A new homogeneous electrochemical immunoassay strategy was developed for ultrasensitive detection of carcinoembryonic antigen (CEA) based on target-induced proximity hybridization coupled with rolling circle amplification (RCA). The immobilization-free detection of CEA was realized by the use of an uncharged peptide nucleic acid (PNA) probe labeled with ferrocene (Fc) as the electroactive indicator on a negatively charged indium tin oxide (ITO) electrode. In the presence of a target protein and two DNA-labeled antibodies, the proximate complex formed in homogeneous solution could unfold the molecular beacon, and a part of the unfolded molecular beacon as a primer hybridized with the RCA template to initiate the RCA process. Subsequently, the detection probe modified Fc (Fc-PNAs) hybridized with the long amplified DNA products. The consumption of freely diffusible Fc-PNAs (neutrally charged) resulted in a significant reduction of the Fc signal due to the fact that long amplified DNA/Fc-PNA products were electrostatically repelled from the ITO electrode surface. The reduction of the electrochemical signal (signal-off) could indirectly provide the CEA concentration. Under the optimal conditions, CEA detection was implemented in a wide range from 1 pg mL−1 to 10 ng mL−1, with a low detection limit of 0.49 pg mL−1. The proposed strategy exhibited advantages of good selectivity, high sensitivity, acceptable accuracy, and favorable versatility of analytes. Moreover, the practical application value of the system was confirmed by the assay of CEA in human serums with satisfactory results.

•A novel enzyme-free electrochemical immunosensor was developed for detection of CEA.•The signal amplification was based on catalytic DNA hairpin assembly and DNA-template-synthesized Pd nanoparticles.•The biosensor could detect CEA down to 0.52 × 10−16 g mL−1 level with a dynamic range spanning 5 orders of magnitude.Novel hybridization proximity-regulated catalytic DNA hairpin assembly strategy has been proposed for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles as signal label. The DNA template-synthesized Pd nanoparticles were characterized with atomic force microscopic and X-ray photoelectron spectroscopy. The highly efficient electrocatalysis by DNA template synthesized Pd nanoparticles for NaBH4 oxidation produced an intense detection signal. The label-free electrochemical method achieved the detection of carcinoembryonic antigen (CEA) with a linear range from 10−15 to 10−11 g mL−1 and a detection limit of 0.43 × 10−15 g mL−1. Through introducing a supersandwich reaction to increase the DNA length, the electrochemical signal was further amplified, leading to a detection limit of 0.52 × 10−16 g mL−1. And it rendered satisfactory analytical performance for the determination of CEA in serum samples. Furthermore, it exhibited good reproducibility and stability; meanwhile, it also showed excellent specificity due to the specific recognition of antigen by antibody. Therefore, the DNA template synthesized Pd nanoparticles based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level.A novel label-free and enzyme-free electrochemical immunoassay based on proximity hybridization-regulated catalytic DNA hairpin assemblies for recycling of the CEA.Download high-res image (169KB)Download full-size image

•Development of a paper-based disposable electrode for nitrite analysis.•Fouling caused by adsorption of oxidation products is effectively avoided.•The paper-based electrode exhibited better electron transfer behavior than commercial electrodes.•The electrode was successfully used to analyse nitrite in environmental and food samples.•The advantages of this approach are accuracy, cheapness, reproducibility, and ease of miniaturization.Electrochemical techniques are attractive for nitrite detection owing to their intrinsic advantages. However, traditional electrochemical sensors often suffer from the effects of fouling due to the adsorption of oxidation products on the electrode surface. In this work, a paper-based, inexpensive, disposable electrochemical sensing platform was developed for nitrite analysis based on a simple and efficient vacuum filtration system. Taking advantage of the physicochemical properties of graphene nanosheets and gold nanoparticles, the mass transport regime of nitrite at the paper-based electrode was thin layer diffusion rather than planar diffusion. In comparison with the electrochemical responses of commercial gold electrodes and glassy carbon electrodes (GCE), a considerably larger current signal was seen at the paper-based sensing interface, which significantly improved its sensitivity for nitrite detection. In particular, the paper-based electrode was a disposable sensing device, so that it effectively avoided the fouling effect arising from the adsorption of oxidation products. Moreover, the paper-based sensing platform made it possible to determine nitrite in environmental and food samples in an accurate, convenient, inexpensive, and reproducible way, indicating that the proposed system is promising for practical applications in environmental monitoring and public health.

•An efficient electrochemical sensor was developed for glucose detection based on the assembly of platinum nanoparticles on carbon nanocubes.•The platinum nanoparticles modified carbon nanocubes exhibited excellent electrocatalytic activity towards glucose oxidation.•The present work implemented the analysis of glucose in clinical serum samples with satisfactory results.In this paper, high-quality and three-dimensional nanocomposites of Pt nanoparticles anchored on carbon nanocubes (PtNPs/CNCs) were synthesized by a simple preparation approach. The surface morphology, structure and composition of the prepared PtNPs/CNCs were examined by transmission electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, thermogravimetric analysis, and Raman spectroscopy. The results showed that the Pt nanoparticles with the diameter of about 3–5 nm were well dispersed on the surface of CNCs. Electrochemical results demonstrated that PtNPs/CNCs efficiently catalyzed the electrochemical oxidation of glucose in 0.1 M phosphate buffer solution (PBS), and exhibited a rapid response time of about 1 s, a broad linear range from 0.05 to 10.0 mM, a low detection limit of 0.02 mM, as well as high stability. Thus, the PtNPs/CNCs based electrochemical sensor was promising for application in direct detection of glucose since the electrochemical detection of glucose was performed at low potential, and the signals from oxidation of interfering species could be effectively declined.

•An efficient electrochemical interface was constructed for the recognition of DNA methylation information.•The conductive graphene nanowall layer altered the mass transport regime of analytes.•The direct potential resolution between cytosine and 5-methylcytosine was 184 mV.•The present work realized the evaluation of DNA methylation level in CpG oligonucleotides.•This method exhibited advantages of rapidity, convenience, accuracy and universal analysis.Herein, an efficient approach was developed for direct electroanalysis of DNA methylation based on the construction of polypyrrole functionalized graphene nanowall interface. Taking advantage of the physicochemical properties of graphene nanowall layer, the mass transport regime of analytes was changed from planar diffusion to thin layer diffusion. As a result, the oxidation signal of 5-methylcytosine (mC) was clearly distinguished from that of cytosine (C) with a direct potential resolution of 184 mV, which paved the way for simultaneous detection with high reliability. Moreover, the proposed method realized the evaluation of DNA methylation level in CpG oligonucleotides in a straightforward, rapid, convenient, label-free and hybridization-free way, suggesting that the system is promising for sensing applications.

An efficient electrochemical approach for the evaluation of DNA methylation level was proposed according to the oxidation signal of DNA bases at an overoxidized polypyrrole (PPyox) directed multiwalled carbon nanotubes (MWNTs) film modified glassy carbon electrode (GCE). The PPyox/MWNTs/GCE exhibited remarkable electrocatalytic activities towards the oxidation of DNA bases due to the advantages of wide potential window, large effective surface area, and excellent antifouling property. As a result, all purine and pyrimidine bases of guanine (G), adenine (A), thymine (T), cytosine (C) and 5-methylcytosine (5-mC) exhibited well identified oxidation peaks at the PPyox/MWNTs/GCE. The direct potential resolution between 5-mC and C was obtained to be 180 mV, which was large enough for their signal recognition and accurate detection in mixture. In particular, the signal interference from T, a great challenge in exploring DNA methylation, was successfully eliminated by an innovative strategy, which was developed based on the stoichiometric relationship between purine and pyrimidine bases in DNA molecular structure. The proposed method was effectively applied to the rapid detection of DNA methylation status in real sample within 45 min with satisfactory results.Highlights► The method realized the rapid detection of DNA methylation information within 45 min. ► The interference from thymine was successfully eliminated by an innovative strategy. ► The peak potential of 5-methylcytosine was clearly distinguished from that of cytosine. ► The assay process was free from bisulfite conversion, hybridization and labeling steps. ► Compared with traditional methods, this approach is convenient, rapid and accurate.