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      Aptamer-modified gold nanoparticles for rapid aggregation-based detection of inflammation: an optical assay for interleukin-6

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          Abstract

          A proof-of-concept aptamer-based optical assay is described for the determination of the immuno signalling molecule interleukin-6 (IL-6), a key marker of acute inflammation. The optical assay is based on the aggregation of gold nanoparticles (AuNP) coated in two complimentary “sandwich-style” aptamers, each with different IL-6 target moieties. IL-6 will recognise the complimentary aptamer pair and bind to it, thereby causing the aggregation of the corresponding functionalised nanoparticles. The aggregation of the AuNPs after exposure to IL-6 induces a visible colour change from red to pink, with a corresponding change in the absorption maximum from 520 to 540 nm. The change in the absorption maximum can be monitored visually, or by using a spectrophotometer or a plate reader. The optimal size and functionalisation of aptamer-coated AuNPs, and the potential assay formats were investigated using UV-vis spectrophotometry, transmission electron microscopy, and dynamic light scattering. The optical assay was applied for detecting mouse IL-6 in a mixed protein solution as a representative biological sample. The assay works in the 3.3 to 125 μg·mL −1 IL-6 concentration range, and the detection limit (at S/ N = 3) is 1.95 μg·mL −1. This study was performed as a proof-of-concept demonstration of this versatile assay design, with a view to developing a similar assay for use in clinical samples in future.

          Graphical abstract

          Schematic representation of the aggregation of aptamer-functionalised nanoparticles in the presence of interleukin-6 (IL-6). The presence of mouse IL-6 in a mixed protein solution leads to a visible colour change, and a change in the absorption spectrum of the nanoparticles.

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          The online version of this article (10.1007/s00604-019-3975-7) contains supplementary material, which is available to authorized users.

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          Most cited references26

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          Kinetically controlled seeded growth synthesis of citrate-stabilized gold nanoparticles of up to 200 nm: size focusing versus Ostwald ripening.

          Monodisperse citrate-stabilized gold nanoparticles with a uniform quasi-spherical shape of up to ∼200 nm and a narrow size distribution were synthesized following a kinetically controlled seeded growth strategy via the reduction of HAuCl(4) by sodium citrate. The inhibition of any secondary nucleation during homogeneous growth was controlled by adjusting the reaction conditions: temperature, gold precursor to seed particle concentration, and pH. This method presents improved results regarding the traditional Frens method in several aspects: (i) it produces particles of higher monodispersity; (ii) it allows better control of the gold nanoparticle size and size distribution; and (iii) it leads to higher concentrations. Gold nanoparticles synthesized following this method can be further functionalized with a wide variety of molecules, hence this method appears to be a promising candidate for application in the fields of biomedicine, photonics, and electronics, among others. © 2011 American Chemical Society
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            Development of DNA aptamers using Cell-SELEX.

            In the past two decades, high-affinity nucleic acid aptamers have been developed for a wide variety of pure molecules and complex systems such as live cells. Conceptually, aptamers are developed by an evolutionary process, whereby, as selection progresses, sequences with a certain conformation capable of binding to the target of interest emerge and dominate the pool. This protocol, cell-SELEX (systematic evolution of ligands by exponential enrichment), is a method that can generate DNA aptamers that can bind specifically to a cell type of interest. Commonly, a cancer cell line is used as the target to generate aptamers that can differentiate that cell type from other cancers or normal cells. A single-stranded DNA (ssDNA) library pool is incubated with the target cells. Nonbinding sequences are washed off and bound sequences are recovered from the cells by heating cell-DNA complexes at 95 degrees C, followed by centrifugation. The recovered pool is incubated with the control cell line to filter out the sequences that bind to common molecules on both the target and the control, leading to the enrichment of specific binders to the target. Binding sequences are amplified by PCR using fluorescein isothiocyanate-labeled sense and biotin-labeled antisense primers. This is followed by removal of antisense strands to generate an ssDNA pool for subsequent rounds of selection. The enrichment of the selected pools is monitored by flow cytometry binding assays, with selected pools having increased fluorescence compared with the unselected DNA library. The procedure, from design of oligonucleotides to enrichment of the selected pools, takes approximately 3 months.
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              Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay.

              The application of antibodies in enzyme-linked immunosorbent assay (ELISA) is the basis of this diagnostic technique which is designed to detect a potpourri of complex target molecules such as cell surface antigens, allergens, and food contaminants. However, development of the systematic evolution of Ligands by Exponential Enrichment (SELEX) method, which can generate a nucleic acid-based probe (aptamer) that possess numerous advantages compared to antibodies, offers the possibility of using aptamers as an alternative molecular recognition element in ELISA. Compared to antibodies, aptamers are smaller in size, can be easily modified, are cheaper to produce, and can be generated against a wide array of target molecules. The application of aptamers in ELISA gives rise to an ELISA-derived assay called enzyme-linked apta-sorbent assay (ELASA). As with the ELISA method, ELASA can be used in several different configurations, including direct, indirect, and sandwich assays. This review provides an overview of the strategies involved in aptamer-based ELASA.
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                Author and article information

                Contributors
                Susan.Giorgi-Coll@mologic.co.uk
                m.marin-altaba@uea.ac.uk
                Journal
                Mikrochim Acta
                Mikrochim Acta
                Mikrochimica Acta
                Springer Vienna (Vienna )
                0026-3672
                1436-5073
                4 December 2019
                4 December 2019
                2020
                : 187
                : 1
                : 13
                Affiliations
                [1 ]ISNI 0000000121885934, GRID grid.5335.0, Department of Clinical Neurosciences, Division of Neurosurgery, , University of Cambridge, ; Box 167, Cambridge Biomedical Campus, Cambridge, CB2 0QQ UK
                [2 ]ISNI 0000 0001 1092 7967, GRID grid.8273.e, School of Chemistry, , University of East Anglia, ; Norwich Research Park, Norwich, NR4 7TJ UK
                [3 ]ISNI 0000 0004 0383 8386, GRID grid.24029.3d, Clinical Microbiology and Public Health Laboratory, , Cambridge University Hospitals NHS Trust, ; Box 236, Addenbrooke’s Hospital, Cambridge, CB2 0QQ UK
                [4 ]ISNI 0000000121885934, GRID grid.5335.0, Department of Clinical Neurosciences, Wolfson Brain Imaging Centre, , University of Cambridge, ; Box 65, Cambridge Biomedical Campus, Cambridge, CB2 0QQ UK
                Author information
                http://orcid.org/0000-0001-8021-5498
                Article
                3975
                10.1007/s00604-019-3975-7
                6892788
                31802241
                28558401-bb4e-47e4-a5a0-632c9a460724
                © The Author(s) 2019

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 10 June 2019
                : 25 October 2019
                Funding
                Funded by: National Institute for Health Research Brain Injury Healthcare Technology Co-operative
                Award ID: n/a
                Funded by: National Institute for Health Research Biomedical Research Centre, Cambridge (Neuroscience Theme; Brain Injury and Repair Theme)
                Award ID: n/a
                Award ID: n/a
                Award Recipient :
                Funded by: National Institute for Health Research Research Professorship
                Award ID: n/a
                Award Recipient :
                Funded by: Academy of Medical Sciences/Health Foundation Senior Surgical Scientist Fellowship
                Award ID: n/a
                Award Recipient :
                Funded by: National Institute for Health Research Biomedical Research Centre, Cambridge
                Award ID: n/a
                Award Recipient :
                Categories
                Original Paper
                Custom metadata
                © Springer-Verlag GmbH Austria, part of Springer Nature 2020

                Analytical chemistry
                cytokines,colorimetry,metal nanoparticles,sepsis,diagnostics,point-of-care assay
                Analytical chemistry
                cytokines, colorimetry, metal nanoparticles, sepsis, diagnostics, point-of-care assay

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