Carbon nanospheres-promoted electrochemical immunoassay coupled with hollow platinum nanolabels for sensitivity enhancement

Abstract

Two nanostructures including carbon nanospheres-graphene hybrid nanosheets (CNS-GNS) and hollow platinum nanospheres (HPtNS) were first synthesized by using direct electrolytic reduction and wet chemistry methods, respectively. Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of carcinoembryonic antigen (CEA) by using HPtNS-labeled horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA) as molecular tags and anti-CEA-assembled CNS-GPS as sensing probes. Compared with pure graphene nanosheets, the presence of carbon nanospheres on the graphene increased the surface coverage of the substrate, and enhanced the immobilized amount of primary antibodies. Several labeling protocols, such as HRP-anti-CEA, solid platinum nanoparticle-labeled HRP-anti-CEA, and hollow platinum nanospheres-labeled HRP-anti-CEA, were investigated for determination of CEA and improved analytical features were obtained with hollow platinum nanosphere labeling. With the HPtNS labeling method, the effects of incubation time and pH on the current responses of the immunosensors were also studied. The strong attachment of biomolecules to the CNS-GPS and HPtNS resulted in a good repeatability and intermediate precision down to 10.2%. The dynamic concentration range spanned from 0.001 ng mL⁻¹ to 100 ng mL⁻¹ CEA with a detection limit of 1.0 pg mL⁻¹ at the 3S_blank level. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of CEA.

Introduction

Sensitive and specific determination of cancer-related biomarkers is very important in the modern medicine and clinic diagnosis (Nie et al., 2009). Usually, the biomarkers are soluble glycoproteins that are found in the blood, urine, or tissues of patients with certain types of cancer. To test for a cancer maker, samples of the patient's blood or urine are oftentimes sent to the analytical laboratory. The marker is usually found by combining the blood or urine with manmade antibodies that react with the marker protein. Immunoassays are chemical tests used to detect or quantify a specific substance, the analyte, in a blood or body fluid sample, using an immunological reaction. Current immunoassay methods mainly consist of immunoprecipitation, particles immunoassays, immunonephelometry, radioimmunoassay, enzyme immunoassays, fluorescent immunoassays and (electro)chemiluminescent immunoassays (Zuiverloon et al., 2012, Ray et al., 2011, Karns and Herr, 2011). Despite many advances in this field, there is still the quest for new schemes and strategies for improvement of the sensitivity and simplicity of clinical immunoassays.

Electrochemical immunoassays have attracted considered attention over the past few years due to its intrinsic advantages, such as good portability, low cost, simple instrumentation, and high sensitivity (Su et al., 2011a, Zhang et al., 2008). Various transducers including amperometry (Wang et al., 2012), electrochemical impedance spectroscopy (Venugopal et al., 2011), potentiometry (Tang et al., 2006), and conductometry (Tang et al., 2011b), have been employed for electrochemical determination of different biomarkers, e.g. alpha-fetoprotein (B. Zhang et al., 2011), carcinoembryonic antigen (Qi et al., 2012), and prostate specific antigen (Wan et al., 2011). Among these methods, sandwich-type amperometric immunoassay is one of the most popular schemes because of the use of a couple of match antibodies (Tang et al., 2008). The method measures the formation of antigen–antibody complexes on the immunosensor, and detects them via an indicator reaction. Usually, the level of cancer markers in serum or urine is relatively low in the early stages of pathogenesis. To enhance the capture probability of the immobilized primary antibodies on the transducer for low level concentrations of the target analyte in the sample, the preparation of the immunosensors is crucial. An effective strategy is to enhance the surface coverage of primary antibodies on the transducer by resorting to bionanotechnology. Graphene, as a single-atom-thick and two-dimensional carbon nanomaterial, has attracted a great deal of attention in the field of electrochemical immunoassays because of its remarkable electrical, mechanical, and thermal properties (Lu et al., 2009). Due to the limitation of surface area of two-dimensional graphene nanosheets, however, the immobilized amount of biomolecules is limited. To tackle this issue, our motivation is to pattern nanoparticle assemblies on the graphene nanosheets. Recently, we synthesized magnetic-graphene hybrid nanosheets for the conjugation of primary antibodies, which were used for simultaneous multiplexed electrochemical immunoassays (Tang et al., 2011a). Unexpectedly, we later found that the doped magnetic beads might decrease the graphene conductivity to some extent, compared with pure graphene nanosheets. The reason might be the fact that iron oxide nanoparticles are semi conductive, but not conductive to electron transfer. In contrast, carbon nanomaterials with intriguing physical properties and remarkable conductivities can be used for the synthesis of hybrid nanostructures (Tang et al., 2010). In comparison with fullerenes or carbon nano-onions, carbon nanoparticles have unclosed graphene layers, and possess unique properties, e.g. low density, high porosity and surface area, and relatively high chemical and thermal stability (Vidal et al., 2008, Amiri et al., 2007). In this regard, we used carbon nanoparticles, rather than magnetic beads, for the synthesis of carbon nanoparticles-graphene hybrid nanosheets in this work. The doped carbon nanoparticles are expected to enhance the immobilized amount of biomolecules and improve the conductivity of the transducer.

Another major issue for the successful development of electrochemical immunoassays is to achieve a high sensitivity. Typical methods involve the use of an indicator system (e.g. enzyme label) that results in the amplification of the measured product. Owing to the limited amount of enzymes on each labeled antibody, many reports were recently focused on nanolabels for signal amplification, encompassing nanoparticles (H. Zhang et al., 2011), nanowires (Su et al., 2011b), nanotubes (M. Yang et al., 2010), nanorods (Du et al., 2011), and alloys (Wei et al., 2011). Various nanomaterials including nanogold hollow microspheres (Tang et al., 2011a), GoldMag nanospheres (Zhang et al., 2012), multiarmed platinum nanowires (Su et al., 2011b), and functional carbon nanotubes (Li et al., 2011) were also exploited for the labels of secondary antibodies in our group. Unfortunately, most nanomaterials were only used as the matrices for the immobilization of biomolecules, and had the inability to catalyze the biochemical reaction for signal amplification. Inspiringly, we recently found that platinum nanoparticles could catalyze the reduction of hydrogen peroxide under the appropriate conditions. Moreover, variously shaped platinum nanostructures displayed different catalytic efficiencies. Hollow platinum nanospheres with excellent loading capability and good biocompatibility can be utilized for the label of biomolecules including enzyme-labeled antibodies. In this case, both platinum nanostructures and enzymes might concurrently catalyze the biochemical reaction with the substrate, and enhance the sensitivity of the electrochemical immunoassays.

Carcinoembryonic antigen (CEA) is a preferred tumor marker to help predict outlook in patients with colorectal cancer. The normal range of blood levels varies between individuals, but levels higher than 3 ng mL⁻¹ are not normal. The higher the CEA level at the time colorectal cancer is detected, the more likely is that the cancer is advanced (Huang et al., 2010, X. Yang et al., 2010). Herein, we combined the merits of carbon nanospheres-graphene hybrid nanosheets with hollow platinum nanospheres, and fabricated a new electrochemical immunosensor for detection of CEA, as a model cancer biomarker. The immunosensors were simply prepared by immobilization of anti-CEA antibodies on carbon nanospheres-graphene hybrid nanosheets-functionalized glassy carbon electrode. Hollow platinum nanospheres-labeled horseradish peroxidase-anti-CEA conjugates were employed as molecular tags with a sandwich-type immunoassay format. The aim of this study is to explore a new electrochemical protocol for sensitive determination of cancer biomarkers.