Dual labeled mesoporous silica nanospheres based electrochemical immunosensor for ultrasensitive detection of carcinoembryonic antigen
Graphical abstract
Schematic Representation of a NiO@Au Nanoparticle Decorated Graphene and Dual Labeled Mesoporous Silica (DLMS) Nanospheres Based Electrochemical Sensor for the Ultrasensitive Detection of Carcinoembryonic (CEA) Antigen in Real Time Samples.
Introduction
Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein (oncomarker) for the diagnostic/therapeutic purposes of several malignancies like a gastrointestinal tumor, ovarian carcinoma, cystadenocarcinoma, lung, and breast cancer [1,2] to name few. The CEA developed in a large proportion in nearly all human carcinomas and the level of more than 2.5 ng/mL (5 ng/mL for cigarette smoker) in human serum is a sign of probable ailment [3,4]. Besides, the level keeps rising constantly in patients with cancer recurrence even after surgery. For example, the CEA has efficiently utilized in the monitoring of breast cancer recurrences for several years and is considered to be one of the two main biomarkers for early detection of breast cancer recurrences well before clinical and radiological signs of the disease occur [5]. Consequently, the development of an efficient diagnostic tool, capable of early detection of recurrences becomes highly desirable. Besides, it should be comprehensive to practice for progression monitoring of CEA in human serum in order to control and reduce the mortality. Among different techniques reported for CEA detection, the electrochemical immunosensor has attracted immense attention among researchers due to its fascinating characteristics such as high sensitivity, rapid response, cost-effective, less diagnostic time, and miniaturization [[6], [7], [8]]. Moreover, the sandwich-type electrochemical immunoassays were broadly utilized for the determination of various tumor oncomarkers due to its detection protocol based on highly specific recognition of antibodies with target antigen [9,10]. However, most of the electrochemical sensors have a limitation of high background signals, interface congestion which results in less sensitive signal responses [[11], [12], [13]]. The labeling of signal antibodies is another significant problem for sandwich-type electrochemical immunoassays. For traditional enzyme-linked immunoassays, the bioactive enzyme was typically used on the label of the secondary antibody. Nevertheless, the possible conjugation of enzyme molecule sites on each antibody has always been reduced. As a result, ongoing efforts have been made worldwide to develop the labeled methods in electrochemical immunoassays using multifunctional nanoscales. For example, Zhao et al. developed an electrochemical biosensor based on lectin as molecular recognition elements for the sensitive detection of CEA [4]. Similarly, the Yang group has reported the streptavidin-functionalized nitrogen-doped graphene (NG) based promising platform for the electrochemical immunosensing of CEA [14] and so on. However, the antibody drawbacks associated with its growth, stability, and manipulation have led researchers to pursue alternatives. Thus, the performance of the immunosensor has been improved efficiently by the exploitation of signal amplification approaches.
The signal enhancement strategies play a vital role in obtaining low detection limits and enhanced sensitivity of sandwich-type electrochemical immunoassays [10,13,15]. The signal enrichment is primarily attributed to the conjugation of different types of labels with secondary antibodies (Ab2) to form antibody-antigen immunocomplex [1,16]. Until now, several signal amplification strategies have been reported to enhance the sensitivity of CEA detection. For example, Chen et al. described the usage of horseradish peroxidase (HRP) as an enhancer and Au@Ag nanoparticles as trace labels for the amplification of electrochemical signals [17]. Similarly, Feng et al. used ferrocene functionalized Fe3O4@SiO2 as labels [18] and Peng et al. described the usage of graphene/chitosan–ferrocene and Fe3O4/Au NPs as labels [19] to enhance the sensitivity of the electrochemical immunosensors. Lv et al. utilized the cubic Au@Pt dendritic nanomaterials functionalized nitrogen-doped graphene loaded with copper ion (Au@Pt DNs/NG/Cu2+) as labels to effectively capture and immobilize secondary anti-CEA [20]. Herein, a mesoporous silica-based immunocomplex was used for signal enhancement strategy. The mesoporous silica (SBA-15 Santa Barbara Amorphous) material has gained intense attention as an ideal support material to improve the material properties due to its structural features such as large specific surface areas (above 1000 m2/g) with a thick pore wall, uniformly arranged pore channels (in range of 4–30 nm), high thermal stability, optical transparency, and high biocompatibility. Due to such beneficial factors, it is widely explored in adsorption, catalysis, optoelectronics, and biosensors [21]. Moreover, SBA-15 can act as an ideal candidate (host) with efficient binding capabilities to a greater number of bio-molecules and thus enhances the sensitivity [22]. However, pristine SBA-15 has poor conductivity and does not possess any catalytic activity as a result of its lower acidic strength. The amalgamation of electroactive metals (such as gold, silver, platinum, and palladium) into the channels of SBA-15 paves the way to enhance its electrochemical performance [23,24]. Several studies have been reported for the integration of metal nanoparticle into the channels of SBA-15.
In this study, we further exploited the application of SBA-15 by entrapping Au NR and HRP to act as a dual signal enhancer for the electrochemical detection of CEA. Besides, the DLMS was synthesized by entrapping Au NR and HRP within the channels of amine-functionalized SBA-15, and then Ab2 was conjugated onto SBA-15. Thus, this work is designed to have the combined advantages of the above various strategies to achieve maximum sensitivity and the approaches used are as follows: 1) the ITO glass was coated with reduced graphene oxide to enhance the conductivity and surface area, 2) Au decorated NiO nanoparticles were deposited on the electrode to enhance the active surface as well as the loading capacity of Ab1 on the electrode surface, 3) to obtain the highest sensitivity, a sandwich-type immunoassay was developed by immobilization of DLMS with Ab1 modified electrode (ITO/rGO/NiO@Au/Ab1). In addition to the above strategies, thionine was dotted over the DLMS to improve the electrochemical performance and also to induce faster electron transport towards an effective reduction of H2O2. With the successful implementation of all the above-mentioned approaches, the sensitivity of the developed biosensor was found to be enhanced drastically. Hence, the proposed immunoassay is foreseen as a potential application towards clinical monitoring of the CEA biomarker.
Section snippets
Experimental section
All the chemicals, details of instrumentation, and characterization of prepared materials were presented in electronic supporting materials (ESM).
Preparation of dual labeled mesoporous silica (Au NR@SBA-15/Ab2-HRP)
The surface of the prepared Au NR@SBA-15 (the preparation methods were discussed in the ESM (Scheme S1) was modified with APTES for the covalent conjugation of HRP and antibody [25]. In brief, 0.5 g of Au NR@SBA-15 was refluxed with 50 mL of toluene (anhydrous) and 0.5 mL of APTES for 24 h. The amine-functionalized Au NR@SBA-15 was separated and washed with methanol to get rid of any physically bounded APTES. Then, 100 mg of APTES functionalized Au NR@SBA-15 was suspended in 2 mL of PBS with
Fabrication of electrochemical immunosensor
The graphical illustration for the electrode fabrication is displayed in Scheme 2. At first, the ITO plates (working electrode) were subjected to successive surface modification prior to treatment with DLMS. The ITO coated glass plates were cleaned by the ultra-sonication process with acetone, methanol, and ultrapure water. As per the earlier reported method, the SAM layer of APTES on the precleaned ITO plates was prepared [26]. The cleaned ITO plates were then treated with a solution
Electrochemical detection of CEA
The sandwich-based immunoassay protocol for CEA detection is presented in Scheme 2. The fabricated immunosensor (ITO/rGO/NiO@Au/Ab1) was utilized for the effective determination of the CEA antigen. In this study, amine-functionalized Au NR@SBA-15 (as a label) and HRP (as signal enhancer) were used to enhance the proficiency of the immunosensor. The adapted ITO plates were initially incubated at 37 °C with 100 μL of PBS solution containing various concentrations of target antigen for 60 min and
Results and discussion
The electrochemical behavior of the immunosensor can be enhanced by i) efficient immobilization of the biomolecules onto the ITO electrode, ii) retainment of their biological activity, and iii) improved electron transfer (electrical conductivity) [27,28]. In this view, graphene has been used in the development of electrochemical immunosensors due to its intriguing physicochemical properties [5]. Moreover, it also acts as a unique functional material for electrochemical sensing platforms with
Electrochemical performance of the immunosensor
The electrochemical performance of the sequentially modified electrode for the detection of CEA was characterized by CV in 0.1 M PBS buffer (pH = 7.0) containing 5 mM [Fe (CN)6]3-/4- (1:1) in 0.1 M KCl at 50 mV/s. The peak-to-peak potential separation ΔEp [ΔEp = EPA (anodic peak potential) - EPC (cathodic peak potential)] of the ferrocyanide [Fe (CN6) −3/−4] redox couple was assessed via CV after sequential surface modification of electrode and are given in Fig. 2a. For bare ITO, the reversible
Detection of CEA antigen
The sensitivity and detection range of the fabricated DLMS based electrochemical immunoassay towards the detection of CEA were evaluated with various concentrations of CEA by DPV analysis. From Fig. 4, the DPV of the developed immunosensor was proportionate to the concentration of CEA under optimal conditions. Specifically, the DPV response was found to be linearly increased along with the increment in the CEA antigen concentration and displayed a virtuous linearity affiliation with the
Selectivity, repeatability, reproducibility and stability studies
In order to suggest the developed immunosensor as an appropriate system for the sensing application of cancer biomarkers, the anti-interference ability was validated with prostate-specific antigens (PSA), bovine serum albumin (BSA), p53, α-fetoprotein (AFP) and their combination with the target antigen. The DPV analysis was performed with 100 ng/mL of interfering proteins (BSA, PSA, p53, IgG, AFP, Uric Acid (UA), Ascorbic Acid (AA) and their mixture with CEA) modified immunosensor and are
Practical application of the developed immunosensor
The feasibility of the developed immunosensor was assessed by the selective detection of CEA in blood (human) samples using a typical spiking technique. Human serum samples were collected from the volunteers with good health. The supernatant solution of the centrifuged serum samples was diluted (fifty times by volume) with PBS (0.1 M). DPV response of retrieval assessments (Fig. S14) was analyzed by adding (spiking) a different quantity of CEA to the as-prepared samples (30 mL). The retrieval
Conclusion
The present work describes the development of an innovative electrochemical immunoassay based on dual-labeled mesoporous silica (DLMS) for the ultrasensitive CEA detection. The features of the developed sensor platform (which includes the enhanced degree of electron transfer, synergistic effect, efficient bio-interface, excellent conductivity, and larger surface area) effectively enhanced the electrocatalytic performance with higher sensitivity. In addition, a signal amplification approach was
CRediT authorship contribution statement
Srinivasan Krishnan: Conceptualization, Methodology, Writing - original draft. Xinxin He: Writing - original draft. Fengjuan Zhao: Writing - original draft. Yuqing Zhang: Writing - original draft. Shanhu Liu: Writing - original draft. Ruimin Xing: Writing - original draft.
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
The authors gratefully appreciate the support from the National Natural Science Foundation of China (21950410531), and Science & Technology Research Project of Henan province (182102410090). Also, we express thanks to Dr. Daibing Luo from the Analytical & Testing Center of Sichuan University for valuable discussion and characterization.
References (41)
- et al.
A novel sandwiched electrochemiluminescence immunosensor for the detection of carcinoembryonic antigen based on carbon quantum dots and signal amplification
Biosens. Bioelectron.
(2017) - et al.
A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles–thionine–reduced graphene oxide nanocomposite film modified glassy carbon electrode
Talanta
(2011) - et al.
Electrochemical lectin-based biosensor array for detection and discrimination of carcinoembryonic antigen using dual amplification of gold nanoparticles and horseradish peroxidase
Sensor. Actuator. B Chem.
(2016) - et al.
Gold and silver bio/nano-hybrids-based electrochemical immunosensor for ultrasensitive detection of carcinoembryonic antigen
Biosens. Bioelectron.
(2019) - et al.
Electrochemical immunosensors – a powerful tool for analytical applications
Biosens. Bioelectron.
(2018) - et al.
CoOOH nanosheets-coated g-C3N4/CuInS2 nanohybrids for photoelectrochemical biosensor of carcinoembryonic antigen coupling hybridization chain reaction with etching reaction
Sensor. Actuator. B Chem.
(2020) - et al.
A “sense-and-treat” ELISA using zeolitic imidazolate framework-8 as carriers for dual-modal detection of carcinoembryonic antigen
Sensor. Actuator. B Chem.
(2019) - et al.
Development of a reliable microRNA based electrochemical genosensor for monitoring of miR-146a, as key regulatory agent of neurodegenerative disease
Int. J. Biol. Macromol.
(2019) - et al.
A label-free and double recognition–amplification novel strategy for sensitive and accurate carcinoembryonic antigen assay
Biosens. Bioelectron.
(2019) - et al.
A high-sensitivity electrochemical aptasensor of carcinoembryonic antigen based on graphene quantum dots-ionic liquid-nafion nanomatrix and DNAzyme-assisted signal amplification strategy
Biosens. Bioelectron.
(2018)
Co-MOF nanosheet array: a high-performance electrochemical sensor for non-enzymatic glucose detection
Sensor. Actuator. B Chem.
Construction of sandwiched self-powered biosensor based on smart nanostructure and capacitor: toward multiple signal amplification for thrombin detection
Sensor. Actuator. B Chem.
Efficient streptavidin-functionalized nitrogen-doped graphene for the development of highly sensitive electrochemical immunosensor
Biosens. Bioelectron.
Functionalization of poly(o-phenylenediamine) with gold nanoparticles as a label-free immunoassay platform for the detection of human enterovirus 71
Sensor. Actuator. B Chem.
Facile synthesis of MoS2@Cu2O-Pt nanohybrid as enzyme-mimetic label for the detection of the Hepatitis B surface antigen
Biosens. Bioelectron.
A dual amplification electrochemical immunosensor based on HRP-Au@Ag NPs for carcinoembryonic antigen detection
Anal. Biochem.
A sandwich-type electrochemical immunosensor for carcinoembryonic antigen based on signal amplification strategy of optimized ferrocene functionalized Fe3O4@SiO2 as labels
Biosens. Bioelectron.
Electrochemical immunosensor for carcinoembryonic antigen based on signal amplification strategy of graphene and Fe3O4/Au NPs
J. Electroanal. Chem.
Enhanced peroxidase-like properties of Au@Pt DNs/NG/Cu2+ and application of sandwich-type electrochemical immunosensor for highly sensitive detection of CEA
Biosens. Bioelectron.
Ultrasensitive electrochemical immunoassay for BRCA1 using BMIM·BF4-coated SBA-15 as labels and functionalized graphene as enhancer
Biomaterials
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