•A proof-of-the-concept on potential-resolved “in-electrode” ECL immunosensor is presented.•Ru-NH2 and AuNPs/g-C3N4 possess different ECL potentials 1.25 V and −1.3 V respectively.•This strategy equates with extending outer Helmholtz plane of the electrode.•This proposed immunosensor realizing simultaneous detection of CA125 and SCCA.Here, a novel potential-resolved “in-electrode” type electrochemiluminescence (ECL) immunosensor was fabricated based on two different types of luminant Ru-NH2 and AuNPs/g-C3N4 to realize simultaneous detection of dual targets. In this strategy, anti-CA1251 and anti-SCCA1 were immobilized on bare gold electrode as capture probes, which could catch the two corresponding target CA125 and SCCA, and the immobilization of the signal tags was allowed via the interaction between antigen and antibody. In this process, (Ru&anti-CA1252)@GO and anti-SCCA2-AuNPs/g-C3N4 could exhibit two strong and stable ECL emissions at 1.25 V and −1.3 V respectively, which could be used as effective signal tags. Taking advantage of “in-electrode” type ECL immunosensor, all the electrochemiluminophores near the outer Helmholtz plane are “effective” in participating in the electrochemical reactions and emitting ECL signals. Therefore, the dual targets CA125 and SCCA could be detected within the linear ranges of 0.001–100 U/mL and 0.001–100 ng/mL, with detection limits of 0.4 mU/mL and 0.33 pg/mL, respectively. All these results demonstrated that the present potential-resolved “in-electrode” type electrochemiluminescence approach provided a promising analytical method for dual targets analysis with the advantages of simple analytical procedure, small sample volume and lower cost, which made the proposed method potential for clinical detection.Download high-res image (123KB)Download full-size image

A new-concept of an “in-electrode” Faraday cage-type electrochemiluminescence immunoassay (ECLIA) method for the ultrasensitive detection of neurotensin (NT) was reported with capture antibody (Ab1)-nanoFe3O4@graphene (GO) and detector antibody (Ab2)&N-(4-aminobutyl)-N-ethylisoluminol (ABEI)@GO, which led to about 1000-fold improvement in sensitivity by extending the Helmholtz plane (OHP) of the proposed electrode assembly effectively.

•Ultrasensitive and visual detection of SCCA based on GLSS method was developed.•The detection signal was amplified by AuNPs@g-C3N4.•The sensitivity of detection was improved about 1000-fold.Based on gold label silver stain (GLSS) method, a sandwich-type immunoassay for visual, ultrasensitive and specific detection of squamous cell carcinoma antigen (SCCA) was constructed using gold nanoparticles@graphite- like carbon nitride (AuNPs@g-C3N4). In this assay, the capture SCCA antibody (Ab1) was immobilized on the silanized slides via the Schiff’s base links, and the AuNPs@g-C3N4 nanocomposite labeled by the detection SCCA antibody (Ab2) acted as the signal probe. The detection mechanism was as follows: Ab1 was immobilized onto the silanized slides in order to catch the target antigen SCCA, and then allowed the immobilization of the signal probe (Ab2) via the interaction between antigen and antibody. Compared with conventional GLSS method in which AuNPs are directly immobilized on Ab2, much more AuNPs corresponded to the antigen SCCA through AuNPs@g-C3N4 because g-C3N4 possessed larger surface area and loaded much more AuNPs than Ab2. Therefore, more AuNPs were exposed to silver ions during staining, more silver ions were reduced, and the sensitivity improved 1000-fold. The average grey values were linearly related to the logarithm of SCCA concentration from 0.001 ng/mL to 100 ng/mL, with high specificity, good reproducibility and long-term stability. Results suggested that the proposed sandwich immunoassay based on GLSS method is a simple, sensitive, selective, and visual tool for the detection of SCCA.

•One-step label-free ECL immunosensor was developed to detect VP.•ECL immunosensor was constructed using multi-functionalized graphene oxide (GO).•Anti-VP and ABEI were simultaneously immobilized on nanoFe3O4@GO.•Multi-functionalized GO captured VP, emitted ECL signal and improved the sensitivity.•VP was detected in the range from 10 to 108 CFU/mL, with a LOD of 5 CFU/mL.A label-free electrochemiluminescence (ECL) immunosensor for ultrasensitive and rapid detection of marine pathogenic bacterium Vibrio parahaemolyticus (VP) in seawater and seafood was developed based on multi-functionalized graphene oxide, which was prepared with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and VP antibody (anti-VP) simultaneously immobilized on the surface of magnetic graphene oxide (nanoFe3O4@GO). ABEI and anti-VP acted as the electrochemiluminophore and the capture device for VP respectively. Good conductivity and two-dimensional structure of the nanoFe3O4@GO enabled all the ABEI, immobilized on GO, electrochemically active and thus improved the detection sensitivity. Under optimal conditions, the ECL intensity decreased with increasing logarithmic concentrations of VP in the range of 10–108 CFU/mL, with a detection limit of 5 CFU/mL for seawater and 5 CFU/g for seafood. This ECL immunosensor showed high specificity, stability and reproducibility for the detection of VP. In addition, the ECL immunosensor has been successfully used to determine the concentration of VP in seawater and seafood rapidly, with a recovery of 94.4–112.0% and RSD 4.1–11.7%. Therefore, the developed immunosensor shows great prospect for practical application.

•A proof-of-the-concept on “in-electrode” ECL immunosensor is presented.•The target is entirely caged by AuNPs/g-C3N4 composites tagged with antibody.•This proposed strategy equates with extending outer Helmholtz plane of the electrode.•The novel amplified assay is used for quantification and identification of SCCA.A novel “in-electrode”-type electrochemiluminescence (ECL) immunosensor for the sensitive detection of squamous cell carcinoma antigen (SCCA) was constructed using magnetic graphene oxide (nanoFe3O4@GO) and Au nanoparticles/graphitic-phase carbon nitride (AuNPs/g-C3N4). The capture probe was prepared by immobilizing the primary antibody of SCCA (Ab1) on the nanoFe3O4@GO, while the AuNPs/g-C3N4 nanocomposites labelled the secondary antibody of SCCA (Ab2), which acted as a signal tag. The recognition scaffold was the following: the capture probe was immobilized onto the magnetic electrode surface that caught the target SCCA and finally allowed the immobilization of the signal tag via the interaction between antigen and antibody. Importantly, a high ECL signal could be obtained due to the unique immunocomplex, which ensured all of the g-C3N4 on the outmost plane were directly fixed onto the electrode surface and became part of the electrode surface. This resulted in an enhanced efficiency of the g-C3N4 for electrochemical luminescence, thus extending the outer Helmholtz plane (OHP) of the proposed electrode and leading to high sensitivity. Taking advantage of both nanoFe3O4@GO and AuNPs/g-C3N4, the ECL intensity was found to increase logarithmically with SCCA concentration in a wide linear range from 0.001 to 10 ng/mL and with a detection limit of 0.4 pg/mL. The proposed “in-electrode”-type ECL immunosensor was used to analyse SCCA in human serum, and satisfactory recoveries were obtained, indicating that the proposed method was promising for practical applications in the clinical diagnosis of SCCA.

Tetracycline selective electrode using molecularly imprinted polymer particles as quasi-ionophore was constructed the first time, and its performance was carefully characterized. Due to the specific recognition of tetracycline by the particles, the selectivity coefficients for routine interferences were less than 10−4. Benefited from the absence of tetracycline in the sensitive membrane and the optimized composition of the inner filling solution, the limit of detection of the electrode was reduced to about 2.5 × 10−8 mol/L. It exhibited a good electrode slope 57.6 mV/decade near the theoretical Nernstian one, with a wide linear working range from 6.0 × 10−8 to 1.0 × 10−3 mol/L. The fabricated electrode should be used in pH 2–4, response time of which was less than 200 s when the concentration of tetracycline was higher than 1.0 × 10−6 mol/L and no more than 30 min at the concentration of 1.0 × 10−8 mol/L.

Based on the perfect ohmic drop compensation by online electronic positive feedback, ultrafast cyclic voltammetry with asymmetrical potential scan is achieved for the first time, with the reduction of anthracene acting as the test system. Compared with the traditional cyclic voltammetry utilizing symmetrical triangular waveform as the excitation one, the new method allows a simpler approach to mechanistic analysis of ultrafast chemical reactions coupled with a charge transfer. And perhaps more important, it also provides a way to eliminate the interference of the adsorbed product in dynamic monitoring.

A HPLC method with UV detection was developed and validated for the simultaneous determination of rivanol and mifepristone in human plasma. Norethisterone was used as the internal standard. Separation was performed by a C18 reversed-phase column maintained at 20 °C. The mobile phase was a mixture of methanol–acetonitrile–0.05% sodium dodecylsulfonate in a 0.05 M phosphate buffer with the pH adjusted to 3.0 (30:30:40, v/v/v) at a flow rate of 0.8 ml/min. Dual wavelength mode was used, with mifepristone monitored at UV 302 nm, while rivanol and norethisterone at 272 nm. A reliable biological sample pre-treatment procedure by means of solid-phase extraction was used, which allowed to obtain good extraction efficiency (>93%) for both of the analytes and the internal standard. The calibration curves were both linear with the correlation coefficient r equal to 0.9999. For rivanol, the assay gave CV% values for precision always lower than 7.8% and mean accuracy values higher than 95.3%. As to mifepristone, precision was always lower than 10.1% and mean accuracy values were higher than 93.8%. The limit of detection for the assay of rivanol and mifepristone was 1.1 and 3 ng/ml, respectively. The method is simple, sensitive and accurate, and allow for simultaneous determination of nanogram levels of rivanol and mifepristone in human plasma. It could be applied to assess the plasma level of rivanol and mifepristone in women undergoing polypharmacy with the two drugs.

An HPLC method was developed and validated for the determination of mifepristone in human plasma. C18 solid-phase extraction cartridges were used to extract plasma samples. Separation was by C18 column; mobile phase, methanol–acetonitrile–water (50:25:25, v/v/v); flow rate, 0.8 ml/min; UV detection at 302 nm. The calibration curve was linear in the concentration range of 10 ng/ml to 20 μg/ml (r = 0.9991). Within- and between-day variability were acceptable. The limit of detection for the assay was 6 ng/ml. Plasma samples were stable for at least 7 days in the state of plasma or residue treated at −20 °C. The method was simple, sensitive and accurate, and allowed to determine ng mifepristone in human plasma. It could be applied to assess the plasma level of mifepristone in women receiving low oral doses of mifepristone.

A new-concept of an “in-electrode” Faraday cage-type electrochemiluminescence immunoassay (ECLIA) method for the ultrasensitive detection of neurotensin (NT) was reported with capture antibody (Ab1)-nanoFe3O4@graphene (GO) and detector antibody (Ab2)&N-(4-aminobutyl)-N-ethylisoluminol (ABEI)@GO, which led to about 1000-fold improvement in sensitivity by extending the Helmholtz plane (OHP) of the proposed electrode assembly effectively.