(795h) Droplet Microfluidic Systems For Single Cell Whole Genome Amplificiation

DROPLET Microfluidic systems FOR SINGLE CELL WHOLE GENOME AMPLIFICIATION

M. Rhee, R.J. Meagher, P. Liu, Y.K. Light, S. Yilmaz, J. Sustarich and A.K. Singh

Sandia National Laboratories, USA

The diversity of microbes in environments
ranging from soil and water to the human body has been long recognized while there
are big challenges for the study of microbial diversity and their roles, including extremely low
cell density and uncultivable microbes. The majority of such microbial organisms are
uncultivable, making it challenging to perform genome sequencing and functional
characterization. Uncultured microbes in the human body may play important
roles in processes related to pathologic development, and uncultured environmental
microbes may also hold key metabolic pathways enabling more efficient biofuel
processing and bioremediation. Even a slight change of this microbial composition
may be a critical indicator of a variety of the progression of diseases;
however, its complex pathway to the change is still uncertain yet. Although Metagenomics has contributed to the discovery of a great
diversity of bacterial species, the complexity of microbial communities is highly
beyond the tractable range of standard metagenomic techniques
where relatively large amounts of starting genetic material is required. In addition, population-averaged
statistical data from metagemonic
study can be hardly used to identify the link between the genotype and
phenotype of each individual cell.  Thus, a
targeted approach to identify, sort and isolate individual bacterial cells of
interest from a pool of microbial samples is required to perform genome
amplification and sequencing without culture. We
present here droplet-based microfluidic tools to isolate
individual microbial cells and amplify their whole genomes in a non-cultivation
manner as a route to single-cell genome sequencing. The system consists of flow
cytometry for identifying bacteria and
on-demand droplet generation for cell encapsulation followed by injection of
amplification reagents.

We have introduced a microfluidic platform for
fluorescence in situ hybridization (FISH) on chips in our previous paper [1].
In order to identify or label specific bacteria with molecular specificity, we
have used fluorescently labeled sequence of 16S rRNA for
hybridization which has long been used for determining phylogenetic
relationships between bacteria [2]. The following fluorescence-activated cell
sorting (FACS) offers a convenient way to physically isolate individual cells. The
individually isolated bacterial cell is then contained in a safe environment where
crosstalk between cells or other contamination is strictly prevented. Aqueous emulsion
droplets in oil phase serves as ideal containers for isolated single cells since
they provide physical water/oil barriers to prevent contamination between
cells. Microfluidic environments provide an ideal platform to generate and
manipulate aqueous droplets in oil for various preparation steps prior to
amplification. Evaporation of oil phase during long incubation time may become
another challenge for successful amplification. Decrease in the amount of
carrier oil due to evaporation can cause significant reduction of physical oil
barriers between droplets or make the droplet exposed to atmosphere, resulting
in non-specific droplet merging or breakage. To minimize evaporation during
amplification, a fast and non-thermal amplification method is preferred. Multiple
displacement amplification (MDA) using ϕ29
polymerase yields relatively uniform coverage when performed in a very small
volume.

We present here a microfluidic processor with
three modules constructed on a single chip, capable of performing (i) fluorescence-based
flow cytometry and cell sorting, (ii) encapsulation of selected cells in sub-picoliter droplets, and (iii) injection of amplification reagents into the droplets for
single-cell genome amplification. Especially, to our best knowledge, this is
the first report in the field that proposes on-demand droplet generation for
single cell sorting. Encapsulation of bacteria in picoliter
plugs in particular allows us to scale down conventional assays into
much smaller reaction volumes better suited to the size of an individual
microbe.  By dramatically reducing
the reaction volume, the effective concentration of template is also increased,
reducing amplification artifacts that often arise in single-cell reactions
carried out at a conventional scale. Fluorescently labeled bacterial cells in the sample stream
are hydrodynamically focused by two aqueous side
sheath streams to minimize the detection area for more accurate reading. Multiple
laser beams with desired wavelengths spot the detection point to determine the
presence of desired cells using computerized PMT based on the fluorescence and
scattered light. Upon the successful detection of bacterial cells, the control
software triggers the microdispensing pump connected
to a dispensing fluid. The dispensing fluid is injected, gets mixed with the
sample stream and forcedly generates a droplet through a small orifice into the
adjacent oil stream. The dispensing is programmed based on calculated delay
time between detection and dispensing and the size of the droplets can be fine-tuned
by dispensing pressure and pumping time. These parameters all together not only
determine whether the cell or particles of interest can be encapsulated in the
generated droplet or escape to the waste collection but also affect the recovery
time for the next droplet generation. Typically, as the size of droplets
decreases, the pump requires less recovery time. For droplets of ~50 µm in
diameter, the full recovery time is approximately 200 ms,
which enables droplet generation at the rate of 5 Hz. This generation rate is
not comparable to that of high-throughput droplet generation systems that
typically produce hundreds to thousands of droplets per second, but the
on-demand nature of our system inevitably has a limitation of speed in
compensation of versatile controls over droplet generation. For our microbial
studies, our candidate samples of interest typically are rare and have a very
low cell density, which makes this platform most suitable for the purpose. In
case of high concentration samples, prior dilution will facilitate single cell
encapsulation.

        An
encapsulated cell in the aqueous droplets is physically protected by water/oil
interface. However, this physical barrier imposes another challenge of adding lysis and amplification reagents into the flowing droplets.
Having a specially designed injection module down the stream, we successfully
demonstrated that various reagents and buffer solutions could be merged into
the cell containing droplets without losing or contaminating encapsulated
cells. When the flowing droplets encounter concurrent reagent stream, by applying
voltage of 100 V at the merging site, the concurrent flow injects an adjustable
amount of reagents into the droplets. Because of internal circulation inside
the injected droplets, almost instantaneous mixing occurs and the merged droplets
become ready for the following amplification. After careful collection of
droplets, the isolated single-cell genomes were amplified by multiple
displacement amplification (MDA).  using high purity ϕ29
DNA polymerase and their 16s rRNA genes were
amplified and sequenced for identification.

Figure Captions

A.     (top) Upon the detection of bacterial cells,
the control software actuates the micro-dispensing pump connected to a
dispensing fluid. (bottom) The dispensing fluid is injected into the channel and forcibly
reroutes the sample stream into adjacent oil stream through a small orifice,
forming an emulsion droplet.

B.     (top) GFP expressing E. coli cells are
detected and encapsulated in aqueous droplets in oil one by one. (middle) Single E. coli cells contained in emulsion droplets flow down
the channel for reagent injection. (bottom) Microdroplets serve as discrete, non-communicating
containers or reaction vessels.  

C.     Injection of Fixed amount of reagents
enabled by con-current pico-injector

D.    
A microscope photo of droplets containing amplified genomic
DNA spread inside the droplet.

REFERENCES

[1]  P. Liu et al., "Microfluidic
fluorescence in situ hybridization and flow cytometry
(mFlowFISH)", Lab
on a Chip, vol. 11, pp. 2673-2679, 2011.

[2]  R.I. Amann
et al., "Combination of 16S rRNA-targeted
oligonucleotide probes with flow cytometry for
analyzing mixed microbial populations", Applied
and Environmental Microbiology, vol. 56, pp. 1919-1925, 1990.

[3]  T.D. Perroud
et al., "Microfluidic-Based Cell Sorting of Francisella
tularensis Infected Macrophages Using Optical Forces",
Analytical Chemistry, vol. 80, pp. 6365-6372,  2008.