Aptasensor based on screen-printed electrode for breast cancer detection in undiluted human serum

Highlights

• Impedimetric aptasensor for the detection of breast cancer biomarker - HER2.

• Two architecture to immobilize DNA aptamers on gold screen-printed electrodes.

• Aptasensors perform within clinical range in PBS, diluted and undiluted human serum.

• EIS provide 172 pg/mL as LOD in a dynamic range from 1 pg/mL to 100 ng/mL.

• Ternary SAM reduce non-specific interaction, improving biosensor performance.

Abstract

Breast cancer remains one of the leading causes of women death. The development of more sensitive diagnostic tests, which could present a faster response, lower cost, and could promote early diagnosis would increase the chances of survival. This study reports the development and optimization of an electrochemical aptasensor for the detection of HER2 protein, a breast cancer biomarker. Two sensing platforms were developed on gold screen-printed electrodes. The first platform is composed of self-assembled monolayer (SAM) made from mixture of thiolated DNA aptamers specific for HER2 and 1-mercapto-6-hexanol (MCH), while the second one is a ternary SAM composed of the same aptamer and 1,6-hexanethiol (HDT). Both platforms were further passivated with MCH and blocked with bovine serum albumin. The biosensors were characterized using electrochemical impedance spectroscopy to detect the target protein from 1 pg/mL to 1 μg/mL in phosphate buffered saline, diluted and undiluted human serum through charge transfer resistance value. The ternary SAM architecture shows a reduction of non-specific attachment to the electrode surface due to the HDT antifouling properties. In addition, this platform exhibits 172 pg/mL as limit of detection and a sensitivity of 4.12% per decade for undiluted serum compared with SAM architecture with the 179 pg/mL and 4.32% per decade, respectively. Electrochemical aptasensors are highly promising for medical diagnostic and ternary layers could improve the limit of detection.

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.