Elsevier

Biosensors and Bioelectronics

Volume 194, 15 December 2021, 113603
Biosensors and Bioelectronics

Simultaneous detection of multiple neuroendocrine tumor markers in patient serum with an ultrasensitive and antifouling electrochemical immunosensor

https://doi.org/10.1016/j.bios.2021.113603Get rights and content

Highlights

  • A new electrochemical sensor was first developed for detecting two NETs biomarkers.

  • Metal ions functionalized PMS were prepared as signal amplification probes.

  • The synergy of PEG and CS endowed the biosensor with excellent antifouling ability.

  • The biosensor can accurately analyze CgA and CgB in patient serum.

Abstract

Neuroendocrine tumors (NETs) are rare heterogeneous tumors that are often misdiagnosed and mistreated. Most NETs patients are diagnosed as advanced. Early on-time detection of NETs is significant for precision therapy. Here, an ultrasensitive and antifouling label-free electrochemical immunosensor was constructed for simultaneous analysis of NETs biomarkers chromogranin A (CgA) and chromogranin B (CgB). The metal ion functionalized porous magnesium silicate/gold nanoparticles/polyethylene glycol/chitosan (PMS-M2+/AuNPs/PEG/CS) composites were employed as the sensing platforms. By combining PEG and CS with good hydrophilicity, the sensing interface exhibited outstanding antifouling ability in complex biological systems. PMS with high surface area and the porous structure can efficiently load Cu2+ and Pb2+, which could directly generate independent electrochemical peak currents that reflected the concentrations of CgA and CgB. Under optimal conditions, this immunosensor can detect CgA and CgB with good linearity from 0.1 pg mL−1 to 100 ng mL−1 as low as 5.3 and 2.1 fg mL−1, respectively. Moreover, this immunosensor can accurately detect CgA and CgB levels in clinical serum, which were well consistent with the enzyme-linked immunosorbent assay (ELISA). This strategy provided a sensitive, simple and low-cost platform for clinical screening and point-of-care diagnosis of NETs.

Introduction

Neuroendocrine tumors (NETs) are a heterogeneous group of rare tumors that originate from neuroendocrine cells distributed throughout the body (Sackstein et al., 2018). Unfortunately, patients with NETs are often misdiagnosed and mistreated (Chauhan et al., 2020). 60–80% of NETs patients are diagnosed at the advanced stages with metastatic progression and poor prognosis (Modlin et al., 2010). So early diagnosis of NETs by the determination of effective biomarkers are critical for improving the efficacy of treatment and survival (Duque et al., 2013). Chromogranin A (CgA) is the most commonly used NETs biomarker in clinical practice (Modlin et al., 2010). The levels of CgA correlate with tumor burden and survival rate of NETs patients. However, the detection of single-analyte CgA is not adequate for the appropriate diagnosis of NETs (Modlin et al., 2014), because the level of CgA is affected by several interfering factors, such as oral use of proton pump inhibitors (PPIs), cardiovascular disease and inflammatory bowel disease, which reduce the specificity of CgA in the diagnosis of NETs (Gut et al., 2016). Chromogranin B (CgB) is not influenced by such factors as PPIs, etc., and can serve as an important NETs biomarker to complement CgA (Stridsberg et al., 2007). In addition, CgB has a higher positive rate than CgA in pancreatic and rectal NETs (Miki et al., 2017), which can make up for the shortcoming of insufficient specificity of CgA, but the sensitivity of CgB assay needs to be improved (Stridsberg et al., 2007). In consequence, the development of a reliable and sensitive analytic technique for simultaneous detection of CgA and CgB to improve the accuracy and efficiency of NETs diagnosis is urgently desirable (Miki et al., 2017; Ramachandran et al., 2015).

Until now, various analytical methods have been applied to simultaneous detection of multiple biomarkers such as surface-enhanced Raman scattering (SERS) (Tang et al., 2016), surface plasmon resonance (SPR) (Malinick et al., 2020), fluorescence (Liao et al., 2019), photoluminescent (He et al., 2017) and electrochemistry (Vargas et al., 2019; Zhu et al., 2020; Zupančič et al., 2021). In particular, electrochemical techniques have drawn extensive interest in multiple biomarkers analysis due to their intrinsic merits such as high sensitivity, rapid response, easy operation, small sample consumption and low sensing cost, which make them a better fit for bedside testing (Filik and Avan, 2019; Lai et al., 2019; Pakchin et al., 2017; Wang et al., 2021). To achieve multiple electrochemical detection, one of the key issues is to acquire appropriate and distinguishable electrochemical signals at different potentials for differentiating the corresponding biomarkers (Wang et al., 2015). Among many electroactive species, metal ions, such as Cd, Zn Cu, and Pb ions, which possess independent peak potentials, would be an ideal choice to realize multi-analyte analysis owing to their highly sensitive electrochemical response and simple detection process (Putnin et al., 2019; Rong et al., 2016; Yuan et al., 2015). Various types of nanomaterials have been used as metal ions nanocarriers for the detection of multi-biomarkers, such as titanium phosphate nanosphere (Feng et al., 2012), magnetic particles (Zhang et al., 2014), reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) (Yuan et al., 2015) and platinum porous nanoparticles (PtPNPs) (Wang et al., 2014). However, there is still a challenge in developing novel functional nanomaterial combining with merits of high surface area, good conductivity, easy modification and facile preparation, which can effectively adsorb multiple metal ions to improve the sensitivity of simultaneous detection (Wang et al., 2016).

Moreover, during the sensing of complex biological samples, nonspecific adsorption of the electrode surface is a ubiquitous phenomenon and can significantly affect the sensitivity and accuracy of quantitative results (Jiang et al., 2020; Río et al., 2019), which were rarely paid attention to by previously reported electrochemical biosensors for multi-biomarker detection. Therefore, it is of utmost importance to design antifouling sensing platforms to resist nonspecific adsorption. Polyethylene glycol (PEG)-based polymers are recognized as one type of the most powerful antifouling materials, which have been successfully applied in numerous electrochemical biosensors (Lin and Li, 2020). However, PEG-modified interface is difficult to completely reject the adsorption of proteins. Besides, PEG is prone to oxidative damage (Campuzano et al., 2019). Thus, there is an urgent need for other materials to compensate for the shortcomings of PEG. Chitosan (CS), as an amino and hydroxyl rich natural polysaccharide, has become an attractive biopolymer due to its excellent biocompatibility, good stability and film-forming ability (Pan et al., 2019; Wang et al., 2021). Furthermore, CS has been widely used in biomedical fields such as antifouling and antimicrobial because of its good hydrophilicity and anti-protein adsorption performance (Huang et al., 2019; Xu et al., 2018). This motivates us to simultaneously introduce PEG and CS to improve the antifouling performance of the sensing interface.

In this work, an ultrasensitive and antifouling label-free electrochemical immunosensor for simultaneous determination of CgA and CgB was developed (Scheme 1). The Cu2+ and Pb2+ were used as electroactive species for their easily distinguishable redox potential at about 0.03 and −0.47 V (vs Ag/AgCl), respectively. For signal amplification, porous magnesium silicate (PMS, Mg3Si2O5(OH)4), a low-cost two-dimension layered silicate with rich functional groups, high surface area and porous structure, which has been demonstrated to show remarkable adsorption capacity for Cu2+ and Pb2+ in our recent research (Huang et al., 2017), was first employed as the metal ions adsorption carrier for immunosensor. The metal ion functionalized porous magnesium silicate/gold nanoparticles/polyethylene glycol/chitosan (PMS-M2+/AuNPs/PEG/CS) composites were used to fabricate the sensing interfaces. Here CS was used as the bridge cross-linker to immobilize the composites onto the electrode surface because of its excellent adhesion and good film-forming ability (Güner et al., 2017; Hu et al., 2018). And capture antibodies for CgA and CgB were respectively covalently bound to AuNPs of the biosensing interface PMS-Cu2+/AuNPs/PEG/CS and PMS-Pb2+/AuNPs/PEG/CS based on the affinity between thiol and gold to form Au–S bond (Duran et al., 2019; He et al., 2015; Hu et al., 2020). The synergy of PEG and CS with good hydrophilicity can effectively resist nonspecific protein adsorption. Once specific recognition occurred between NETs biomarkers and antibodies, the quantitative detection of CgA and CgB was realized by observing the decrease in current signals from Cu2+ and Pb2+. The designed biosensor has been successfully applied to simultaneously detect CgA and CgB with desirable performance. In addition, the biosensor was applicable to accurately analyze the levels of CgA and CgB in clinical serum samples from NETs patients. As far as we know, this is the first electrochemical biosensor for the simultaneous determination of multiple NETs biomarkers.

Section snippets

Reagents and apparatus

Human chromogranin A protein (CgA), human chromogranin B protein (CgB), anti-CgA monoclonal antibody, anti-CgB monoclonal antibody, human CgA enzyme-linked immunosorbent assay (ELISA) kit and human CgB ELISA kit were purchased from Abcam (USA). The clinical serum samples were obtained from China-Japan Friendship Hospital. Other chemicals were analytical grade and shown in the Supporting information S1. Doubly distilled water (DDW) was employed throughout to prepare aqueous solutions.

Characterization of PMS and PMS-M2+

The PMS was characterized as demonstrated in Fig. 1. The scanning electron microscopy (SEM) image in Fig. 1A displays the PMS self-assembles into a flower-like structure with a diameter of about 200 nm. And transmission electron microscopy (TEM) image (Fig. 1B) shows the interwoven PMS nanosheets form a larger network structure, laying the foundation for the high capacity and rapid adsorption of metal ions. The elemental composition of PMS was analyzed with energy dispersive X-ray spectrometer

Conclusions

We demonstrated a novel ultrasensitive and antifouling electrochemical biosensor based on label-free immunoassay for simultaneous determination of CgA and CgB. In this design, the PMS-M2+/AuNPs/PEG/CS composites were employed as the sensing platform for the attachment of antibodies for CgA and CgB. PMS with abundant functional groups and the porous structure can load a large number of metal ions, thereby greatly amplifying the detection signals. The introduction of PEG and CS endowed the

CRediT authorship contribution statement

Xuejiao Liu: Conceptualization, Methodology, Writing – original draft. Yuanliang Li: Formal analysis. Li He: Investigation, Validation. Yongjun Feng: Resources, Supervision. Huangying Tan: Resources, Supervision. Xu Chen: Data curation, Writing – review & editing, Supervision. Wensheng Yang: Project administration, Visualization.

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.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (21874005 and 21521005), the Fundamental Research Funds for the Central Universities (XK1901) and Research projects on biomedical transformation of China-Japan Friendship Hospital (No. PYBZ1833).

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