Dr Swee Lim

Hepatitis B core antigen with the mutation of Y132A (HBcAg-Y132A) was successfully expressed in Escherichia coli. The mutant HBcAg-Y132A forms dimers and is unable to self-assemble into virus-like particles (VLPs). Hence, it is a potential antigen used in the antibody-responsive biosensor for the detection of anti-HBcAg in patients infected with hepatitis B virus. The aim of this study was to establish a direct purification strategy to recover HBcAg-Y132A dimer from the E. coli feedstock using SepFast¿ Supor DEAE pre-packed column. The performance of this anion exchange chromatography was optimized in terms of the buffer composition (for adsorption and elution steps) and the mode of elution (i.e., step or gradient). The highest adsorption of HBcAg-Y132A dimer in the DEAE column was achieved with the buffer composed of 50 mM Tris-HCl (pH 8.4). The step elution using 50 mM Tris-HCl elution buffer (pH 8.4) supplemented with 1 M NaCl resulted in 1.2-fold increase in the purity of HBcAg, as compared to the gradient elution mode. In addition, it was found that the optimized 3-step elution is not directly applicable to elute the self-assembled HBcAg VLPs, as only 24.7% of the particles were recovered due to the limitation of size effect.

Hydrogel based on poly(acrylic acid) (PAAc) polymers was successfully fabricated as the biosensor for detecting hepatitis B core antigen (HBcAg). Specifically, the pendant HBcAg and the anti-hepatitis B core antigen (anti-HBc) antibody were first immobilized on the PAAc, which were then covalently cross-linked via radical polymerization to form the HBcAg-sensitive (HBPAAc) hydrogel. The non-covalent affinity binding between the immobilized HBcAg and anti-HBc would be disrupted by the presence of free HBcAg in the HBPAAc hydrogel. The competitive binding of free HBcAg on the immobilized anti-HBc triggered the swelling of HBPAAc hydrogel. The equilibrium swelling ratio and the oscillatory swelling-deswelling kinetics of the HBPAAc hydrogel in response to protein concentration were studied. The swelling ratio of HBPAAc hydrogel increased along with an increase in HBcAg concentration until equilibrium was achieved at 4¿mg/mL HBcAg. The HBPAAc hydrogel did not exhibit swelling/deswelling behavior when interacted with the negative control, i.e., bovine serum albumin (BSA). Based on the result of oscillatory swelling-deswelling of HBPAAc hydrogel, the minimum duration for HBcAg detection by the HBPAAc hydrogel was ~20¿min, whereas the regeneration of HBPAAc hydrogel took about 60¿min. These results confirmed the reversibility and the reusability of this HBcAg-sensing HBPAAc hydrogel. Besides, the hydrogel demonstrated zero cross-reactivity to the hepatitis B surface antigen, a common serological marker for hepatitis B patients. The high sensitivity of HBPAAc hydrogel to the HBcAg was successfully demonstrated with quartz crystal microgravimetry. The magnitude of resonance frequency was inversely correlated with the swelling of HBPAAc hydrogel, which is governed by the concentration of analyte HBcAg. The application of HBPAAc hydrogel as a biosensor component in the detection and surveillance of hepatitis B holds great promises.