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Protein detection using electrocalytic-sensing systems

Dans le document Marisa Maltez da Costa (Page 22-26)

Between the several biomarkers with interest for medical diagnostics, proteins represent a significant group since they can be associated with many diseases, manifested through their malfunction or increased/decreased expression by human organs, tissues or cells. Therefore their detection and quantification stands out as an important tool for screening, early diagnostics, prognostics and also to monitor the outcomes of therapies. [3]

The most used methods for the detection of protein biomarkers are enzyme-linked immunosorbent assay (ELISA), western blot assays, immuno-precipitation, immunoblotting techniques and immunofluorescence. ELISA assays is the most usual assay employed in protein sensing for diagnostics, it relies in the sandwich-type immunoassays that have high specificity and sensitivity because of the use of a couple of match antibodies [3]. But besides being time-consuming and labor-intensive, requiring highly qualified

Chapter1. General introduction

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personnel, this and other traditional protein detection methods fail to achieve the very low limits of detection that are required for several biomarkers (like certain cancer biomarker). More cost-effective methods requiring simple/user-friendly instrumentation that can provide an adequate sensitivity and accuracy would be ideal for point-of- care diagnosis. For this reason, there is a high demand for simple, fast, efficient and user-friendly alternative methods for the detection of protein markers.

The recent development of immunoassay techniques aims in most cases at decreasing analysis times, improving assay sensitivity, simplification and automation of the assay procedures. [39] Among other types of immunosensors, electrochemical immunosensors are very attractive tools and have gained considerable interest. [40] Unlike spectroscopic-based techniques, electrochemical methods are not affected by sample turbidity and fluorescing compounds commonly found in biological samples. Furthermore, the required instruments are relatively simple and can be miniaturized with very low power requirements.[41]

Immunosensors, usually used for quantitative determination of protein biomarkers, are important analytical tools based on the detection of the binding event between antibody and antigen. Finding antibodies for specific protein biomarker is not always easy and this represents a drawback of the traditional immunoassays. Therefore other synthetic biomolecules, such as aptamers, have been synthetized and studied to achieve their inclusion in protein detection assays. Several elucidating reviews can be found, that cover all the steps between the synthesis and final application in biosensor systems.

[42–44]

Aptamers are synthetic single stranded DNA or RNA molecules that fold up into 3D structures with high affinity for their target molecules (for example proteins, and cell receptor molecules) that retain their binding properties after immobilization. Since they are synthetized for a specific portion of a protein, they can be used as bio-recognition elements in several types of protein biosensors, and in particular for the detection of small proteins. A label-free bioelectronic detection of aptamer-thrombin interaction, based on electrochemical impedance spectroscopy (EIS) technique was reported by Kara et al. [45] Multiwall carbon nanotubes (MWCNTs) composite was used as modifier of screen-printed carbon electrodes (SPCEs), and the aptamer was then immobilized on the modified electrode through covalent attachment.

The binding of thrombin was then monitored by EIS in the presence of [Fe(CN)6]3−/4− redox pair. The MWCNT modified electrodes showed improved characteristics when compared to the bare ones. This study exemplifies an alternative electrochemical biosensor for the detection of other proteins. Even though most of the reported aptamers were applied for the detection of

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based biosensor. [46] They used QCM sensor system for the detection of immunoglobulin E (IgE) in human serum to test both bio-receptors and although both operated in the same protein range, the aptamer-based achieved a lower limit of detection. The aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity, and could support regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity.

Aptamers can also be conjugated to nanoparticles and used for a variety of applications. As for example two specific aptamers conjugated to silica-coated magnetic and fluorophore-doped silica nanoparticles for magnetic extraction and fluorescent labeling allows to detect and extract targeted cells in a variety of matrixes [47]. This work illustrates the overall enhanced sensitivity and selectivity of the two-particle assay using an innovative multiple aptamer approach, signifying a critical feature in the advancement of this technique.

Yang’s group [21] developed an ultrasensitive and simple electrochemical method for the fabrication of a sandwich-type heterogeneous electrochemical immunosensors using mouse IgG or PSA antigen as target. Fig.2 shows a typical fabrication procedure of DNA-free electrochemical immunosensor. An IgG layer was formed on an ITO electrode via a stepwise assembly process (Fig. 2). First, partially ferrocenyl tethered dendrimer (Fc-D) was covalently immobilized to the ITO electrode onto the phosphonate self-assembled monolayer. Some of the unreacted amines of Fc-D were modified with biotin groups to allow the specific binding of streptavidin. Afterward, biotinylated antibodies were immobilized to the streptavidin-modified ITO electrode. An IgG-nanocatalyst conjugate was also prepared via direct adsorption of IgG onto AuNPs. This conjugate and the immunosensing layer sandwiched the target protein. Signal amplification was achieved by catalytic reduction of p-nitrophenol to p-aminophenol using gold nanocatalyst labels and the chemical reduction of p- quinone imine by NaBH4. This novel DNA-free method could attain a very low detection limit (1 fg mL−1).

Chapter1. General introduction

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Figure 2(a) Schematic representation of the preparation of an immunosensing layer. (b) Schematic view of electrochemical detection of mouse IgG or prostate specific antigen.

As aforementioned, nanoparticles can act as electrocatalytic labels for several reactions involving other species in solution. Inspired by the last explained example that uses a gold nanoparticle conjugated to an antibody

Cancer diagnostic is one of the main application areas of biomarkers. Cancer biomarkers include proteins overexpressed in blood and serum (Fig. 3) or at the surface of cancer cells (Fig.4), and their low levels at the initial stages of the disease are most important for an early intervention in the cancer progression. Therefore there is a high demand of fast and sensitive detection systems that can overcome the existent limitations.

Figure 3. Schematic representation of several tumor markers and their tumor origin.

A simple and sensitive label-free electrochemical immunoassay electrode for detection of carcinoembryonic antigen (CEA) was developed by Yao’s group.

[48] CEA antibody (anti-CEA) was covalently attached on glutathione monolayer-modified AuNPs and the resulting anti-CEA-AuNPs bioconjugates were immobilized on Au electrode by electrcopolymerization with

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increased the electron-transfer resistance of [Fe(CN)6]3−/4− redox pair at the poly-OAP/anti-CEA-AuNPs/Au electrode. The immunosensor could detect the CEA with a detection limit of 0.1 ng mL−1 and a linear range of 0.5–20 ng mL−1. The use of anti-CEA/AuNP bioconjugates and poly-OAP film could enhance the sensitivity and anti-nonspecific binding of the resulting immunoassay electrode.

3.4. Cancer cell detection using electrocatalytic sensing systems

Dans le document Marisa Maltez da Costa (Page 22-26)