Quantum dots as nanolabels for breast cancer biomarker HER2-ECD analysis in human serum
Graphical abstract
Introduction
The incidence of cancer has increased considerably worldwide, and affects the general population, resulting in significant mortality rates [1,2]. Screening and early detection allow appropriate treatments according to the stage of the disease and the availability of healthcare resources. Most European countries have national cancer screening programmes for colorectal [3,4], cervical [5,6] and breast [7,8] cancer. Breast cancer is an important public health concern with considerable impact for the patients, families and society as a whole. Its incidence and prevalence reveals slight differences among developed and developing countries, with the former presenting a global reduction in mortality due to a combination of improvements in prevention, detection and treatment [1,9]. In fact, some studies indicate that mortality rates have been diminishing in places where active screening programmes (e.g. mammography) were implemented [[7], [8], [9], [10]]. Therefore, new analytical tools for the point-of-care (POC) detection of this disease at the early stages, as well as during its management and follow-up, are widely demanded. Moreover, the development of portable equipment for in situ breast cancer analysis could also be very useful in less developed regions, remote access areas or even for patients with reduced mobility.
The guidelines established by the European Group on Tumor Markers (EGTM) on the use of breast cancer biomarkers for decision-making regarding the treatments to be administered were recently updated. Despite the large number of new biomarkers that have been reported, only three are mandatory for all patients diagnosed with invasive breast cancer: oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) [11]. The latter is established as an important diagnostic biomarker and is recommended for testing since this protein is overexpressed in 15–20% of primary invasive breast cancers and is related with the most aggressive phenotypes [12,13]. HER2 is a transmembrane protein located on the cell surface and has distinct domains: an extracellular domain (ECD), an intracellular tyrosine kinase domain and a transmembrane lipophilic segment. ECD can be cleaved by metalloproteases and released into the bloodstream. Consequently, HER2 can be measured in serum, which is key for the development of quantitative analytical strategies for breast cancer detection [14,15].
Currently, the two main types of tests accepted for the evaluation of HER2 overexpression in clinical use are immunohistochemistry and in situ hybridisation (ISH), which are not suited for POC diagnostics [11]. Hence, a considerable effort in the development of new alternative methodologies has been carried out. The use of “lab-on-a-chip” and biosensor technologies are currently providing remarkable tools for POC analysis. The possibility of miniaturization, portability, fast response and low cost are some of the electrochemical biosensors' features that make them attractive for the development of new analytical devices [16,17]. Electrochemical-based approaches for the detection of HER2-positive breast cancer have already been proposed. Different biosensing strategies were reported, which include nanomaterial-based sensing- and/or detection platforms [[18], [19], [20], [21], [22], [23], [24]], nanoelectrode ensembles (NEEs) [25], magnetic-based immunoassays [26,27], a sandwich-type immunoassay based on nanobodies [28], sensing strategies based on affibody/antibody recognition events [29], a MIP-based sensor [30], an impedimetric biosensor based on a zwitterionic hydrogel [31], an impedimetric immunosensor based on the use of single-chain fragment variable antibody fragments [32], a cellulase-linked sandwich assay [33], an inkjet-printed electrochemical platform [34], a polycytosine DNA-based immunosensor [35], and a microfluidic device based on a nanoshearing method [36]. While all of these methods have demonstrated suitable analytical performances, their application is still limited because of extensive and laborious protocols. The use of screen-printed electrodes (SPE) as the biosensor's transducer, without any surface modification prior to the functionalization with the biorecognition element, would considerably simplify the methodology. To date, only one electrochemical immunosensor for HER2 detection without prior modification of the electrode surface has been published [37]. In this work, a sandwich assay with enzymatic labelling allowed to achieve a low detection limit (4 ng/mL). However, a total assay time of 8 h revealed to be its major disadvantage because it is incompatible with a POC sensing strategy.
Nanoparticle-based signal amplification has attracted considerable interest in the development of electrochemical methods. Distinct signal amplification methodologies can be achieved using nanomaterials. Their application as labels can greatly improve the signal transduction and simplify the detection strategy. Quantum Dots (QDs) revealed to be promising candidates as such labels, since they can be conjugated to antibodies and other proteins [[38], [39], [40], [41]]. In addition, they can be synthesized with different compositions or with distinct core-shell structures, which can be useful for multiplexed sensing. The commonly used QDs are composed of a CdS or CdSe core with an external shell to provide functional groups for bioreceptor immobilization with inert and biocompatible coatings [[38], [39], [40]]. Comparing to laborious enzymatic methodologies, the use of QDs eliminates the need for substrate addition, which can contribute to the reduction of the analysis time by applying a straightforward process consisting of: QD dissolution to release the metal ions, electrochemical deposition (preconcentration) of the released ions and a potential (stripping) scan to detect the deposited metal. The obtained electrochemical signal can then quantitatively be related to the analyte concentration. Furthermore, working with electroactive labels also surpasses thermal instability aspects inherent to the nature of enzymes, the main difficulties in their use as labels [42,43].
To the best of our knowledge, this is the first electrochemical immunosensing strategy based on QDs as electrochemical label for in situ detection of HER2-ECD.
Section snippets
Apparatus and electrodes
Electrochemical measurements were carried out with a potentiostat/galvanostat (Autolab PGSTAT204, Metrohm Autolab) controlled by the NOVA software package v.1.10 (Metrohm Autolab). Disposable screen-printed carbon electrodes (with a 4-mm working electrode, a silver pseudoreference electrode and a carbon counter electrode, all made of conducting ink (SPCE, DRP-110)) as well the specific connector to interface the electrodes (DRP-CAC) were supplied by Metrohm DropSens.
Reagents and solutions
Rabit IgG monoclonal
Optimization of experimental conditions
Non-invasive analysis of cancer biomarkers can be performed in biological fluids such as serum. Bearing in mind the complex matrices of biological samples, it is mandatory to ensure that background interferences, due to the nonspecific adsorption of biomolecules, are minimized. Hence, the blockage of nonspecific adsorptions on the sensor platform after the modification with the Ab-C is of great importance for efficient and specific biomarker detection. The Ab-C concentration (25 μg/mL) was
Conclusions
A novel and efficient electrochemical immunosensor for the analysis of the breast cancer biomarker HER2-ECD, with a total time assay of 2 h, was developed. This work highlights the simplicity of the assay, with an actual hands-on-time of less than 30 min, without resorting to laborious electrode surface modifications. An electroactive QD label was employed and a limit of detection of 2.1 ng/mL was achieved, corresponding to the detection of 1.18 fmol of the analyte (sample volume = 40 μL). The
Acknowledgements
The authors are grateful for the financial support from the Fundação para a Ciência e a Tecnologia (FCT)/the Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through national funds (Portugal) (UID/QUI/50006/2019). This work was also supported by the European Union through projects Norte-01-0145-FEDER-000024 and Norte-01-0145-FEDER-000011, co-funded by FEDER in the scope of CCDR-N and NORTE2020 Partnership Agreement. Maria Freitas is financially supported by FCT through the doctoral
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