University of Groningen

A droplet-based microfluidic platform for on-chip viral-fusion studiesMashaghi Tabari, Samaneh

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13

CHAPTER1

Microdroplet‐basedsyntheticbiology,chemistryand

nanofabrication

ABSTRACT

The ability to perform laboratory operations on small scales using miniaturized devices

provides numerous benefits, including reduced quantity of reagents and waste as well as

increased portability and controllability of assays. Most of these operations involve

reaction components in the solution phase and as a result, their miniaturization requires

microfluidic approaches. In particular, droplet microfluidics provides a high-

throughput platform for a wide range of assays and approaches in chemistry, biology

and nanotechnology. We highlight recent advances in the application of droplet

microfluidic-chip-based technologies, such as single-cell analysis tools, small-scale cell

cultures, in-droplet chemical synthesis, high-throughput drug screening, and nanodevice

fabrication.

S. Mashaghi, A. Abbaspourrad, D. A. Weitz, A. M. van Oijen

Manuscript in preparation (2015)

14

1.1. Introduction

More than a century ago, the transition from chemistry-in-a-tube to chemistry-in-a-tank

approaches resulted in a revolution in chemical industry. This transformation was a

challenging one and chemists and engineers were faced with fundamental questions regarding

plant design and scaling [1]. Their efforts were highly successful and chemical manufacturers

gained the capability of generating drugs, textiles and other valuable chemicals at large scales.

Today, we are witnessing a revolution in the opposite direction in length scale. This new trend

is fueled by the need to manipulate precious and rare materials and fabricate portable

measurement devices involving liquid-phase reactions. For example, detecting a biomolecule

within a small sample from a patient, screening a large library of molecules for a certain

function, and the need for mobile health devices naturally led to the emergence of wet

chemistry on a chip.

One can immediately identify fundamental differences between large-scale and small-scale

chemistry. The former requires more reagents and generates more products; therefore, it is

easy to have a sufficient amount of sample for analytical methods. For very large and

inhomogeneous systems, in some cases, many samples might be needed for accurate

characterization. It is also costly to set the reaction parameters, such as temperature and

mixing [2]. At small scales, the cost of the reagents and the amount of toxic waste that must

be handled is greatly reduced. In addition to the differences between large-scale and small-

scale chemistry, some tasks that are trivially performed by chemists when working with test

tubes are no longer trivial at small or large scales, e.g., mixing reagents and adding a new

reagent pose challenges at both large or small scales.

Manipulation of liquids at small length scales is challenging. The behavior of liquids at the

microscale, typically called microfluidics, enters a regime defined by the Reynolds number

(Re). The Reynolds number, a term first coined by Arnold Sommerfeld (while working on the

Reynolds theory of lubrication) [3], refers to the ratio of inertial forces to viscous forces, and

in microfluidics, Re is equal to or less than unity. Experimental realization of flow in this

regime dates to the early 1950s when efforts to dispense sub-nanoliter amounts of liquids

were made, providing the basics of current ink-jet technology [4]. The invention of the ink jet

was followed by the development of HLPC and the introduction of microvalves and

micropumps during the 1980s. To generate small reaction volumes, a fluid stream has to be

split to make small droplets; alternatively, the liquid can be split into micro/nano wells. The

advantage of the former is that it is easy to transport the liquid drop due to the lack of Taylor

15

dispersion, i.e., the absence of a dispersion in the fluid medium as it flows through a tube [4].

The breakup of a continuous flow in a confined environment, such as a microfluidic system,

has been realized, and efficient droplet generation protocols have been designed [5].

The physics behind droplet formation has been widely studied for a long time. For droplet

formation at macroscopic length scales, the relative effect of viscous forces versus surface

tension (defined as the capillary number Ca) governs the breakup. Droplet microfluidics is,

however, governed by different physics. In microfluidic systems, confinement [6, 7] is a

determining factor in droplet formation and has to be considered along with the capillary

number [8]. Dominant interfacial and surface tensional forces at small scales enable the

precise generation and spatial stabilization of droplets. Droplet-based microfluidic systems

can be fundamentally categorized into two basic designs: channel-based microfluidics in

which the actuation occurs via liquid flows within microfabricated devices and the planar-

surface approach where the actuation occurs through electrowetting or dielectrophoresis [4,

9]. The planar-surface approach, also called digital microfluidics, enables manipulation of

discrete droplets on an array of electrodes [10]. In this chapter, I will focus exclusively on

recent advancements in channel-based systems.

1.2. Technical aspects of droplet engineering

Chemical reactions in nature occur in an aqueous environment. Thus, setting up

biologically relevant reactions at a small scale requires the generation of aqueous

microdroplets. Standard methods exist to generate droplets, either in a hydrodynamically

driven manner (e.g., flow focusing, co-axial and T-junction geometries) or in a manner driven

by an external force (e.g., droplet generation based on an electromagnetic valve that provides

the ability to produce a droplet on demand [11] or droplet generation by pneumatic

micropumps) [12]. Recently, new methods have been introduced to increase the rate of

generating and producing droplets, for example, by introducing gradient confinement [13] or

by parallelizing conventional structures [14]. Such developments in droplet-generation

technologies are fundamental to the development and application of droplet-based

approaches. Surface chemistry is an important issue for droplet controllability in

microfluidics chips. For full control of the generation, size and movement of aqueous droplets

in an organic continuous phase, the surface must be wetted by the continuous phase.

In organic chemistry, reactions are typically performed in non-aqueous environments.

Recently, a simple technique that enables precise loading of droplets of both wetting and non-

wetting liquids was proposed. This technique relies on the use of a combination of

16

compressed inert gas and gravity to exert driving and retracting forces on the liquid [15].

Performing biological reactions, particularly enzymatic reactions, in organic solvents is of

interest to industry. Efforts have been made to increase the stability and catalytic activity of

certain enzymes by dissolving them in a non-aqueous solvent [16, 17].

1.3. Droplet-based cell studies: cell and synthetic biology

Droplet microfluidics opens up new opportunities in biology. More specifically, it

facilitates screening and allows for dramatically increased throughput. While bioassays can be

miniaturized by using microwells with a picoliter volume, such approaches are technically

challenging due to capillary effects and liquid evaporation. Droplet-based cell assays have

some advantages over conventional methods, e.g., using droplets as a picoliter reaction vessel

to encapsulate cells prevents dilution and evaporation. Biological assays might be cell-based

or cell-free assays. In this section, we focus on cell-based assays, and in the subsequent

section, cell-free assays will be discussed. Cell-based assays include single-cell assays, assays

with microculture and assays with synthetic cells.

1.3.1. Cell biology and tissue engineering

Engineering droplets that contain single or multiple cells with precise control of the cell

count is an emerging technology. To incorporate cells within droplets, a bulk liquid

containing cells is typically dispersed into droplets. Here, the probability of finding a certain

number of cells inside a droplet follows the Poisson distribution [18]. To avoid droplets that

lack cells, one should typically have 106 cells/ml at the start for a droplet size of

approximately 100 micrometer [19]. Prior removal of multi-cellular aggregates leads to a

better encapsulation efficiency than that for Poisson encapsulation statistics [20]. In some

applications, the encapsulation of two cells is desirable. A Poisson distribution would limit

the percentage of droplets containing exactly two cells to approximately 13%. Recently,

through a combination of droplet microfluidics with inertial microfluidics, a nearly fivefold

improvement in the Poisson co-encapsulation of droplets was demonstrated [21].

Cells trapped in droplets can then be subjected to analysis or can be manipulated. If the

droplet contains enough nutrients, cells can be cultured within droplets [22] (Figure 1.1).

Interestingly, the gas exchange needed for culturing can be readily provided, typically by the

use of fluorinated oils with high oxygen permeability or through the device itself. These small

cultures can be sorted based on the expression of a certain fluorescent protein or based on the

number of cells within a droplet in a label-free manner. The latter has been recently achieved

usin

allow

sens

cells

cells

freq

impo

A

expo

can

the a

F

cells

envi

drop

merg

s, th

lase

meta

seco

Pr

drop

ng real-time

w for fast (

sitivity [24].

s. Moreover,

s, a strategy

quency cance

ortant for the

Analysis of ce

ose intracellu

be chemical

addition of ly

Figure 1.1. D

s and culture

ironment wh

plets from th

ged with ano

he reaction b

er/photomulti

abolite of in

ond. The figu

reservation o

plet-based m

image-based

(> 100 Hz),

This metho

, using this m

y that is pa

er cells at the

erapeutic dec

ells often re

ular biomole

lly achieved

ysis buffer to

Droplet-base

e media are g

en capped.

he syringe ar

other set of d

begins in the

iplier tube

terest is qua

ure is reprodu

of biologica

microfluidic p

d droplet cla

label-free d

od also allow

method, rare

articularly r

e very early s

cisions.

quires some

ecules to the

using a pul

o the droplets

ed microfluid

generated an

The syringe

re reinjected

droplets cont

e merged dro

system. Con

antified. This

uced, with pe

al materials

platform rem

17

assification [

detection of

ws for discri

e cell types m

relevant to

stage of canc

preparation

detection ass

lsed laser m

s during enc

dics and cel

nd collected

is placed in

into another

taining fluore

oplets. The d

nsequently,

s system can

ermission, fr

and medical

mains a relati

23]. Electric

f cells withi

mination be

may be sorte

medical onc

cer or after c

n and handlin

say. On-chip

icrobeam, us

apsulation [2

ll biology. I

in a syringe

n an incubato

r microchan

escent enzym

droplet fluor

the extrace

n screen app

rom Ghaderi

l samples af

ively unexpl

cal impedanc

n a droplet

tween viable

ed from a lar

cology, whe

chemotherap

ng, such as l

p lysis of cell

sing electrop

25].

Initially, dro

that provide

or for cell c

nel structure

matic assay r

rescence is m

ellular conce

roximately 1

et al. [22].

fter they are

lored area. V

ce measurem

with single

e and non-v

rge pool of o

ere finding

py/radiothera

lysing the ce

ls within dro

poration or u

oplets contai

es a microaer

culture. Then

e, where they

reagents. Afte

measured usi

entration of

1 to 2 clones

e processed

Various meth

ments

e-cell

viable

other

low-

apy is

ell to

oplets

using

ining

robic

n, the

y are

er 30

ing a

f the

s per

in a

hods,

18

including cryopreservation, chemical fixation, and freeze drying, have been developed to

preserve biological samples after they are processed with bulk methods. These techniques can

likely be adapted to preserve droplets containing biological materials. For live cells within

droplets, it would be important to design protocols for freezing droplets with cells inside and

recovering the cells.

1.3.2. Synthetic cells

Droplet microfluidics is rapidly becoming an important tool in studies that aim to develop

synthetic cells. Various approaches exist to arrive at synthetic cells. Some researchers have

explored the possibility to incorporate cellular components into giant lipid vesicles [26].

Others have tried to reconstitute cellular machinery into microfabricated solid wells. For

example, microtubule asters and cellular divisomes have been assembled in microwells, and

their dynamics visualized (Figure 1.2a) [27, 28]. Alternatively, one could also use a droplet as

a platform in synthetic biology. Several approaches could be considered: 1) Water in an oil

droplet. Phospholipids are incorporated in the water-oil interface to mimic the cytoplasmic

leaflet of cellular membranes. Cellular components can be added to the droplet interior. 2)

Multiphase droplets containing an internal water phase surrounded by a thin layer of the oil

phase and residing in an aqueous carrier fluid. The advantages of a droplet platform compared

to a microfabricated solid platform are the following. 1) The former is a soft platform, which

is mechanically closer to the physiological situation. 2) At least for some cell types, a droplet

can be a model with a 3-dimensional shape that mimics that of the cell. 3) By studying the

interaction of two droplets, one can study the interaction of synthetic cells [29, 30]. Droplet

microfluidics is thus a useful platform for synthetic cell research.

1.4. Droplet-based biochemistry and chemical biology

Microfluidics platforms are well suited for biochemistry. Enzymatic reactions in particular

have been studied in microchannels, and both detection and analysis techniques have been

adapted from bulk biochemistry to microchannel biochemistry. For example, Stone et al.

adapted the Michaelis-Menten kinetic theory to model the kinetics of an enzymatic reaction in

a system made of two merging channels; one channel carried the enzyme, and the other

carried the substrate [31]. Diffusion and flow rates are included in the new model. Detection

techniques, particularly fluorescence methods [32, 33] and more recently vibrational

spectroscopy (e.g., ATR-FTIR [34]), have been adapted to extract chemical information from

the contents of microfluidic channels in the presence of flow.

19

Biochemistry in microdroplets is an emerging field with promising applications in industry,

such as digital PCR, directed evolution approaches, and new drug-screening strategies. High-

throughput droplet-based microfluidic platforms have, for example, been used to study the

directed evolution of CotA laccase. This technique allows for the evaluation of the

distribution of enzymatic activity within a large library, rapid enrichment of a library in active

variants at a high-throughput rate, and precise selection of variants depending on their

enzymatic activity [35]. Similar to the case of biochemistry in microfluidics, there is a need to

demonstrate the applicability of detection and analysis techniques for monitoring the reactions

that occur in dynamic droplets.

A combination of droplet-based microfluidics and a reductionist biochemistry approach can

be applied to study both virus-host cell interactions and the interaction of a virus with

intracellular organelles and to the development of a high-throughput antiviral drug screening

assay. With aqueous droplets as the reaction chamber, the virus-host interaction can be

studied within each droplet. Such an assay can be performed by encapsulating host-like

liposomes in droplets and then merging the droplets with a second group of droplets

containing fluorescently labeled viruses and a fusion inhibitor at a range of doses. Fusion of

the merged droplet can be triggered after the appropriate incubation time. The fusion

phenomenon can be studied by fluorescence microscopy, and information regarding the

mechanism and kinetics of the fusion in addition to the efficiency of fusion can be extracted

by tracking each droplet. Moreover, one can learn about the mechanism of action of an

inhibitor by determining the stage at which the fusion process is arrested (this thesis, Chapter

4).

F

exte

comp

33 m

the

their

spon

phas

Figure 1.2. D

ensible micro

mpressed betw

min is overla

absence of A

r movement

ntaneously a

se exhibits s

Droplet-base

otubule bun

ween chambe

aid on a brig

ATP, passive

is minor dr

adsorb onto

streaming fl

d microfluid

ndles exhibit

er surfaces.

ght-field drop

e droplets ex

rift. (3) Fluo

the oil–wat

lows, indicat

20

dics and a sy

t spontaneou

A droplet tr

plet image. T

xert no inter

orescence im

ter interface

ted with blu

ynthetic cell

us autonom

rajectory reco

The scale bar

rnal forces,

mage of acti

e. The result

ue arrows. T

l. (a) 1. Dro

ous motility

orded over a

r for 1 and 2

and the only

ive microtub

ting active l

The red arro

oplets contai

y when part

a time interv

2 is 80 µm. (

y contributio

bule bundles

liquid crysta

ow indicates

ining

tially

val of

(2) In

on to

that

alline

s the

21

direction of the instantaneous droplet velocity. The image is focused on the droplet surface

that is in contact with the coverslip. Scale bar, 100 µm. (4) The image of a droplet taken at a

midplane indicates that the droplet interior is largely devoid of microtubule bundles. Scale

bar, 100 µm. The figure is reproduced, with permission, from Sanchez et al. [28]. (b)

Reconstruction of the interaction of viruses with a host cell within a microdroplet.

Microaliquots of a library of drugs and the corresponding titrations are assembled in a

droplet that contains the virus. After incubation, the droplets form a complex with the

droplets that contain a synthetic host membrane. This step is followed by a second incubation

period. The droplets are then sent to the detection module, where the kinetics of binding,

hemi-fusion and pore formation are monitored. Finally, the droplets are sorted based on

fusogenicity (Figure 1.2b).

1.5. Droplet-based chemical synthesis

The development of droplet microfluidics has significantly influenced chemical synthesis,

micro/nano fabrication and synthetic biology. Compared to continuous-flow chemistry where

channel fouling could lead to poor product control and reactor failure, droplet chemistry

prevents fouling by isolating the reaction from the channel walls [36]. Droplets can be used as

chambers within which synthesis occurs; they can be used as a structure that directs agents to

generate complex nanostructures [37] and they can be used to generate cell-like structures.

Performing reactions within droplets can have a profound impact on the reaction kinetics. For

example, when imine synthesis was performed in emulsion droplets, the apparent equilibrium

constant and the forward rate constant were found to be inversely proportional to the droplet

radius [38]. Compartmentalization affects the reaction thermodynamics at the mesoscale,

although there exists no confinement on the molecular scale, a phenomenon that can be

exploited to improve unfavorable reactions.

Most synthesis reactions that have been performed within droplets are single-step synthesis

reactions [39-43]. One major issue in complex synthesis is that multiple reaction steps are

involved, new reagents have to be added to the droplet, and some reagents may require

removal. These requirements can be partly met by the fusion of droplets containing the new

reagents with the reaction droplet or by the injection of the new reagent into the reaction

droplet [36] (Figure 1.3).

An essential requirement for droplet-based chemistry and chemical synthesis is the ability

to monitor the content of the reaction chamber over time. Quantitative and qualitative analysis

of the droplet content is crucial in the development and application of droplet microfluidics.

Vari

imag

capi

dete

Fo

drop

mas

drop

the m

an

spec

desi

spec

F

the e

dyed

strea

or O

strea

and

repr

ious analytic

ging-based

illary electro

ection [44].

or many an

plets can be

s spectrome

plet into a ca

mass spectro

electrospray

ctroscopy, th

red signal. T

ctroscopic an

Figure 1.3. D

experimenta

d solvent) int

am of blue

ODE/PFPE/A

am of red-dy

after the add

roduced, with

cal detection

methods, la

ophoresis, m

nalytical met

subjected to

etry, a comm

arrier phase v

ometer. Alter

y ionization

he droplet m

The compone

nalysis is con

Droplet-based

l setup used

to a two- an

-dyed ODE

Ar (QR2/ Qc/

yed ODE (QR

dition of the

h permission

techniques

aser-based m

mass spectro

thods, prepa

o analysis. Fo

mon approac

via diffusive

rnatively, the

n-mass spe

medium is oft

ent of interes

nducted [46].

d microfluidi

to compare

d three-phas

droplets is

/ Qg) through

R2) into the f

red-dyed sol

n, from Night

22

have been u

molecular sp

ometry, NM

aratory steps

or instance,

ch is to tran

e exchange. T

e droplet can

ectrometry

ten IR active

st is then tran

.

dics and chem

the efficiency

se droplet flo

first produ

h a junction

flowing drop

lvent for the

tingale et al.

used to analy

pectroscopy,

MR, absorpti

s are needed

when introdu

sfer the com

The carrier p

n be extracted

detection s

e and will n

nsferred to an

mical synthes

cy of the dire

ow (blue drop

uced by flo

n. Then, a T

plets. (b–e) I

two- and thr

[36].

yze droplet co

, electroche

ion and che

d before the

ucing drople

mponent of i

phase will th

d and introdu

system [45]

ot allow for

n IR-transpar

sis. (a) Sche

ct addition o

oplets). A two

owing ODE/P

T-junction is

Images of the

ree-phase flo

ontent, inclu

emical detec

emiluminesc

e content of

ets to analys

interest from

hen be direct

uced directly

]. For infr

r detection o

rent liquid be

ematic presen

of a reagent

o- or three-p

PFPE (QR2/

used to inje

e droplets be

ows. The figu

uding

ction,

cence

f the

is by

m the

ed to

y into

frared

of the

efore

nting

(red-

phase

/ Qc)

ect a

efore

ure is

23

1.6. Droplet-based nanotechnology

Droplets are used in nanotechnology for different reasons. Droplets can be used as (i)

containers for material transport, (ii) reaction chambers to fabricate nanomaterials, and (iii)

building blocks to make structures [47].

One major area of droplet-based nanotechnology involves the transport of liquid cargo in

the form of oil-in-water or water-in-oil emulsion droplets. These platforms move the droplets

along various types of tracks, including microfluidic channels, nanotubes and organic planar

tracks, such as graphene strips. Microtubules are biological nanotubes that act as the skeleton

of cells and play the role of linear tracks for the movement of molecular motors and the

associated cargos. These rigid biological polymers have been widely used in bio-

nanotechnology and microfluidics. In microfluidics, the system was employed to sort,

transport and concentrate molecules [48, 49]. However, droplet technology and microtubule-

based technologies have only been recently combined to generate lab-on-a-chip devices.

Bottier et al. used a hybrid platform in which kinesin motors actively carry oil-in-water

droplets along microtubules [50]. The transport of droplets along organic tracks has recently

attracted attention. Yin et al. discovered that voltages on the order of a few millivolts can be

produced by moving a droplet of seawater or ionic solution over a strip of monolayer

graphene under ambient conditions [51].

When aqueous droplets come in contact with a hot surface, the Leidenfrost effect produces

an insulating vapor layer that keeps that water from boiling rapidly. This effect has been

recently employed to use droplets as platforms for green chemistry and nanoparticle

fabrication [52]. For example, the technology was used to fabricate nanoporous black gold,

which acts as a plasmonic wideband superabsorber. Another demonstrated application of this

technology is the synthesis of superhydrophilic and thermally resistant metal–polymer hybrid

foams.

Water-in-oil droplets can be assembled to form networks. These networks can be designed

to perform useful tasks. Lipids and surfactants are typically used to avoid the merging of the

contacting droplets [53]. Despite being thin, bilayers form robust interfaces, allowing for

flexible network architectures. One can readily excise a droplet from a network and replace it

with another one without disrupting the integrity and functioning of the rest of the network

[54]. Droplet networks have been designed to act as light sensors, batteries and electrical

systems [54, 55]. Networks of aqueous droplets with distinct chemical compositions have also

been

com

curr

The

inclu

recti

A

biolo

laser

cavi

and

mole

total

pote

mole

com

work

coul

F

a dr

n realized [

mmunication

rent through

se pores ca

ude the fabri

ifiers [54, 55

An important

ogical molec

r consists of

ity and pump

the optical

ecules in the

l loss in the

ential as bio

ecules in the

mposition of t

ks utilize mi

ld be tuned u

Figure 1.4. D

roplet of fluo

[29]. At the

between dro

these pores

n be engine

ication of de

5].

t droplet-bas

cules are inc

f three main c

ping. The ph

feedback in

e cavity are

e cavity, and

osensors. Dr

e gain mediu

the gain med

icrofabricate

using digital

Droplet-based

orescein diso

e contact si

oplets [54].

s via the app

eered for ne

evices such a

sed nanotech

orporated in

components:

hotons emitt

nduces stimu

excited by p

d laser oscil

riven by a

um can be al

dium and can

ed lasers, inc

microfluidic

d microfluidi

odium salt in

24

ites, pores

Inserting el

plication of

etworks with

as current lim

hnology is o

nto the gain m

: a gain medi

ted from the

ulated emiss

pumping, the

llation build

variety of

ltered. The o

n thus be use

cluding a mi

cs [58].

ics and nano

n gelatin. (b)

can be eng

ectrodes into

f desired vol

h specific u

miters, half-w

optofluidic la

medium [56]

ium in the flu

gain mediu

sion. When

e available g

ds up. Optof

biochemical

output laser c

ed as probes

icrofluidic dy

otechnology.

Optofluidic

gineered to

o droplets c

ltages across

uses; interest

wave rectifie

asers, where

] (Figure 1.4

uidic environ

m are trappe

a sufficient

ain becomes

fluidic lasers

l processes,

characteristic

for the reac

ye laser [57]

(a) Stimulate

laser based

allow chem

can modulate

s the pores

ting applica

ers and full-w

e biochemic

4). An optoflu

nment, an op

ed by the ca

number of

s greater than

s have enorm

the numbe

cs depend on

ction. Many o

] and a laser

ted emission f

d on a distrib

mical

e the

[54].

ations

wave

al or

uidic

ptical

avity,

gain

n the

mous

er of

n the

other

r that

from

buted

25

feedback grating embedded in a microfluidic channel. The periodic structures form a pair of

virtual mirrors for resonant light to bounce back and forth to provide optical feedback. (c)

Optofluidic laser using an evanescently coupled ring resonator. The resonant light circulates

along the circumference to provide optical feedback. (d) Optofluidic laser using dye

microdroplets and an integrated Fabry-Pérot cavity formed by two reflectors coated on

optical fiber tips. The resonant light bounces back and forth between the two reflectors to

provide optical feedback. The figure is reproduced, with permission, from Fan et al. and

references therein [56].

1.7. Conclusions

Much of the world’s technology requires fluid manipulation. Micro- and nanotechnology,

which aim at miniaturizing current technologies and developing new ones, thus involve

extending those manipulations to small volumes while maintaining precise dynamic control

over the droplet properties, such as the position, concentration, temperature and stability.

When droplet-based lab-on-chip technologies become widespread, new challenges will

emerge. How can droplets be preserved and stored? How can droplets be sent to other

laboratories? How can large numbers of droplets be packaged, and how can the barcoding of

droplets be standardized? Overcoming these challenges in the field promises much-improved

platforms for laboratory operations and environment-friendly practices.

26

1.8. References

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