A novel fluorescent and electrochemical dual-responsive immunosensor for sensitive and reliable detection of biomarkers based on cation-exchange reaction

Highlights

• The dual signal outputs showed an attractive self-correcting ability, which could make the detection result more accurate.

• CER could release thousands of Cd²⁺, which could obtain hundreds of times signal amplification.

• The Ab₂-CdSe NCs-PAMAM-CNTs conjugate was adopted as amplification label, which could further enhance the detection signal.

• The CdSe NCs were successfully functionalized with carboxyl groups, making them effectively conjugate with PAMAM.

• Magnetic Fe₃O₄ NPs was used to conjugate the primary antibody, which provided an easy way to separate the immunocomplex.

Abstract

Sensitive and reliable detection of biomarkers is of vital importance in tumor early detection and clinical therapy. A novel fluorescent/electrochemical dual-responsive immunosensing platform for reliable and sensitive quantification of biomarkers was designed based on cation-exchange reaction. To construct such a versatile platform, the model analyte, carcinoembryonic antigen (CEA), was captured by magnetic Fe₃O₄ nanoparticles bound with primary antibodies (Fe₃O₄-Ab₁) and then recognized by the detection antibodies conjugated complex containing poly(amidoamine) (PAMAM), carbon nanotube (CNT) and carboxyl functionalized CdSe nanocrystals (NCs) (CNT-PAMAM-CdSe NCs-Ab₂). Via ligand exchange, the stable CdSe nanocrystals were easily functionalized with carboxylate ion (CdSe–COO^-) and showed high hydrophilicity. The CdSe–COO^- was effectively and densely conjugated to CNT coated dendrimer PAMAM that possesses large specific surface area. Finally, the target CEA was detected based on cation-exchange reaction (CER) by adding Ag⁺ to release thousands of cations Cd²⁺, which were detected by fluorescence and electrochemistry simultaneously. The electrochemical measurement was performed by directly detecting Cd²⁺ through square wave voltammetry (SWV), which displayed an excellent correlation with CEA from 5 pg/mL to 50 ng/mL, with a limit of detection (LOD) of 1.7 pg/mL. The fluorescence detection was implemented since free Cd²⁺ could trigger the weak fluorescence metal-sensitive dyes (Rhod-5N) to generate extremely high fluorescence signal. The fluorescence results showed the LOD for CEA detection was 0.25 pg/mL with a calibration curve range from 1 pg/mL to 20 ng/mL. The dual signal outputs showed an attractively self-correcting ability, which provides the capability of avoiding false positive signal and making the detection result more reliable. The proposed dual-responsive platform holds great promises for biomarkers detection in clinical diagnostics and therapy.

Introduction

Globally, cancer is one of the leading causes of mortality and nearly 1 in 6 deaths is due to cancer. Despite cancer research has received considerable attention and funding, the 5-year relative survival rates of sufferers who are diagnosed with advanced cancer are still extremely low (e.g. 3% for lung cancer). Fortunately, researches reveal that the survival rate can be significantly improved when cancers are discovered at an early stage (50% for lung cancer) [1]. Therefore, early detection of cancer is of great significance to reduction mortality. One of the most important methods for early detection is to measure available blood tumor markers (biomarkers). These biomarkers are generated by either tumor itself or human body in response to the tumor growth, thus they could be good indicators for the presence, growth, and metastasis of tumors. Generally, various immunoassays have been reported to assay biomarkers, including colorimetric immunoassay [2,3], enzyme-linked immunosorbent assay [4], chemiluminescence immunoassay [[5], [6], [7]], surface plasmon resonance immunoassay [8,9], electrochemical immunoassay [[10], [11], [12], [13], [14]], electrochemiluminescent immunoassay [15] and fluorescence immunoassay [16,17]. In comparison to other counterparts, electrochemical immunoassay (EIA) has captured lots of attention due to its low-cost, high sensitivity and miniaturization, thus showing promise in clinical application [18]. However, the major drawback of EIA is that they lack enough stability, which limits its successful clinical applications.

Dual-responsive assay strategies can solve this problem, because two types of analytical platforms can offer two types of signal outputs simultaneously to ensure the accuracy and persuasiveness of the detection results [19]. Up to now, many dual-responsive assay platforms have been developed, such as fluorescent-colorimetric [20,21], Surface enhanced Raman scattering-fluorescent [22], magnetic-fluorescent [23] and electrochemical-fluorescent [24]. Among them, electrochemical-fluorescent (EC-FL) dual-responsive assay has gained much attention because this method could provide sensitive electrochemical signal and stable fluorescence response simultaneously. For example, Lapos et al. have reported an EC-FL dual-responsive detection of various analytes on electrophoresis microchip [24]. Kevin et al. assembled copper nanoblocks capable of sensitively detecting multiple RNA samples with flexible FL and EC readouts. However, thus far, EC-FL dual-responsive assay for immunoassay biomarkers has not been reported.

In this study, we developed a sensitive EC-FL dual-responsive immunoassay based on cation exchange reaction (CER) for the early detection of biomarkers. CER was first reported by Alivisatos and co-workers [25], which is a gentle, fast response and high efficiency method to generate divalent cations. CdSe was adopted in the current study because its CER mechanism has been demonstrated [25] and a large amount of synthesis methods and various surface modification strategies have been developed, which facilitates their applications in bioassays [[26], [27], [28]]. The CER equation was shown in Fig. S1, in which Ag⁺ exchanged CdSe NCs to Ag₂Se and a large number of Cd²⁺. The formation of Ag₂Se is due to the thermodynamic driving force, the Ag₂Se is more thermodynamically favored compared with CdSe [29]. If we combine CER with metal-sensitive dyes, thousands of Cd²⁺ can be released from each NC and the Cd²⁺ can trigger the non- or weakly fluorescence dye molecule to obtain hundreds of folds increase in FL signal intensity [30]. Such a CER system is a simple and highly sensitive method, which could be developed for immunoassay biomarkers [31,32].

Herein, several kinds of nanomaterials were combined in constructing the immunosensor. Fe₃O₄ nanoparticles (NPs) are widely used in immunosensors since the magnetic NPs can provide an easy way to separate the immunocomplex. Carbon nanotubes (CNTs) play a leading role in nanomedicine and biosensing due to their large specific surface area. The water-soluble carboxylate ion functionalized CdSe NCs were obtained by ligand exchange. Poly(amidoamine) (PAMAM) dendrimers exhibited a high density of functional groups, excellent solubility and high branched units, making them perfect candidates for linking CNTs, CdSe NCs and recognition antibody. Carcinoembryonic antigen (CEA) was selected as the model biomarker which is widely used to evaluate many kinds of malignancies, such as lung cancer, colorectal cancer and ovarian cancer. The immunosensor was constructed as follows (Scheme 1): CEA was firstly captured by magnetic Fe₃O₄ nanoparticles bound primary antibodies (Fe₃O₄-Ab₁) and then recognized by the detection antibodies conjugated compound containing PAMAM, CNTs and carboxylate ion functionalized CdSe nanocrystals (CNTs-PAMAM-CdSe NCs-Ab₂). Finally, the target CEA was detected by adding Ag⁺ to release thousands of Cd²⁺, which were detected by fluorescence and electrochemistry. The nanomaterials (PAMAM coated CNTs) combining CER could extremely amplify the detection signal, and the fluorescence and electrochemistry dual-response could guarantee a more convincing result. Therefore, the proposed immunosensor exhibited promising future in early detection of various biomarkers in clinical diagnostics and therapy.