SERS and electrochemical impedance spectroscopy immunoassay for carcinoembryonic antigen
Introduction
Many of the existing cancers have a high chance of cure when early diagnosed [1]. An earlier identification may result in a higher probability of survival, as well as a more effective and less expensive treatment. To this end, researchers have identified specific biomarkers that circulate in the blood and may signal, in a non-invasive and inexpensive way, the presence of cancer. These biomarkers include cell-free DNA, cell-free RNA, tumour proteins and extracellular vesicles [2,3] and may be detected in early stages of the disease, in people who have no symptoms [4].
Carcinoembryonic antigen (CEA) is a cancer biomarker that is currently employed in clinical context. CEA is a glycoprotein of 200 kD that has been associated mostly with colon and gastric cancer diseases [5], but may also be linked to breast, ovarian and lung cancers. Normal CEA levels range 3–5 ng/mL, but these may reach 10 ng/mL in specific benign diseases and CEA levels above 20 ng/mL before therapy are commonly linked to metastatic state [6]. Thus, the routing monitoring of CEA may contribute to the early detection of cancer diseases, while also contributing to follow-up the therapy.
It is therefore clear that quick and low cost methods are required for CEA screening. Conventional methods to this end include enzyme-linked immunosorbent assay (ELISA) (Human CEA ELISA Kit) as, radioimmunoassay (RIA) [7] and fluoroimmunoassay [5]. These methods are reliable and accurate, but require much time, trained personal, expensive equipment, being therefore expensive for routing analytical procedures.
Alternative methods include electrochemical immunosensors, which combine the quick response of electrochemistry methods with the great selectivity of antibodies, yielding sensitivity, accurate and inexpensive analysis [8,9]. These methods have been currently employed in the detection of a wide variety of biomolecules, including cancer biomarkers [10] and specifically CEA. Different approaches are employed for this purpose. This includes using different electrodes, as gold [11], transparent oxides [12,13] or carbon-based [14] materials. Different chemical entities are also employed to improve the detection features, such as poly(sulfanilic acid), chitosan, thionine, toluidine blue, methylene blue [[15], [16]–17], as well as titanium dioxide nanomaterials, gold nanoparticles, porous nanogold, Janus nanoparticles of gold/silica [18], magnetic DNA nanoprobes [19] and different composites of organoclay nanogold [20] or graphene/Nafion [21].
In terms of detection, electrochemical immunosensing of CEA may be established by the direct reading of the antigen binding, usually leading to changes in the electrical current of a standard redox probe [22]. However, if another protein in a complex sample would adsorb non-specifically to the sensing surface, it would also contribute to the detected signal, thereby leading to a positive error. This may be minimised by combining electrochemical immunosensors with sandwich approaches [23], in which labelled antibodies in a solution are added after antigen binding and prior to detection. Labels used for this purpose that contribute for the electrochemical detection include metallic/redox-based compounds or labelled enzymes (horseradish peroxidase), or a combination of both [17,24,25]. CEA has a single polypeptide chain with an NH terminal domain of 107 amino acids, in addition to three highly homologous domains of 178 amino acids. Most likely, these homologous domains are responsible for the presence of repetitive epitopes in CEA, meaning that one antigen molecule can bind simultaneously to more molecules of the same monoclonal antibody [26].
The two stages of incubation (the antigen and the labelled antibody) required in sandwich-based methods are an excellent opportunity to establish dual detection approaches. This would further validate the analytical readings, thereby ensuring the desired accuracy of these sandwich-based methods in highly complex samples. The first stage could remain of electrochemical nature, as this is non-destructive. The second stage could rely on Surface-Enhanced Raman Scattering (SERS), a versatile approach that is attracting much attention in the field of biosensing, including for CEA detection [27].
In general, Raman spectroscopy is known as a non-destructive and reliable analytical technique, and the use of SERS further allows enhancing the Raman scattering of molecules that are in close proximity to a suitable plasmonic metal [28,29] Different approaches may be established for this purpose [29]. The most simple one consists in labelling the second antibody with a suitable Raman probe linked to a gold/silver nanostructure. Herein, the metal support should be a suspended nanoparticle that may come in a wide variety of shapes, including nanorods, nanocubes, nanospheres, nanotriangles, nanowires, nanoplates, and nanostars [30]. Higher signal enhancement is linked to nanostars, due to their higher number of ‘hotspots’ per particle, generated by the multiple sharp branches that create a “lightning rod” effect and enhance intensely the local electromagnetic field [30]. As gold nanostars (AuNS) have been found highly attractive materials in photocatalysis and sensing [31,32], these nanostructured materials were selected herein as signal enhancer.
Overall, this work reports for the first time an immunosensor combining electrochemical and SERS sequential readings on the same sensing surface, thereby yielding quantitative/qualitative data and further contributing to the accuracy of the data so obtained. Some research work has been reported in the literature describing the integration of electrochemical and SERS techniques for testing the interaction between anticancer drugs and DNA [33,34] but it was never used before for the immunosensing of tumour markers. To this end, the immunosensor was assembled and optimised on a commercial Au-SPE. The first detection stage is obtained after CEA binding and concerns electrochemical signals produced by electrochemical impedance spectroscopy (EIS). Then, the labelled antibody linked to Au-NS with 4-ATP (yielding a specific and well-established Raman signal) was incubated on the working electrode and SERS data were collected. The analytical performance of immunosensor was tested with spiked serum.
Section snippets
Equipment
Electrochemical measurements were made in a potentiostat/galvanostat from Metrohm Autolab, equipped with an impedimetric module and controlled by NOVA 2.0 software. Commercial Au-SPEs were used (DropSens, 220AT), combining working and counter electrodes made of gold, and reference electrode and electrical contacts made of silver. The switch box interfacing these SPEs and the potentiostat was obtained from BioTID, Portugal. Raman spectra were collected in a Thermo Scientific DXR Raman with a
Immunosensor assembly
The immunosensor assembly followed the several stages described in Fig. 1A. In general, these include (i) cleaning stage, (ii) preparation of the electrode to receive the antibody, (iii) antibody binding, and (iv) the blocking the non-specific interaction. Each of these stages was monitored by EIS, in order to monitor and control the progresses of each stage of modification upon the immunosensor assembly.
Conclusions
The experimental results showed that EIS was an adequate technique to follow the performance of the immunosensor. In general, this immunosensor was developed in a simple way, showing its potential for clinical applications. This sensor was used to determine CEA in a real serum matrix, showing sensitive response at concentrations within the physiological levels.
There are reports in the literature where the detection method manages to go to lower values than those presented in this work. However,
Credit author statement
Yuselis Castaño-Guerrero: investigation, methodology, validation, visualization, formal analysis, data curation, and witting - original draft. Felismina T.C. Moreira: methodology, formal analysis, supervision, and writing – review & editing. Ana Sousa-Castillo: investigation, methodology, and data curation, involving gold nanostars. Miguel A. Correa-Duarte: conceptualization, funding acquisition, supervision, and writing – review & editing. Goreti Sales: conceptualization, supervision, funding
Declaration of Competing Interest
None.
Acknowledgements
Authors acknowledge funding to FEDER/COMPETE 2020 and Fundação para a Ciência e Tecnologia through the projects OligoFIT (ERA-NET Cofund, JPCOFUND2/0001/2019), RamSERS (NORTE-01-0247-FEDER-017834, call no. 33/SI/2015) and STRIP2SENSE (NORTE-01-0145-FEDER-024358-SAICT-POL/24358/2016). MINECO-Spain CTM2017-84050-R and Xunta de Galicia (IN607A 2018/5 and Centro Singular de Investigación de Galicia, Acc. 2016-2019) are also acknowledged. YCG also acknowledge the PhD grant reference
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