Discrimination between protein glycoforms using lectin-functionalised gold nanoparticles as signal enhancers.

Glycoforms (and other post-translational modifications) of otherwise identical proteins can indicate pathogenesis/disease state and hence new tools to detect and sense a protein's glycosylation status are essential. Antibody-based assays against specific protein sequences do not typically discriminate between glycoforms. Here we demonstrate a 'sandwich' bio-assay approach, whereby antibodies immobilised onto biolayer interferometry sensors first select proteins, and then the specific glycoform is identified using gold nanoparticles functionalised with lectins which provide signal enhancement. The nanoparticles significantly enhance the signal relative to lectins alone, allowing glycoform specific detection as low as 0.04 µg mL-1 (1.4 nM) in buffer, and crucially there is no need for an enrichment step and all steps can be automated. Proof of concept is demonstrated using prostate specific antigen: a biomarker for prostate cancer, where glycoform analysis could distinguish between cancerous and non-cancerous status, rather than only detecting overall protein concentration.

Electrochemical Immunosensor for the Simultaneous Determination of Two Main Peanut Allergenic Proteins (Ara h 1 and Ara h 6) in Food Matrices.

Efficiently detecting peanut traces in food products can prevent severe allergic reactions and serious health implications. This work presents the development of an electrochemical dual immunosensor for the simultaneous analysis of two major peanut allergens, Ara h 1 and Ara h 6, in food matrices. A sandwich immunoassay was performed on a dual working screen-printed carbon electrode using monoclonal antibodies. The antibody-antigen interaction was detected by linear sweep voltammetry through the oxidation of enzymatically deposited silver, which was formed by using detection antibodies labeled with alkaline phosphatase and a 3-indoxyl phosphate/silver nitrate mixture as the enzymatic substrate. The assay time was 2 h 20 min, with a hands-on time of 30 min, and precise results and low limits of detection were obtained (Ara h 1: 5.2 ng·mL-1; Ara h 6: 0.017 ng·mL-1). The selectivity of the method was confirmed through the analysis of other food allergens and ingredients (e.g., hazelnut, soybean and lupin). The dual sensor was successfully applied to the analysis of several food products and was able to quantify the presence of peanuts down to 0.05% (w/w). The accuracy of the results was confirmed through recovery studies and by comparison with an enzyme-linked immunosorbent assay. Tracking food allergens is of utmost importance and can be performed using the present biosensor in a suitable and practical way.

Electrochemical immunosensor towards invasion-associated protein p60: An alternative strategy for Listeria monocytogenes screening in food.

This work reports the development of an electrochemical immunosensor for rapid, specific and decentralized detection of the invasion-associated protein p60 secreted by Listeria monocytogenes, a life-threatening foodborne pathogen. A disposable screen-printed electrode was used as transducer surface and monoclonal and polyclonal antibodies that specifically recognize Listeria monocytogenes p60 protein and Listeria spp. p60 proteins, respectively, were used as the sandwich immuno-pair. The reaction was detected with the aid of an additional secondary antibody conjugated with the enzyme reporter (alkaline phosphatase) and using 3-indoxyl phosphate/silver ions as the mixture substrate. The analytical signal was acquired through the voltammetric stripping of the enzymatically deposited silver, which was directly correlated to p60 concentration in the sample. In optimized conditions, a limit of detection and quantification of 1.5 ng mL-1 and 5.1 ng mL-1 were achieved, respectively, in a useful time (<3 h). As proof-of-concept, the proposed immunosensor was successfully applied to spiked milk samples, demonstrating to be a suitable device for further use in real sample detection of Listeria monocytogenes in food products.

Quantum dots as nanolabels for breast cancer biomarker HER2-ECD analysis in human serum.

Early detection of cancer increases the possibility for an adequate and successful treatment of the disease. Therefore, in this work, a disposable electrochemical immunosensor for the front-line detection of the ExtraCellular Domain of the Human Epidermal growth factor Receptor 2 (HER2-ECD), a breast cancer biomarker, in a simple and efficient manner is presented. Bare screen-printed carbon electrodes were selected as the transducer onto which a sandwich immunoassay was developed. The affinity process was detected through the use of an electroactive label, core/shell CdSe@ZnS Quantum Dots, by differential pulse anodic stripping voltammetry in a total time assay of 2 h, with an actual hands-on time of less than 30 min. The proposed immunosensor responded linearly to HER2-ECD concentration within a wide range (10-150 ng/mL), showing acceptable precision and a limit of detection (2.1 ng/mL, corresponding to a detected amount (sample volume = 40 µL) of 1.18 fmol) which is about 7 times lower than the established cut-off value (15 ng/mL). The usefulness of the developed methodology was tested through the analysis of spiked human serum samples. The reliability of the presented biosensor for the selective screening of HER2-ECD was confirmed by analysing another breast cancer biomarker (CA15-3) and several human serum proteins.

Miniaturized analytical instrumentation for electrochemiluminescence assays: a spectrometer and a photodiode-based device.

Herein, a new miniaturized analytical instrumentation for electrochemiluminescence (ECL) assays is presented. A photodiode integrated in an ECL cell combined with a potentiostat/galvanostat, all integrated in a one-piece instrument (µSTAT ECL), was developed. In addition, a complementary micro-spectrometer integrated in a similar ECL cell for luminescence spectra recording is also proposed. Both cells are intended to be used with screen-printed electrodes and all the devices are portable and small sized. Their performance was corroborated with two innovative proofs-of-concept that centered on the luminol transduction chemistry: a first time reported ECL assay based on the enzymatic reaction between an indoxyl substrate and the enzyme alkaline phosphatase, and the electrochemiluminescence resonance energy transfer (ECL-RET) process triggered by the electro-oxidized luminol to the acceptor fluorescein. The photodiode system revealed to be more sensitive than the spectrometer device in collecting the light; however, with the latter, it is possible to discriminate different luminescent species according to their maximum wavelength emission, which is extremely useful for carrying out simple and simultaneous ECL multiplex analyzes. The spectrometer device works as an excellent accessory to couple with the µSTAT ECL instrument, complementing the experiments. Graphical abstract Schematic representation of the ECL-RET: from luminol-H2O2 system to fluorescein, the micro-spectrometer for the light collection and the 3D representation of the ECL-RET reaction.

An electrochemical deamidated gliadin antibody immunosensor for celiac disease clinical diagnosis.

The first electrochemical immunosensor (EI) for the detection of antibodies against deamidated gliadin peptides (DGP) is described here. A disposable nanohybrid screen-printed carbon electrode modified with DGP was employed as the transducer's sensing surface. Real serum samples were successfully assayed and the results were corroborated with an ELISA kit. The presented EI is a promising analytical tool for celiac disease diagnosis.

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes.

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.