Development of a bio-nanogate-based electrochemical immunosensing strategy for the detection of anti-hepatitis B surface antigen antibody
An area requiring real-time analysis is the diagnosis of infectious disease and monitoring of vaccination efficiency against the disease to determine immunity level particularly among high risk group including immunocompromised patients against infectious diseases in screening for immunization p...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/104586/1/FBSB%202022%2012%20IR.pdf |
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| Summary: | An area requiring real-time analysis is the diagnosis of infectious disease
and monitoring of vaccination efficiency against the disease to determine
immunity level particularly among high risk group including
immunocompromised patients against infectious diseases in screening for
immunization program including hepatitis B virus (HBV). However, the
conventional methods require laborious work and tedious fabrication of
sensing platforms which limit the efficiency in upscaling the screening
tests. These further impede the analysis of large cohort of clinical samples.
Therefore, an effort is needed in order to improve the outcome of this labbased
technology. Generally, a bio-nanogate system involves the use of
synthetic or natural molecules as a ‘gate’ towards bioreceptors and ideally,
the gating mechanism should respond only upon the presence of external
stimuli i.e. targeted analytes in a nanoscale dimension. The versatility of
polyamidoamine (PAMAM) dendrimers to form conjugates with proteins
can be utilized to form a bio-nanogate. PAMAM interaction with protein
bioreceptor and the ability of a bio-nanogate-based immunosensing
strategy in detecting an antibody i.e. anti-hepatitis B surface antigen (anti-
HBsAg) antibody electrochemically were of interest in this study.
An antigenic determinant (aD) region of HBV fused with maltose binding
protein (MBP-aD) was synthesized to form a specific bioreceptor for anti-
HBsAg antibody in the bio-nanogate system. The bio-nanogate interaction
was further analysed for its binding affinity, thermal stability, and
thermodynamic analysis. Following that, a proof of concept utilizing
displacement immunosensing strategy was conducted electrochemically,
where the MBP-aD was immobilized on the screen-printed carbon
electrode (SPCE) platform, and further sandwiched with electroconductive
PAMAM encapsulated gold nanoparticles (PAMAM-Au), forming the ‘gate’. PAMAM-Au here also functions as a monitoring agent capable of
generating a signal response upon a displacement event in the presence
of anti-HBsAg antibody in differential pulse voltammetry (DPV) analysis.
Finally, the PAMAM-Au displacement efficiency was further improved via
implementation of acoustic mixing on modified SPCE platform coupled
with piezoelectric transducer.
The synthesized MBP-aD was confirmed with western blotting technique.
The interaction study revealed that the interaction of MBP-aD with anti-
HBsAg antibody has a higher thermal stability and binding affinity (KA = 1.6
x10-5 Lmol-1) as compared to its interaction with PAMAM (KA = 2.9 x 10-6
Lmol-1). Thermodynamic parameters also demonstrated that the bionanogate
components interact through van der Waals and hydrogen
bonding. Based on the interaction study, it was hypothesized that the
interactions among the bio-nanogate components may be able to be
manipulated at the nanoscale level for the detection of anti-HBsAg
antibody. Under optimal conditions, the hypothesis was proven that the
high specificity of anti-HBsAg antibody towards MBP-aD displaced
PAMAM-Au, in a range of 1mIU/mL to 1000 mIU/mL with a detection limit
(LOD) of 2.5 mIU/mL. The results also showed high specificity and
selectivity of the immunosensor platform in detecting anti-HBsAg antibody
both in spiked buffer and human serum samples. Furthermore, the
incubation/reaction time for detecting anti-HBsAg antibody has been
reduced from an initial incubation time of 20 min to 8 min via the
improvement of PAMAM-Au displacement efficiency under acoustic
streaming effect. The newly developed immunosensor platform utilizing the
manipulation of lower interaction between PAMAM-Au (gate) and the
candidate bioreceptor (anchor) would ultimately eliminate the need of
having specifically designed and labelled analogues which has been
commonly used in displacement-based immunoassays. |
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