Elsevier

Electrochimica Acta

Volume 366, 10 January 2021, 137377
Electrochimica Acta

SERS and electrochemical impedance spectroscopy immunoassay for carcinoembryonic antigen

https://doi.org/10.1016/j.electacta.2020.137377Get rights and content

Highlights

  • Combining electrochemical and surface enhanced Raman spectroscopy in the same spot.

  • Immunosensor for CEA, assembled on a gold screen-printed electrode.

  • First reading is of electrochemical nature, and followed by an iron redox probe.

  • Second reading is obtained after antibody binding with the Raman reporter 4-aminothiophenol.

  • Dual-signals of different nature allows one to double check the analytical result, with expected improvement of the accuracy.

Abstract

This work describes an innovative dual detection approach, combining electrochemical and surface-enhanced Raman scattering (SERS) sequential readings, on the same sensing surface. This was achieved by establishing (i) an antibody binding stage on a suitably modified screen-printed electrode (Au-SPE), to produce an electrochemical signalling system, followed by (ii) a second antibody binding stage, on the same sensing surface, comprising gold nanostars (AuNS) with a suitable Raman reporter, and acting as a second signalling system (SERS). This simple principle is applied herein to carcinoembryonic antigen (CEA) detection.

The first layer of antibodies was assembled on the Au-SPE previously modified with a cysteamine layer. Binding to CEA was allowed for 30 min. Electrochemical impedance spectroscopy (EIS) readings followed the several stages of Au-SPE modification and generated analytical data. After, the AuNS were modified with 4-aminothiophenol (4-ATP)/Ab-CEA, and incubated on the same sensing surface, to provide SERS data.

The analytical features were checked for both EIS and SERS. In EIS, the sensor showed linear response range from 0.25 to 250 ng/mL, with a linear correlation coefficient of 0.991, evaluated in serum. It also demonstrated good selectivity against creatinine and glucose. Using the SERS as signalling system, the spectra confirmed differentiated signals from the background within 0.025 ng/mL to 250 ng/mL of CEA. As expected, the Raman signal of the reporter increased with increasing CEA concentrations, and contributed to confirm the accuracy of the analytical data.

Overall, this approach is simple, and may be adaptable to new multiplexing devices, also being adaptable to mass production.

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

References (52)

  • L. Truta et al.

    A dye-sensitized solar cell acting as the electrical reading box of an immunosensor: application to CEA determination

    Biosens. Bioelectron.

    (2018)
  • X.M. Pei et al.

    Sandwich-type immunosensors and immunoassays exploiting nanostructure labels: a review

    Anal. Chim. Acta

    (2013)
  • Y.G. Wang et al.

    Sandwich-type electrochemical immunosensor for CEA detection based on Ag/MoS2@Fe3O4 and an analogous ELISA method with total internal reflection microscopy

    Sens. Actuat. B-Chem.

    (2018)
  • Y. Lin et al.

    Detection of CEA in human serum using surface-enhanced Raman spectroscopy coupled with antibody-modified Au and gamma-Fe2O3@Au nanoparticles

    J. Pharmaceut. Biomed. Anal.

    (2016)
  • A. Sousa-Castillo et al.

    Hybrid plasmonic nanoresonators as efficient solar heat shields

    Nano Energy

    (2017)
  • H. Ilkhani et al.

    Nanostructured SERS-electrochemical biosensors for testing of anticancer drug interactions with DNA

    Biosens. Bioelectron.

    (2016)
  • M. Meneghello et al.

    Using electrochemical SERS to measure the redox potential of drug molecules bound to dsDNA – a study of mitoxantrone

    Electrochim. Acta

    (2016)
  • L.M. Fischer et al.

    Gold cleaning methods for electrochemical detection applications

    Microelectron. Eng.

    (2009)
  • A.R. Bizzarri et al.

    Surface enhanced Raman spectroscopy based immunosensor for ultrasensitive and selective detection of wild type p53 and mutant p53(R175H)

    Anal. Chim. Acta

    (2018)
  • F.T.C. Moreira et al.

    Protein-responsive polymers for point-of-care detection of cardiac biomarker

    Sens. Actuat. B-Chem.

    (2014)
  • F.T.C. Moreira et al.

    Novel sensory surface for creatine kinase electrochemical detection

    Biosens. Bioelectron.

    (2014)
  • N.S. Ferreira et al.

    Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

    Biosens. Bioelectron.

    (2014)
  • J.T. Andersen et al.

    Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal fc receptor with different affinities that reveal distinct cross-species differences in serum half-life

    J. Biol. Chem.

    (2012)
  • G.S. Bumbrah et al.

    Raman spectroscopy – basic principle, instrumentation and selected applications for the characterization of drugs of abuse

    Egypt. J. Forensic Sci.

    (2016)
  • F. Bray et al.

    Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries

    CA – Cancer J. Clin.

    (2018)
  • J. Marrugo-Ramirez et al.

    Blood-based cancer biomarkers in liquid biopsy: a promising non-invasive alternative to tissue biopsy

    Int. J. Mol. Sci.

    (2018)
  • Cited by (22)

    • Design of highly selective, and sensitive screen-printed electrochemical sensor for detection of uric acid with uricase immobilized polycaprolactone/polyethylene imine electrospun nanofiber

      2023, Electrochimica Acta
      Citation Excerpt :

      Bare QD SPCE did not exhibit a semi-circle radius while PCL/PEI/UOx/QD SPCE (5.3381 × 104 ± 4.2444 Ω) exhibited broader radius than PCL/PEI/MB/UOx/QD SPCE (1.8768 × 104 ± 1.7778 Ω). In comparison to Castano–Guerrero et al. [17], the binding of the mediator decreased the charge transfer resistance which consequently dropped the semicircle diameter. Furthermore, it is regulated by the redox properties of MB as a redox shuttle which is negatively charged.

    View all citing articles on Scopus
    View full text