(265f) A Handheld Optical Detection Method for Detection of Vibrio Cholerae in Environmental Water Samples

Environmental pathogen
detection presents unique
challenges in the development of novel biosensors due to the exceedingly low
concentrations of pathogens in their native environments. For example, despite surviving at
only 100 cells/mL in environmental water sources, the Vibrio cholerae (V. cholerae)
bacteria that causes the devastating diarrheal disease cholera, leads to over
150,000 deaths worldwide each year. Further, the current gold standard for the
detection of V. cholerae
in water sources is an >8-hour process involving bacteria enrichment and
culture followed by polymerase chain reaction (PCR). Despite being one of the
most sensitive laboratory detection methods, PCR is not robust enough to directly
detect V. cholerae
from the environment; as it requires purification of DNA prior to amplification  Hence,
there is a need for a biosensor that can rapidly detect pathogens, such as V. cholerae,
in their native matrix (environmental water sources).

In
this work, we develop a highly accurate and sensitive biosensor for the rapid
detection of V. cholerae
in environmental water sources by pairing loop-mediated isothermal
amplification (LAMP) with particle diffusometry (PD)
(Figure 1A). We have developed a LAMP protocol for V. cholerae that is efficient (under 30-minute
amplification), specific (targeting 6 different regions of the cholera toxin
gene), and robust (usable in non-pretreated environmental water sources). When V. cholerae
DNA is present in the solution, the LAMP assay polymerizes DNA targets into a
variety of base pair lengths up to 25 kilobases. This
polymerization causes an increase in the visocity of
the sample solution that can be measured via particle diffusometry
(PD). In particle diffusometery we can calculate the
change in the diffusion of nanoparticles in the sample using correlation-based
algorithms of the particle images (Figure 1B). In this work, we show the
applicability of PD-LAMP to detect the presence of V. cholerae. We demonstrate that PD-LAMP can
be used to detect as few as 1 V. cholerae cells in a 25 μL sample with only a
20-minute amplification period and an 8-second optical measurement time. Further,
PD is a robust measurement system because it can detect amplified V. cholerae
DNA from whole cells in environmental water without any additional enrichment
or sample preparation steps. This detection method is 100-fold more sensitive
than current gold standard fluorescence detection of nucleic acid
amplification. We additionally demonstrate that detection of V. cholera from
environmental water sources can be detected via PD using a novel handheld
biosensor platform in as little as 30 min.

Figure 1. Illustration
of PD-LAMP set-up. (A) The LAMP assay is performed in the presence of V. cholerae
DNA (left). LAMP amplicons combined with polystyrene
fluorescent particles (middle) are imaged under fluorescence microscopy
(right). (B) Relationship of particle motion and viscosity. Particles undergo
Brownian motion in a solution (left). In the presence of LAMP amplicons, the viscosity of the solution increases and
particles experience hindered motion, indicating the presence of V. cholerae
DNA in the sample (right).