Development of diazonium functionalized electrochemical aptasensor for simultaneous detection of tuberculosis antigens
In the management of tuberculosis (TB), prompt and accurate diagnosis is of the utmost significance for the purpose of life-saving and transmission cessation. Despite being time-consuming and having a low sensitivity, traditional smear microscopy and culture procedures remain the mainstay of TB a...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2023
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/113982/1/113982.pdf |
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| Summary: | In the management of tuberculosis (TB), prompt and accurate diagnosis is of the utmost
significance for the purpose of life-saving and transmission cessation. Despite being
time-consuming and having a low sensitivity, traditional smear microscopy and culture
procedures remain the mainstay of TB antigen detection modality. To overcome this
limitation, this study highlights the fabrication of the first amperometric dual aptasensor
for the simultaneous detection of Mycobacterium tuberculosis CFP10 and MPT64-
secreted antigens to facilitate better TB diagnosis and control. The proposed sensor
utilized the aptamer–antibody sandwich assay that would be measured by
chronoamperometry via electrocatalytic reaction between peroxidase-conjugated
antibodies, hydrogen peroxide (H2O2), and hydroquinone (HQ). The aptamer-target
binding ability for CFP10 and MPT64 was first reassessed using enzyme-linked
oligonucleotide assay (ELONA), which showed a statistically significant difference
between the wells incubated with the target antigen compared to other control well with
a p-value <0.0001, indicating the sensitive and selective behavior of the selected aptamer
towards the target antigen. The aptamers were then immobilized via carbodiimide
covalent chemistry over the disposable screen-printed carbon electrodes (SPCE)
modified with 4-carboxyphenyl diazonium salt. The successful deposition of the
diazonium layer was verified with several methods, including X-ray photoelectron
spectroscopy (XPS), Fourier-transform infrared (FTIR), contact angle, and
electrochemical analysis, i.e., cyclic voltammetry (CV) and electrochemical impedance
spectroscopy (EIS). Analysis of the diazonium-modified surface by FTIR showed a new
sharp band at ~1703 cm–1 attributed to the C=O stretching of the terminal carboxylic
groups that was absent in the bare SPCE surface. Additionally, the appearance of
moderate peak intensity at ~1255 cm–1 and ~1367 cm–1 contributed to the C–O stretching
and O–H band, respectively, confirming the existence of the 4-carboxyphenyl diazonium
salt on the carbon surface. For the XPS analysis, there was a significant increase in the
oxygen content from 4.85% (bare) to 18.81% (diazonium-grafted), suggesting the
surface enrichment with oxygen corresponding to the oxygen belonging to the carboxylic
functionality of the 4-carboxyphenyl diazonium. Further high-resolution scan of C 1s
components also produced a unique peak around 289.5 eV assigned to the carboxylic
group, H–O–C=O. This peak is a typical signature of a surface modified with carboxylic
functionality. CFP10 and MPT64 single aptasensors were initially fabricated and
optimized to validate their analytical and diagnostic performance before being adapted
to the dual detection platform. The stepwise assembly was characterized using CV and
EIS techniques. Under optimal conditions, the CFP10 single aptasensor exhibited a linear
relationship with the increasing CFP10 antigen concentration of 5 to 500 ng mL–1. The
detection (LOD) and quantification limit (LOQ) of the single CFP10 aptasensor was
estimated to be 1.22 ng mL–1 and 1.93 ng mL–1, respectively. The MPT64 single
aptasensor, on the other hand, achieved a LOD and LOQ of 1.11 ng mL–1 and 1.402 ng
mL–1, respectively, with an increasing trend in the current response with the increase in
antigen concentrations from 5 to 200 ng mL–1. Both single aptasensors also showed
excellent current reproducibility with a relative standard deviation (RSD) of 1.39%
(CFP10) and 1.52% (MPT64) when testing with a series of five modified electrodes
under the same preparation batch. The CFP10 single aptasensor demonstrated good
storage stability without significant current difference for up to two months when stored
at 4°C under a dry environment. Meanwhile, the MPT64 aptasensor displayed slight
attenuation by 8% from the initial current (day 0) after day 45 of storage. Clinical
evaluation using TB-positive [TB (+)] and non-TB sputum samples [TB (–)] revealed
satisfactory results for every aptasensor. Surprisingly, the diagnostic sensitivity and
specificity between both sensors were found to complement each other, thus making
them ideal candidates to be combined on a dual simultaneous detection platform. The
CFP10 single aptasensor produced 100% sensitivity and 81.8% specificity, while MPT64
single aptasensor achieved 88% and 100% for diagnostic sensitivity and specificity,
respectively. Furthermore, exceptional analytical performances were obtained upon
applying both detections on a dual SPCE, as demonstrated by the detection limit of 1.62
ng mL–1 (CFP10) and 1.82 ng mL–1 (MPT64) with no significant reaction when incubated
with other non-target reagents. A linear dependence of the amperometric signal was
observed between the corresponding target antigens concentration in the range of 0.5 to
100 ng mL-1 and 0.75 to 250 ng mL–1 for CFP10 and MPT64, respectively. An RSD
value of 2.6% and 2.99% for CFP10 and MPT64 working electrodes suggested a reliable
performance of the fabricated when tested with the amperometric technique. The dual
aptasensor also demonstrated good storage stability for up to 35 days at 4°C. In the
clinical study, the MPT64 working sensor was the least sensitive (91.7%), followed by
the CFP10 working electrode (95.8%). Overall, a combined CFP10 and MPT64 detection
achieved a perfect score for TB diagnosis when evaluated on 24 TB (+) and 13 TB (–)
sputum samples, thus indicating the readiness of the developed assay to be used
clinically. In conclusion, the developed CFP10-MPT64 dual electrochemical aptasensor
is a potentially sensitive, specific, and easy-to-apply assay for TB. Therefore, it would
be a promising alternative to conventional microscopy and TB culture. |
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