolayer techniques for fast screening cardiac biomarkers in point-of-care (POC). For this purpose, Myoglobin (Myo) was selected as target analyte and its plastic antibody imprinted over a glutaraldehyde (Glu)/cysteamine (Cys) layer on the gold-surface. The imprinting effect was produced by growing a reticulated polymer of acrylamide (AAM) and N,N * -methylenebisacrylamide (NNMBA) around the Myo template, covalently attached to the biosensing surface. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) studies were carried out in all chemical modification steps to confirm the surface changes in the Au-SPE.The analytical features of the resulting biosensor were studied by different electrochemical techniques, including EIS, square wave voltammetry (SWV) and potentiometry. The limits of detection ranged from 0.13 to 8 µg/mL. Only potentiometry assays showed limits of detection including the cut-off Myo levels. Quantitative information was also produced for Myo concentrations ≥0.2 µg/mL. The linear response of the biosensing device showed an anionic slope of ∼70 mV per decade molar concentration up to 0.3 µg/mL. The interference of coexisting species was tested and good selectivity was observed. The biosensor was successfully applied to biological fluids.

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“…The currently used techniques are in principle based on these studies and they have been applied to a variety of IgG molecules (Kato et al 1976, Karyakin et al 2000, Cho et al 2007, Pereira and Lai 2008, Cho et al 2009, Billah et al 2010, Ho et al 2010, Hu et al 2010, KausaiteMinkstimiene et al 2010, Wu et al 2014). Another of their advantages is that in cases when the amount of antibody is limited, it enables the reduction and/or conjugation procedures to be tested using other IgG antibodies at first.…”

Section: Introductionmentioning

confidence: 99%

“…One of them is following the established protocol (Hermanson 2008) not taking the antibody type into consideration (Karyakin et al 2000, Billah et al 2010, Ho et al 2010, Hu et al 2010. The second one is tailoring the reduction conditions to particular antibodies resulting in many rather widely distributed adaptations of the original protocol (Kato et al 1976, Cho et al 2007, Pereira and Lai 2008, Kausaite-Minkstimiene et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Considerations in producing preferentially reduced half-antibody fragments

Makaraviciute

Jackson

Millner

et al. 2016

Journal of Immunological Methods

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Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

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Directed immobilization of reduced antibody fragments onto a novel SAM on gold for myoglobin impedance immunosensing

Cited by 46 publications

References 35 publications

The development and optimisation of nanobody based electrochemical immunosensors for IgG

The development and optimisation of nanobody based electrochemical immunosensors for IgG

Novel biosensing device for point-of-care applications with plastic antibodies grown on Au-screen printed electrodes

Considerations in producing preferentially reduced half-antibody fragments

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