Development of paper-based biosensor for point-of-care- nucleic acid testing / Choi Jane Ru
Nucleic acid testing (NAT), a molecular diagnostic technique that involves nucleic acid extraction, amplification and detection, conventionally relies on well-established laboratories, high-end instrumentation and highly trained operators, limiting its use in resource-poor settings, where most di...
| Main Author: | |
|---|---|
| Format: | Thesis |
| Published: |
2016
|
| Subjects: | |
| Online Access: | http://studentsrepo.um.edu.my/6703/ http://studentsrepo.um.edu.my/6703/4/jane_ru.pdf |
| Summary: | Nucleic acid testing (NAT), a molecular diagnostic technique that involves nucleic acid
extraction, amplification and detection, conventionally relies on well-established
laboratories, high-end instrumentation and highly trained operators, limiting its use in
resource-poor settings, where most diseases exist. With advances in point-of-care
testing (POCT), lateral flow assays (LFA) have been explored for nucleic acid detection.
However, as biological samples are generally complex and contain low amounts of
target nucleic acids, a substantial off-chip extraction and amplification process (e.g.,
tube-based extraction and polymerase chain reaction (PCR)) is normally required prior
to lateral flow detection. Additionally, the applications of LFA have been currently
limited by their low sensitivity and poor functionality. Herein, it was demonstrated for
the first time a novel fully integrated paper-based sample-to-answer biosensor
incorporating nucleic acid extraction, amplification and sensitive lateral flow detection.
The optimum concentration of reagent and environmental conditions (i.e., temperature
and relative humidity) for LFA were determined. Paper-based LAMP was integrated
into LFA, coupled with a handheld battery-powered heating device for nucleic acid
amplification in POC settings, which showed a comparable result to that of
conventional tube-based platform. A fully integrated paper-based sample-to-answer
biosensor was then developed, which could successfully detect Escherichia coli in
spiked drinking water, milk, blood, and spinach with a detection limit of as low as 10-
1000 CFU/mL, and Hepatitis B virus (HBV) in clinical blood sample, highlighting its
potential use in medical diagnostics, food safety analyses and environmental monitoring.
The sensitivity of biosensor was further enhanced by incorporating a piece of paperbased
shunt and a polydimethylsiloxane (PDMS) barrier into the strip to achieve
optimum fluidic delays for LFA signal enhancement, resulting in 10-fold signal
iv
enhancement over unmodified LFA. This strategy could successfully detect HBV with
concentrations of as low as ~102 IU/mL in clinical blood samples, which was
comparable to that of conventional detection strategy and even more sensitive than the
existing biosensors, demonstrating its ability to detect acute HBV infections. The
phenomena of fluidic delay were also evaluated by mathematical simulation, through
which the fluid movement throughout the shunt and the tortuosity effects in the
presence of PDMS barrier were revealed, which significantly affect the detection
sensitivity. The proposed fully integrated biosensor offers great potential for highly
sensitive detection of various targets for wide applications in the near future. |
|---|