An Electrochemical Sandwich Immunosensor for the Detection of HER2 using Antibody-Conjugated PbS Quantum Dot as a label

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Abstract

A facile electrochemical sandwich immunosensor for the detection of a breast cancer biomarker, the human epidermal growth factor receptor 2 (HER2), was designed, using lead sulfide quantum dots-conjugated secondary HER2 antibody (Ab2-PbS QDs) as a label. Using Ab2-PbS QDs in the development of electrochemical immunoassays leads to many advantages such as straightforward synthesis and well-defined stripping signal of Pb(II) through acid dissolution, which in turn yields better sensing performance for the sandwiched immunosensor. In the bioconjugation of PbS QDs, the available amine and hydroxyl groups from secondary anti-HER2 and capped PbS QDs were bound covalently together via carbonyldiimidazole (CDI) acting as a linker. In order to quantify the biomarker, SWV signal was obtained, where the Pb2+ ions after acid dissolution in HCl was detected. The plated mercury film SPCE was also detected in situ. Under optimal conditions, HER2 was detected in a linear range from 1–100 ng/mL with a limit of detection of 0.28 ng/mL. The measures of satisfactory recoveries were 91.3% to 104.3% for the spiked samples, displaying high selectivity. Therefore, this method can be applied to determine HER2 in human serum.

Introduction

The major role of HER2, which is expressed in many tissues, is to facilitate excessive or uncontrolled cell growth and tumorigenesis [1]. HER2 is a gene that acts as a receptor on the surface of cells, and is very sensitive to hormonal or chemical ‘growth’ signals. If the cancer tumor cells receive more ‘messages’ to grow and divide more than a normal cell, more of these HER2 proteins will be produced. The HER2 biomarker is one of the cancer biomarkers that show overexpression of about 25-30 % breast cancer amplification [3]. Breast cancers can have up to 25–50 copies of the HER2 gene, and up to a 40–100-fold increase in HER2 protein, resulting in 2 million receptors expressed at the tumor cell surface [1]. This protein can also be overexpressed in other types of cancer such as ovarian, gastric, colorectal, pancreatic, endometrial, bladder, lung, colon, and head and neck cancers [1,2]. Early detection of this cancer biomarker can increase the success rate of treatment [3]. Therefore, a fast and sensitive technique to detect the HER2 biomarker is necessary to increase patient survival rate.

A number of methods have been used for breast cancer detection and treatment in the medical field such as the mammogram, magnetic resonance imaging (MRI), molecular breast imaging (MBI), and biopsy [4]. Mammogram is often used at the early stage of the development of cancer, where symptoms have not yet arisen. With computer-aided detection and algorithm-based programs, the process to detect any abnormalities in the breast tissue has become easier. However, this method increases the chance of possible radiation risk to frequent users [5]. Meanwhile, the biopsy test is often used as a diagnostic test after the symptoms of breast cancer have been confirmed. The test requires a breast tissue sample to be taken using a needle and analyzed in the lab. In order to minimize the false negative result, the biopsy test is generally done together with clinical breast cancer and breast imaging [6].

Current clinical diagnostics used for quantification of the breast cancer biomarker, HER2, are widely dependent on the Enzyme-Linked Immunosorbent Assay, also known as ELISA, together with different signal transduction approaches, such as surface Plasmon resonance (SPR) [7], fluorescence [8,9], chemiluminescence [10], surface-enhanced Raman scattering (SERS) [11], and opto-fluidic ring resonator [12]. Despite their high selectivity of biological recognition of the target analyte, these biosensors are expensive, time consuming and inaccurate measurements. Thus, there is a growing interest in the development of HER2 detection using alternative techniques to overcome these limits. The electrochemical technique has emerged as an alternative to HER2 breast cancer detection. This is because it is now possible to miniaturize modern microelectronics, which allows for the building of microelectrodes that are useful for multiplexing. Furthermore, the method lends itself well to the detection of very small volumes of samples ranging from microliters to nanoliters. In addition, the electrochemical approach is favorable due to its low cost, and the large-scale production of electronic devices results in high-throughput analysis [13]. Due to these favorable characteristics, the past few years have seen the development of electrochemical immunosensors for quantification of the HER2 biomarker [[14], [15], [16], [17], [18]]. The method works by converting the interaction of antibody-antigen complexes into an electrical signal. The electrochemical detection uses amperometric, potentiometric, and conductometric transducers.

The inclusion of nanomaterials in the electrochemical immunosensor has been widely studied as a new tool that can improve the performance of the sensor [19]. ELISA requires higher incubation times, but the addition of nanoparticles will decrease this incubation time to only a few minutes [20]. Apart from that, nanomaterials offer a simple, sensitive, rapid, and cost-effective method for the detection of cancer [21]. In recent work, a polycytosine DNA-based immunosensor for electrochemical detection was developed and tested in the detection of HER2, utilizing gold nanoparticles (AuNPs) as the supporting matrix to immobilize polycytosine DNA sequence (dC20) for electrochemical current generation and anti-HER2 antibodies. A sandwiched immunocomplex was formed between a peptide specific to HER2 immobilized on the gold electrode and the anti-HER2 antibodies on the AuNPs in the presence of the target HER2. The HER2 captured by the sensor was detected by the electrochemical current generated from the reaction of the dC20 phosphate backbone with molybdate, forming a redox molybdophosphate precipitate [22]. Another work developed a novel impedance aptasensor for the detection of HER2 by immobilizing a HER2-specific single stranded DNA aptamer onto a 3-mercaptopropionic acid self-assembled on a gold nanoparticle electrode. The aptasensor showed excellent sensitivity, while the specificity of the aptasensor provided a good potential sensing platform for non-invasive early detection of breast cancer biomarkers [23].

Similar to nanoparticles, quantum dots (QDs) have now become an alternative method to replace the use of metal NP labels in a sandwich immunoassay. QDs are an excellent candidate for label application in biosensors, as they provide unique physical and optical properties. The surface of QDs has the possibility to attach to various biomolecules [24]. Many reported studies show the utilization of QDs via the covalent attachment of biomolecules such as the use of streptadivin malemaide or the formation of QD-biomolecule conjugate from the EDC/NHS coupling method [[25], [26], [27]]. PbS QDs were employed for the sensitive detection of Staphylococcal enterotoxin B (SEB) using glassy-carbon electrode (GCE), which gave a limit of detection of 0.01 ng/mL [28]. A novel electrochemical immunosensor for multiplexed detection using multiple QDs was also reported. In the study, CdS and ZnS were used as the label tags to detect CA 125, CA 15-3, and CA 19-9. A sandwich format was adopted followed by an acid release procedure by utilizing the mercury film electrode. This work enabled the detection of simultaneous biomarkers in one run [29].

The novelty of this work centers on the use of PbS QDs as a label for the quantitative detection of HER2 cancer biomarker (Fig. 1). This approach has not been attempted in any previous works. PbS QDs were initially synthesized with thioglycerol (TGL) and dithioglycerol (DTG) as a capping agent to produce PbS QDs with hydroxyl surfaces. The as-synthesized PbS QDs was conjugated with antiHER2 antibody by using carbonylimidazole (CDI) as a linker (Fig. 1(a)). A screen-printed carbon electrode (SPCE) was used as the platform for the immunosensor development, as shown in Fig. 1(b). In order to improve the electrode interface, the transducer surface was functionalized. The surface of SPCE was electrochemically pre-treated to form a carboxyl functional group, which was then activated with the EDC/NHS linker for immobilization of primary anti-HER2 (Ab1). The quantification of the HER2 biomarker was performed through acid dissolution of the bioconjugated PbS QDs. The released cations (Pb2+) were transferred to an electrochemical cell and measured using square wave voltammetry (SWV) with in-situ plated Hg films (Fig. 1(c)).

Section snippets

Materials and reagents

Polyclonal Anti-HER2 and Active HER-2 were purchased from BioVision Inc, United States. 2-(N-morpholino)ethanesulfonic acid (MES) buffer, human serum (from male AB clotted whole blood, USA origin), bovine serum albumin (BSA), thioglycerol (TGL), phosphate buffered saline (PBS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 2,3-dimercapto-1-propanol (DTG), and 1,1'-carbonyldiimidazole (CDI) were purchased from Sigma Aldrich, Germany. Sulfuric acid was purchased from Baker

Characterizations of PbS QDs and bioconjugation (Ab2-PbS QDs)

The morphology and size distribution of PbS QDs and Ab2-PbS QDs were characterized by HRTEM. As can be observed from Fig. 2(a), the PbS QDs in the solution has a uniform and quasi-spherical shape with a size ranging from 5 nm to 6 nm. After bioconjugation with the antibody, well-dispersed cores of the QDs could be seen. The good dispersion of Ab2-PbS QDs is likely attributed to the increased spacing in each PbS QD because of the formation of large biomolecule conjugates on the surface.

Conclusions

In this work, a new sandwich electrochemical immunoassay, based on Ab2-PbS QDs as the label, was designed to detect breast cancer HER2. This method combined low-cost carbon-printed electrodes and leveraged on the simplicity of quantum dots and biomolecule conjugation synthesis. PbS QDs provided a highly accessible surface area for conjugation of the antibody loading that increased the sensitivity of the immunosensor. The acid dissolution method of the Pb2+ ions offered an attractive way to

Novelty statement

An electrochemical sandwich immunosensor based on lead sulfide quantum dots-conjugated secondary HER2 antibody (Ab2-PbS QDs) as a label was developed to detect HER2. Although it is based on well-known assay format using Cd-based quantum dots. The detection principle is new which use Pb-based quantum dots detection. Under optimal conditions, HER2 was detected in a linear range from 1–100 ng/mL with a low-limit detection of 0.28 ng/mL. The measures of satisfactory recoveries were 91.3% to 104.3%

Acknowledgements

This research was funded by UNIVERSITI PUTRA MALAYSIA, grant number GP-9480600, Ministry of Higher Education (MOHE) (Fundamental Research Grant Scheme (5540049)), Graduate Research Fellowship, UPM for funding the researcher.

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