Aptasensor based on screen-printed electrode for breast cancer detection in undiluted human serum
Introduction
Breast cancer is the leading cause of death among women, consisting in 34% of all cancers in women. In 2018, more than 2 million new breast cancer cases were diagnosed, and an estimated 627,000 women died [1]. Thus, it is crucial that the diagnosis occurs in its early stages. Quantitative cancer diagnoses are mostly made using tumor markers, which are macromolecules present in cells, blood or other biological fluids, whose appearance or changes in their concentrations are related to the genesis and growth of neoplastic cells. Biomarkers for breast cancer are normally proteins synthesized through specific genes. One example is Human Epidermal Growth Factor Receptor 2 (HER2), which is a gene responsible for the production of HER2 protein. The HER2 protein consists of three domains: intracellular, transmembrane and extracellular, which act as receptors, helping to control cell growth, repair and division. A fragment of the extracellular domain can be found in the circulatory system in a concentration of 2–15 ng/mL. However, in 25% of breast cancers, HER2 protein is overexpressed (15–75 ng/mL) [2], leading to uncontrolled growth of breast cells and also neoplastic cells [3]. These cancers are classified as HER2 positive and tend to grow and spread more aggressively [4].
The detection of HER2 protein (or its gene) in patients is mainly performed by two techniques: immunohistochemistry, which quantifies the amount of HER2 genes, and fluorescence in situ hybridization (FISH) [5]. Both techniques depend on patient's biopsy, being invasive, and require trained professionals. A less invasive approach is possible through biosensors development. Biosensors are chemical sensors in which recognition events are based on biochemical reactions between molecules such as enzymes and specific analyte [6].
Electrochemical biosensors are promising due to their high sensitivity, rapid response, portability, low cost, simple instrumentation and easy operation [7], especially for point of care [8]. Miniaturized electrochemical sensors are possible using screen-printed electrodes (SPE), which are often functionalized with self-assembled monolayers (SAM) to create an organized and oriented layer for different applications [9]. However, SPE have a high surface roughness affecting the SAM formation and increasing non-specific interactions on the surface [10]. Campuzano et al. proposed a new surface architecture by co-immobilizing hexanedithiol (HDT) with thiolated single strand DNA at gold electrodes and using 6-mercapto-1-hexanethiol (MCH) as diluent, creating a ternary SAM to increase the biosensor performance [11]. The same design was tested by Miodek et al. on the top of gold SPE to create an electrochemical aptasensor [12]. Different from MCH, HDT has a horizontal configuration on the surface, creating a bridge between the surface irregularities and, at the same time, reducing the adsorption of non-specific proteins [11].
Here we report the expansion of these ideas to develop an electrochemical DNA aptasensor for HER2 detection comparing two platforms developed on gold screen-printed electrodes: a mixed SAM with thiolated aptamer and a ternary SAM with aptamer, HDT and MCH. Electrochemical impedance spectroscopy (EIS) technique was used to perform the characterization. Quantitative measurements for different HER2 concentrations in phosphate buffered saline (PBS), diluted and undiluted commercial human serum were performed.
Section snippets
Materials and reagents
HPLC purified synthetic DNA aptamer (5′-SH-(CH2)6-ATTAAGAACCATCA CTCTTCCAAATGGATATACGACTGGG-3′) was purchased from Sigma-Aldrich in lyophilized form and prepared in TE buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0). This HER2 aptamer sequence was reported by Liu et al. [13], where the authors have developed it by SELEX technology. HER2 protein was purchased from R&D Systems and prepared in PBS, pH 7.4. All other chemicals used were purchased from Sigma–Aldrich. All aqueous solutions were
Surface characterization of the fabricated biosensor
AFM micrographs were used to characterize different surfaces, giving information as surface roughness that can corroborate the EIS results. Fig. 1 shows the AFM images of clean gold SPE (a), SPE modified with SAM (b), SAM binding with 10 ng/mL of HER2 (c), SPE modified with ternary SAM (d) and ternary SAM binding with 10 ng/mL of HER2 (e). The surface morphology of clean electrode (a) with 76 nm surface high change to 108 and 159 nm (b and c respectively) for aptamer modified surface, which
Conclusion
Screen-printed electrodes have been used to produce portable and non-expensive biosensors that can be easily applied in clinics and hospitals. However, their high surface roughness affects the monolayer formation, increasing the undesired non-specific interactions. In this study, two different aptamer immobilization strategies have been demonstrated for the detection of HER2 protein biomarker in PBS, diluted and undiluted serum using gold screen-printed electrodes. The two platforms, SAM and
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
D.C.F. was funded by FAPESP (2017/13161-3). M.R.B. was funded by FAPESP (2013/26133-7). B.B.J. was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Brasil (CAPES - Finance Code 001). M.M. was funded by FAPESP (2017/24201-6) and CNPq (308713/2018-4).
Author Contributions
D. C. Ferreira and B. B. Jr carried out the experimental work under the supervision of M. R. Batistuti and M. Mulato. M. R. Batistuti and D. C. Ferreira wrote the paper with contributions from B. B. Jr and M. Mulato. All authors checked and approved the final manuscript.
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