A Sensitive Capacitive Biosensor for Protein a Detection Using Human IgG Immobilized on an Electrode Using Layer-by-Layer Applied Gold Nanoparticles.

Kosin Teeparuksapun,Kosin Teeparuksapun,Martin Hedström,Bo Mattiasson

doi:10.3390/s22010099

Kosin Teeparuksapun, Kosin Teeparuksapun + Show 2 more

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https://doi.org/10.3390/s22010099

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Abstract

A capacitive biosensor for the detection of protein A was developed. Gold electrodes were fabricated by thermal evaporation and patterned by photoresist photolithography. A layer-by-layer (LbL) assembly of thiourea (TU) and HAuCl4 and chemical reduction was utilized to prepare a probe with a different number of layers of TU and gold nanoparticles (AuNPs). The LbL-modified electrodes were used for the immobilization of human IgG. The binding interaction between human IgG and protein A was detected as a decrease in capacitance signal, and that change was used to investigate the correlation between the height of the LbL probe and the sensitivity of the capacitive measurement. The results showed that the initial increase in length of the LbL probe can enhance the amount of immobilized human IgG, leading to a more sensitive assay. However, with thicker LbL layers, a reduction of the sensitivity of the measurement was registered. The performance of the developed system under optimum set-up showed a linearity in response from 1 × 10−16 to 1 × 10−13 M, with the limit detection of 9.1 × 10−17 M, which could be interesting for the detection of trace amounts of protein A from affinity isolation of therapeutic monoclonal antibodies.

Highlights

The recent expansion of biopharmaceutical processes has created a large demand for rapid and sensitive analytical techniques for the detection of trace concentrations of impurities [1]
The image ex nique patterned withwith photoresist lithography is shownisinshown image hibits a uniform pattern, corresponding to a spherical gold cluster, with a diameter o a uniform pattern, corresponding to a spherical gold cluster, with a diameter of approxapproximately
The sensitivities of the calibration curves of four electrodes were investigated and found to be 7.71 ± 0.47, 7.69 ± 0.50, 7.62 ± 0.31, and 7.79 ± 0.34 −nF/cm2 /logarithm of concentration (log M). These results show that the preparation of electrodes using layer-by-layer of TU and AuNPs provides an immobilization strategy with good reproducibility

Summary

Introduction

The recent expansion of biopharmaceutical processes has created a large demand for rapid and sensitive analytical techniques for the detection of trace concentrations of impurities [1]. It was sufficient to state the percentage of the pure product; recent concerns have arisen about the identity and quantification of the remaining impurities. Host-cell proteins [2,3], endotoxins [4], nucleic acids, and viruses [5] constitute such impurities, together with reagents from the downstream processing. Contamination of monoclonal antibody preparations by protein A (PA) leaching from the affinity adsorbent is such an example [6,7]. Products contaminated with PA can cause immunogenic [8] and mitogenic effects [9,10]. The high purity of immunoglobulin for clinical applications is required [6]

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