Rawiwan Laocharoensuk

Ultrasensitive electrochemical immunosensor based on dual signal amplification process for p16(INK4a) cervical cancer detection in clinical samples.

The p16(INK4a) (p16) is a cyclin-dependent kinase inhibitor, which has been evaluated in several studies as a diagnostic marker of cervical cancer. Immunostaining using p16 specific antibody has confirmed an over-expression of p16 protein in cervical cancer cells and its association with disease progression. This article reports an ultrasensitive electrochemical immunosensor for specific detection of p16 and demonstrates its performance for detection of solubilized p16 protein in cell lysates obtained from patients. Sandwich-based immunoreaction couple with double signal amplification strategy based on catalytic enlargement of particle tag was used for high sensitivity and specificity. The conditions were optimized to create an immunoassay protocol. Disposable screen-printed electrode modified with capture antibodies (Ab1) was selected for further implementation towards point-of-care diagnostics. Small gold nanoparticles (15 nm diameter) conjugated with detection antibodies (Ab2) were found to better serve as a detection label due to limited interference with antigen-antibody interaction. Double signal enhancement was performed by sequential depositions of gold and silver layers. This gave the sensitivity of 1.78 μA mL(ng GST-p16)(-1) cm(-2) and detection limit of 1.3 ng mL(-1) for GST-p16 protein which is equivalent to 0.49 ng mL(-1) for p16 protein and 28 cells for HeLa cervical cancer cells. In addition to purified protein, the proposed immunosensor effectively detected elevated p16 level in cervical swab samples obtained from 10 patients with positive result from standard Pap smear test, indicating that an electrochemical immunosensors hold an excellent promise for detection of cervical cancer in clinical setting.

Extrinsic surface-enhanced Raman scattering detection of influenza A virus enhanced by two-dimensional gold@silver core–shell nanoparticle arrays

A surface-enhanced Raman scattering (SERS) based biosensor using a direct immunoassay platform is demonstrated for influenza A detection. The nucleoprotein of influenza A virus, which is one of the most conserved and abundant structural proteins on the virion, was used as a target. In this study, highly sensitive biosensors were realized by combining specific recognition of antibody–antigen interactions and high signal enhancement of the SERS effect. SERS probes were fabricated by decorating PEGylated, 4,4′-thiobisbenzenethiol (TBBT)-labeled gold nanoparticles (NPs) with influenza A antibodies. To improve the sensitivity, a SERS immunoassay was performed on two-dimensional (2D) arrays of gold@silver core–shell (Au@Ag) NPs, which work as SERS substrates. The SERS signal of TBBT was utilized to detect the selective nucleoprotein–antibody recognition. The SERS signal was enhanced ∼4 times by using the SERS substrates instead of a flat Au film. These results indicate that using a well-tuned Au@Ag 2D array as a SERS substrate is an effective way of improving sensitivity of SERS-based biosensors. Our SERS immunoassay system revealed high selectivity and good reproducibility with a sample-to-sample variation of 4.6% (relative standard deviation). To demonstrate the applicability of our SERS immunoassay system to real biological samples, the detection of influenza A using infected allantoic fluid was also performed. The linear relation between the concentration of infected allantoic fluid and the SERS signal was obtained in the range of 5 to 56 TCID50 per mL (R2 = 0.96 for the TBBT Raman bands at 1565 cm−1) with the lowest detection limit of 6 TCID50 per mL. These findings demonstrated the potential of this SERS immunosensor platform for the highly sensitive and specific detection of target molecules in a complex matrix commonly found in clinical specimens.

SERS-based immunoassay on 2D-arrays of Au@Ag core–shell nanoparticles: influence of the sizes of the SERS probe and sandwich immunocomplex on the sensitivity

We report a sandwich-type SERS-based immunoassay using a two-dimensional (2D) array of gold core@silver shell (Au@Ag) nanoparticles (NPs) as the SERS substrate and antibody-conjugated gold NPs labeled with 4-mercaptobenzoic acid (MBA) as the SERS probes. To achieve highly sensitive detection, the size of the SERS probes was first optimized for the immunoassay of Human-IgG (H-IgG), where the Au core size of the SERS probes was varied from 26 to 110 nm in diameter. The maximum SERS intensity was observed at an Au core size of 53 nm. Then, the influence of the size of the sandwich immunocomplexes on the sensitivity was examined by performing sandwich SERS immunoassays for H-IgG and prostate-specific antigen (PSA) using SERS probes with 53 nm Au core size. The sensitivity improvement by using the SERS substrate (2D-array of Au@Ag NPs) instead of an Au evaporated film, which was used as a reference substrate, was evaluated for each immunoassay. The sensitivity improvement for H-IgG and PSA detection was 2.3-fold and 6.4-fold, respectively. The larger sensitivity improvement for the PSA system can be attributed to the smaller immunocomplex of PSA; the shorter separation distance between the SERS probes and the SERS substrate induces stronger plasmon coupling. This result indicates that the sensitivity of the sandwich-type immunoassay performed on the SERS substrate increases with decreasing size of sandwich immunocomplex, suggesting that the sensitivity can be improved by adopting an antibody-fragment with the same affinity for the target antigen as that of the antibody.

Electrochemical detection of c-reactive protein based on anthraquinone-labeled antibody using a screen-printed graphene electrode

In this present work, a novel electrochemical immunosensor employing a screen-printed graphene electrode (SPGE) for a simple and highly sensitive determination of C-reactive protein (CRP) in a sandwich-type format was proposed. The sensor comprised of two CRP-specific antibodies: an unlabeled capture primary antibody (Anti-1°Ab) and an electrochemically detectable anthraquinone-labeled signaling secondary (AQ-2°Ab) antibody. The Anti-1°Ab was first covalently anchored onto an L-cysteine/gold-modified disposable SPGE (L-Cys/Au/SPGE) to create the anti-CRP surface. After binding of the CRP and the AQ-2°Ab, the electrochemical signal response was measured using differential pulse voltammetry (DPV). In the presence of CRP, the sensor exhibited a significant increase in the AQ current at AQ-2°Ab compared to the negative control. The CRP concentration was detected in the range of 0.01-150 µg/mL, and the limit of detection (LOD) (S/N = 3) and limit of quantitation (LOQ) (10 SD/Slope) were 1.5 ng/mL and 10 ng/mL, respectively. This sensor exhibited very high sensitivity in determining CRP and was successfully applied to detect CRP in certified human serum with satisfactory results. The developed sensor is suitable as an alternative method for determination of CRP and the same principle may be further applied to determine other clinically important target molecules.

A Novel Electrochemical Sensor for the Simultaneous Determination of Fat-Soluble Vitamins Using a Screen-Printed Graphene/Nafion Electrode

In this work, a novel electrochemical sensor was proposed for the simultaneous determination of fat-soluble vitamins (A, D, E, K) using a screen-printed graphene/Nafion electrode (SPGNE). The scanning electron microscopy was used for morphological characterization of the electrode surface. The electrochemical behaviors of fat-soluble vitamins have been studied in a mixture of ethanol and sodium perchlorate monohydrate using square-wave voltammetry (SWV). The results obtained indicated that the oxidation peak of each fat-soluble vitamin appeared at different potentials leading to the possibility for the simultaneous detection. The influences of experimental parameters such as the effects of proportions of ethanol, potential increment, amplitude, frequency and quiet time were examined. Under the optimized conditions, the linearity between oxidative currents and concentrations of fat-soluble vitamins ranged from 0.1 μg mL-1 to 5 μg mL-1 for vitamin A, 0.08 μg mL-1 to 5 μg mL-1 for vitamin D and E, and 0.2 μg mL-1 to 1.6 μg mL-1 for total vitamin K, with the limits of detection of 0.018, 0.013, 0.012 and 0.004 μg mL-1, respectively. These developed sensors provide high sensitivity in detection and offer high potential to apply them for the simultaneous determination of fat-soluble vitamins in dietary supplements.

A sensitive electrochemical immunosensor based on poly(2-aminobenzylamine) film modified screen-printed carbon electrode for label-free detection of human immunoglobulin G

Abstract This work focuses on fabricating poly(2-aminobenzylamine)-modified screen-printed carbon electrode as an electrochemical immunosensor for the label-free detection of human immunoglobulin G. To selectively detect immunoglobulin G, the anti-immunoglobulin G antibody with high affinity to immunoglobulin G was covalently linked with the amine group of poly(2-aminobenzylamine) film-deposited screen-printed carbon electrode. The selectivity for immunoglobulin G was subsequently assured by being challenged with redox-active interferences and adventitious adsorption did not significantly interfere the analyte signal. To obviate the use of costly secondary antibody, the [Fe(CN)6]4-/3- redox probe was instead applied to measure the number of human immunoglobulin G through the immunocomplex formation that is quantitatively related to the level of the differential pulse voltammetric current. The resulting immunosensor exhibited good sensitivity with the detection limit of 0.15 ng mL−1, limit of quantitation of 0.50 ng mL−1 and the linear range from 1.0 to 50 ng mL−1. Given those striking analytical performances and the affordability arising from using cheap screen-printed carbon electrode with label-free detection, the immunosensor serves as a promising model for the next-step development of a diagnostic tool.