Applications of bioluminescence in biotechnology and beyond

Abstract

Bioluminescence is the fascinating natural phenomenon by which living creatures produce light. Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent systems but the luciferin–luciferase pairs of only 11 systems have been characterised to-date, whilst other novel systems are currently under investigation. The different luciferin–luciferase pairs have different light emission wavelengths and hence are suitable for various applications. The last decade or so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is now routinely used for gene assays, the detection of protein–protein interactions, high-throughput screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to control neurons. There has also been an increase in blue-sky research such as the engineering of various light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This review focuses on the past, present, and future applications of bioluminescence. We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist.

Full text

Applications of bioluminescence in biotechnology
and beyond†
Aisha J. Syed *‡and James C. Anderson
Bioluminescence is the fascinating natural phenomenon by which living creatures produce light.
Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme
luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent
systems but the luciferin–luciferase pairs of only 11 systems have been characterised to-date,
whilst other novel systems are currently under investigation. The different luciferin–luciferase pairs have
different light emission wavelengths and hence are suitable for various applications. The last decade or
so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed
luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is
now routinely used for gene assays, the detection of protein–protein interactions, high-throughput
screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo
imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the
applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of
small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to
control neurons. There has also been an increase in blue-sky research such as the engineering of various
light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This
review focuses on the past, present, and future applications of bioluminescence. We aim to make this
review accessible to all chemists to understand how these applications were developed and what they rely
upon, in simple understandable terms for a graduate chemist.
Cite this: DOI: 10.1039/d0cs01492c
Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK. E-mail: Aisha_asrar@hotmail.com
†Electronic supplementary information (ESI) available. See DOI: 10.1039/d0cs01492c
Aisha J. Syed
Aisha J. Syed received her BSc in
Chemistry from the University of
Birmingham in 2015. She then
studied for a PhD in Organic
Chemistry and Chemical Biology
at University College London with
Prof. Jim Anderson on the
synthesis and bioluminescence
properties of infraluciferins
which she received in 2019. She
is currently a Research Fellow in
the labs of Prof. Andrew Wilson
and Prof Adam Nelson at the
University of Leeds. Her research
interests include using synthesis and related chemical tools for
visualising and manipulating biological processes including
molecular imaging and protein–protein interactions.
James C. Anderson
Jim Anderson studied at Imperial
College, receiving his PhD in 1990
working with Professor S. V. Ley
FRS. After postdoctoral study with
Professor D. A. Evans at Harvard
University, he started his
independent career at the
University of Sheffield in 1993.
He moved to the University of
Nottingham in 1999 and to his
current position at University
College London in 2009. His
work has been mainly concerned
with asymmetric reaction
methodology and total synthesis. At UCL these have been applied
to the rational design of red shifted luciferins, which has led to the
development of infraluciferin.
‡Present address: School of Chemistry, University of Leeds, Woodhouse Lane, LS2 9JT, Leeds, UK.
Received 30th November 2020
DOI: 10.1039/d0cs01492c
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1. Introduction
Bioluminescence is the fascinating natural phenomenon by
which living creatures produce and emit light. In nature this
hasbeenevolutionarilyconservedprimarily in marine organisms,
some species of bacteria, fungi and terrestrial insects for various
purposes such as to hunt prey, ward-off predators, and attract
mates.
1–3
Since 1667 when one of the earliest scientific records of
the study of bioluminescence was made by Robert Boyle, many
researchers have contributed to our understanding of the
mechanisms that govern bioluminescence and how this natural
phenomenon can be tailored for targeted use as a powerful tool in
biotechnology for the visualisation, imaging, and control of
biochemical processes.
4
Naturally occurring light originates from two main kinds of
systems – bioluminescent systems which comprise of distinct
luciferase enzymes and luciferin moieties, and photoproteins
in which the light-emitting chromophore is part of the protein
itself and light emission is triggered by changes in the protein’s
environment. The discovery of the mechanisms underpinning
photoproteins such as the green fluorescent protein (GFP),
aequorin, kaede and pholasin and their subsequent applications
have been previously reviewed and will not be discussed in this
review.
5–8
Whilst there are more than 40 known bioluminescent systems,
the structures of the luciferin and luciferase have only been
elucidated for 11 of them. The quest for the mechanistic
characterisation of luciferin and luciferase pairs is an active
area of research as is the search for new pairs.
9–11
The bio-
luminescent reaction generally requires a luciferase enzyme, its
luciferin substrate and an oxidant which is often molecular
oxygen. Some systems require energy in the form of ATP or
NADH as well. One of the earliest luciferin structures to be
elucidated were those of D-luciferin found in fireflies, reported
in the mid 1900s.
12
About twenty years later the luciferin
coelenterazine and its luciferase were discovered from the
deep-sea shrimp Oplophorus gracilirostris.
13
Since then, due to the low toxicity, high quantum yield and
high sensitivity of both of these reactions, these molecules and
their luciferase enzyme partners have found wide use as in vitro
reporters of analytes and metabolites – for example the firefly
luciferase (FLuc) and D-luciferin system are widely used in
biochemical assays to determine ATP levels. The last decade
or so has seen luciferins and luciferases to reach previously
uncharted applications fuelled by great advances in protein
engineering, synthetic chemistry, physics and light capture
technology. We will first cover some of the basic qualities and
characteristics of the most widely used natural luciferin/luciferase
pairs, with a brief mention of their uses as well as their
shortcomings that make them less than ideal candidates for
certain applications. This then leads up to the developments in
synthetic luciferins and engineered luciferases and their
improved properties and uses. The applications of bioluminescence
are then extensively discussed including ATP sensing, hygiene
control in the fish and milk industries, mapping pollution in
ecosystems using bioluminescence based assays, culture and
heritage in the form of art work preservation, the sensing of pH,
metal ions, membrane potential, drug molecules, other
metabolites, gene assays, the detection of protein–protein
interactions, high-throughput screening in drug discovery and
in vivo imaging of tumours as well as infections. Apart from
sensing applications, the discussion will then move to how new
applications are now trying to make use of the light from
bioluminescence for various purposes such as to effect healing
in the form of bioluminescence based photodynamic therapy
(PDT)
14
or using the light for bioluminescence-induced photo-
uncaging of small molecules,
15,16
and the use of bioluminescence
to control neurons.
17
Exciting recent blue-sky research is also
discussed such as the engineeringofalightemittingplantsof
various types.
18
We aim to make this review accessible to all
chemists to understand how these applications were developed
and what they rely upon, in simple understandable terms for a
graduate chemist. Hence, a stepwise journey across the various
applications of bioluminescence has been taken, and how
protein-engineering, synthetic chemistry and chemical biology
tools have fed into the development of novel, state-of-the-art
applications.
Whilst bioluminescence has found utility in various fields
including medicine, biology, physics and engineering and led
to exciting multidisciplinary science across all of them, we also
discuss the current limitations in the state of the art, as well as
prospects and future directions for the research community for
the future development of applications of the luciferin–luciferase
reaction from a chemical biology tool to something more widely
used in industry, daily life or in a clinic.
2. Natural luciferin, their luciferases
and mechanisms
The structures of D-luciferin and coelenterazine, and their
respective luciferases were some of the earliest luciferin–luciferase
pairs to be elucidated and their enzymes synthetically
expressed.
12,19–25
Hence, these bioluminescence systems are used
in the vast majority of applications of bioluminescence by virtue
of our greater understanding of their mechanisms of action and
ease in preparing the reagents needed for their assays.
In this section the mechanisms of bioluminescence with
respect to D-luciferin, coelenterazine and bacterial luciferin
have been looked at in greater detail as these luciferins and
their respective luciferases have found the most utility in
various applications. The remaining eight known luciferin–
luciferase pairs have been included and discussed briefly
towards the end of the section, as their understanding and
inclusion would help foster future applications.
2.1 D-Luciferin
Although each bioluminescent insect species has a distinct
luciferase enzyme, they all share the same substrate D-luciferin
which is found in around 40 different species of fireflies, click-
beetles and the rail-road worms.
12,26–33
In the bioluminescence
reaction, the 62 kDa insect luciferase (Fluc)
19
catalyses the
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oxidation of D-luciferin 1in two distinct steps – the activation of
the carboxyl group of 1through adenylation, followed by the
oxidation of the luciferyl-adenylate 2to form oxyluciferin 4as an
excited state anionic species through a dioxetanone intermediate
3(Fig. 1).
34–36
The excited state oxyluciferin species 4relaxes to
its ground state 5by giving off a photon of light. Around 20% of
luciferyl adenylate undergoes a dark-side oxidation to form H
2
O
2
and dehydro-luciferyladenylate 6which is a strong inhibitor of
luciferase (K
i
=3.80.7 nM),
37
andthislimitsthequantumyield
of the bioluminescence reaction.
38
Protonated 5, oxyluciferin itself
is also an inhibitor of the luciferase enzyme (K
i
= 500 30 nM).
37
Due to the dark-side reaction and other energy losses, firefly
luciferase catalyses light emission from D-luciferin with a
maximum quantum yield of 41% at pH = 8.5.
39
The activation of D-luciferin by ATP prior to oxidation is a
unique feature of D-luciferin bioluminescence that is not
shared by other luciferins such as coelenterazine. This feature
bestows a variety of unique benefits upon the D-luciferin/firefly
luciferase system that are not enjoyed by other bioluminescent
systems. For example, as D-luciferin requires activation in the
form of adenylation, it is less susceptible to auto-oxidation and
more stable in solution, leading to less background
chemiluminescence.
40
ATP is widely known as the ‘energy
currency’ of a cell and found in varying amounts in virtually
all eukaryotic and prokaryotic cells.
41
As the D-luciferin/Fluc
assay produces ATP dependent light output, this assay has been
widely adapted to measure ATP concentration in the double
digit nanomolar region in various systems for both quality
control and hygiene, often to determine the extent of bacterial
contamination.
42,43
It is also used to monitor both ATP-forming
reactions
44–47
and ATP-degrading reactions.
48–50
With the advent
of more sensitive luminometers and improved assay techniques,
ATP levels as low as attomole levels – corresponding to a single
bacterial cell – can be routinely detected now using commercially
available ATP bioluminescence reagents and kits.
42
More details
on these assays are discussed in Section 4.1.
It is known that D-luciferin has greater aqueous solubility
51
and lower toxicity than coelenterazine.
52
Moreover D-luciferin
has the most impressive quantum yield amongst all known
luciferin/luciferase systems (41.0 7.4% compared to 15–30%
for most luciferase/luciferin pairs)
39,53
and the longest emission
wavelength amongst them (l
max
=558nm
39
), it is more ideal for
imaging applications where blood is involved. Haemoglobin and
tissues absorb light most strongly of wavelength o500 nm.
54
Mammalian cells and other biological entities of interest such as
bacteria, fungi, protozoa and viruses can be genetically encoded
with the luciferase gene of interest
55,56
and introduced in to a
small mammal for proliferation, tracking and imaging. From
naturally occurring luciferins and luciferases, D-luciferin and the
firefly luciferase from the North American firefly,Photinus pyralis
are the most common pair used for in vivo imaging (Table 1).
57
D-Luciferin is commercially available in its carboxylic acid
form (CAS Number: 2591-17-5) as well as its sodium salt
Fig. 1 The mechanism of firefly bioluminescence.
Table 1 Properties of firefly luciferase (Fluc) from the American firefly
Photinus Pyralis
PpyLuc
Wavelength 558 nm
Optimum pH 7.8
Optimum temperature 28 1C
Quantum yield
39
41 7% at pH 8.5
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(CAS Number: 103404-75-7). It can also be readily prepared
in up to 450 mg scale batches using multi-step organic
synthesis – preparations up to 50 mg scale have been reported
although it can be envisaged that larger batches of 4200 mg
could be readily prepared too.
58,59
The plasmids and vectors
used to express firefly luciferase are also readily available.
Although the light from D-luciferin bioluminescence is
red-shifted compared to that from other naturally occurring
luciferins, it is still strongly absorbed by blood and tissue.
Near infra-red light (650–900 nm) has better penetration
through blood and tissue.
60
Whilst there is a portion of light
in the broad emission spectrum of D-luciferin within this
desirable range of wavelengths, more red-shifted light would
allow more sensitive imaging.
54
There are other factors as well that make D-luciferin a less
than ideal candidate for bioluminescence imaging. For example,
D-luciferin has modest cell permeability
61
and hence has to be
dosed in large amounts for in vivo experiments.
62
Studies in
which the
14
C-labelled radioactive D-luciferin substrate has been
used have also demonstrated the inhomogeneous bio-
distribution of the substrate in rodents.
63
Moreover, there is
poor uptake of the probe in some organs of interest such as the
brain.
64
Whilst D-luciferin is capable of emitting light of different
wavelengths on interaction with various mutants of Fluc;
65
the
range of wavelengths emitted does not render it suitable for
in vivo multi-parametric imaging particularly in deep tissue
imaging because the most red-shifted l
max
obtained by D-luciferin
and Fluc mutants is 620 nm.
66,67
The light at this wavelength
suffers from absorption, attenuation and scatter in tissues making
spectral unmixing from different luciferases more challenging.
68
Some of these set-backs have been overcome with the design,
synthesis and testing of novel D-luciferin analogues, which have
led to brighter, red-shifted emission in some cases.
69
Details on
these can be found in Section 3.1.
There has also been a strong case for engineering the firefly
luciferase enzyme to obtain better properties more suited for
various applications. For example, in order to detect a variable
ATP concentration, it is essential that the luciferase concentration
remains constant during the duration of the measurement and
luciferase is not inactivated by other factors such as pH, temperature,
the concentration of metal ions, detergents and other soluble
proteins such as bovine serum albumin, in the assay mixture.
The optimum pH for wild-type luciferase activity is pH 7.8.
70,71
A workable pH of 6–8 can be used for analytical applications
using the wildtype enzyme.
46
However, the colour of emission
from some wild-type firefly luciferases such as P. pyralis and
H. parvula changes from yellow-green to red when the pH is
lowered below optimum.
72–74
The reason for this has been
proposed to be the disruption of a key hydrogen-bonding
network involving key water molecules and amino acid residues
in the enzyme’s active site around the oxyluciferin emitter. It is
believed that disruption at lower pH enhances the delocalisation
of the phenolate negative charge that increases red shifted
emission.
75
This rendered these wild-type luciferases not as
useful for comparative bio-analytical applications where often
photons of a specific wavelength of light are being quantified.
Moreover, most wild-type luciferases are thermolabile at even
room temperature, whilst work on mammalian cells often
requires temperatures of around 37 1C.
76
Indeed, wild-type
luciferase has a half-life of only 3–4 h in mammalian cells due
to both thermal inactivation and proteolysis.
77
Consequently,
there has been significant work done in protein engineering to
create mutant firefly luciferases of greater pH stability, thermo-
stability and proteolytic stability. Indeed, a small change in the
sequence of the enzyme can lead to changes significant changes
in the emission and properties of the enzyme. For example, the
mutant S286N of the Japanese Firefly Luciferase has red-shifted
emission at l
max
= 605 nm compared to the wild-type at l
max
=
560 nm (Fig. 2A and B). This is because Ser 286 is involved in a
key hydrogen-bonding network that is disrupted when it is
mutated to an asparagine residue. Moreover, this also causes a
change in the conformation of Ile 288 close to the emitter
making the hydrophobic pocket more flexible (Fig. 2C and D).
For further details on engineered Fluc enzymes please see
Section 3.1.
2.2 Coelenterazine
Coelenterazine is found in several marine creatures including
copepods and the deep-water shrimp, and is the other most
commonly used luciferin.
13,78
Coelenterazine is the substrate
for around 15 different naturally occurring luciferases.
24,25,79–84
From these, the luciferases from Renilla luciferase (Rluc),
Gaussia luciferase (Gluc) and Metridia longa luciferase (Mluc)
were one of the earliest to be cloned and have found most
utility in biotechnological applications. The bioluminescent
reaction of coelenterazine also involves an enzymatically
catalysed oxidation. Coelenterazine 7is converted to an excited
state coelenteramide oxyluciferin 10 through a dioxetanone
intermediate 9. The excited state oxyluciferin relaxes to its
ground state to emit a photon of blue light of wavelength
454–493 nm, dependent upon the enzyme (Fig. 3). This
bioluminescent reaction is not dependent on ATP.
Coelenterazine is commercially available (CAS Number:
55779-48-1) and can be synthesised in up to 4200 mg scale
batches using multi-step synthesis.
85
However, coelenterazine
is a larger molecule than D-luciferin, has poor water solubility,
greater toxicity and is also susceptible to auto-oxidation leading
to chemiluminescence in solution as it does not need activation
in the form of adenylation.
40
Moreover, coelenterazine has also been
reported to be transported into cells through other mechanisms.
For example, the multidrug resistance P-glycoprotein (MDR1 Pgp)
was reported to mediate the transport of coelenterazine into
cell lines. This led to greater amounts of coelenterazine being
transported into cancer cells expressing greater quantities of
MDR1 Pgp. This could lead to an inaccurate representation of
tumours in small mammal in vivo imaging i.e. tumours that do
not express MDR1 PGp would not be detected.
86
Coelenterazine
gives out blue light which is strongly absorbed by blood and
tissue, making it a poor candidate for in vivo imaging
when used alone without the red-shifting effects of BRET.
54
To overcome these short-comings, several synthetic coelentera-
zine analogues have been prepared, of which some are
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commercially available. Details of these analogues are presented
in Section 3.2.
In terms of size, both Rluc (B34 kDa), Gluc and Mluc (both
B20 kDa) are smaller than Fluc (B62 kDa). This makes them
more suitable for applications involving small vectors and/or
proteins. In the sea pansy, Renilla reformis the luciferase Rluc
is closely associated with a green fluorescent protein (GFP) and
the blue light emitted by the luciferase is coupled through
resonance energy transfer to the fluorophore of the GFP
allowing it to form an excited-state species which emits a
photon of green light (l
max
510 nm).
87
This principle of
resonance energy transfer led to the development of
bioluminescence resonance energy transfer (BRET) and several
associated applications where the BRET light emission has be
used as a measure for the spatial proximity of two proteins.
88
Most coelenterazine utilising luciferases possess several
disulphide bonds in the protein structure which often help in
protein-folding and confer these enzymes with greater thermal
stability than firefly luciferases. However, these disulfide bonds
also make the enzymes sensitive to any reducing agents in
buffer solutions as it is vital for the cysteine residues to be in
the correct oxidation state for native protein folding. Moreover,
the optimum conditions for activity of these luciferases often
mimic their natural marine environment. So, most of these
luciferases are halophilic reflecting the saltiness of seawater
and some forms – for example the Mluc2 isoform of Metridia
longa luciferase – is psychrophilic i.e. has a very low optimum
temperature reflecting the low temperatures at the bottom of
the ocean (Table 2).
89
Copepod luciferases Gluc and Mluc are
the only known luciferases that are naturally secreted from
Fig. 2 (A) The co-crystal structure of the Japanese firefly luciferase (Luciola cruciata) with a high-energy intermediate analogue, 50-O-[N-(dehydro-
luciferyl)-sulfamoyl]adenosine (DLSA) – PDB code: 2D1S. (B) Overlay of complex of Luciola cruciata luciferase with DLSA (PDB code: 2D1S – coloured in
light orange) with mutant S286N complexed with DLSA (PDB code: 2D1T – coloured in cyan). The side-chains of the residues of the key mutation are
shown as sticks and highlighted in the white circle. (C) The conformation of Ile 288 (green spheres) in wild-type Luciola cruciata. (D) The changed
conformation of Ile 288 in the S286N mutant (red spheres), leading to a more flexible hydrophobic pocket.
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eukaryotic cells.
90
Theseuniquepropertiesmakethempotentially
useful for a unique set of applications such as high throughput
studies as cell-lysis is not required.
2.3 Bacterial luciferin
Besides firefly bioluminescence and coelenterazine based marine
bioluminescence, bacterial bioluminescence has also received
considerable attention in terms of applications over the past half
century or so.
57,94,95
All known bioluminescent bacteria are
Gram-negative, facultative anaerobic
94
and most are symbiotic
and found in their host organisms such as the Hawaiian
Bobtail squid (Aliivibrio fischeri)
96
or terrestrial roundworms
(Photorhabdus luminescens).
97
Although some are free-living
species such as Vibrio harveyi
98
and Alteromonas hanedai.
99
Although bioluminescent bacteria belong to various species and
genre, the biochemical machinery of bacterial light emission is
globally conserved between species. In bacteria, all the enzymes
required for bioluminescence are completely genetically encoded
for in the luxCDABEG operon.
100
An operon is a functioning unit
ofDNAcontainingaclusterofgenesunderthecontrolofasingle
promoter.
101
A promoter is a sequence of DNA to which proteins
bind that initiate transcription of a single RNA from the DNA
downstream of it.
102
The luxA and luxB genes encode for the
40 kDa asubunit and 37 kDa bsubunit of the bacterial luciferase
respectively. The luxC,luxD and luxE genes encode enzymes
involved in the synthesis of the aldehyde co-factor, and luxG
encodes for flavin reductase which participates in flavin mono-
nucleotide (FMN) turnover.
94,103
The mechanism of bacterial bioluminescence is well
understood (Fig. 4).
104
Flavin mononucleotide (FMN) reductase
reduces oxidised FMN 12 to form reduced FMN 13 which reacts
with molecular oxygen to form an FMN-hydroperoxide species
14, possibly through a single electron transfer (SET) process as
suggested for adenylated D-luciferin and oxygen.
35,105,106
In the
absence of long-chain aldehyde 15, intermediate 14 is fairly
stable and was characterised by UV-vis absorption and NMR
spectroscopy.
107,108
It has been proposed that the long-chain
aldehyde 15 adds to 14 to form the FMN-4a-peroxyhemiacetal
species 16 which collapses to form the carboxylic acid 17 and
FMN-4a-hydroxide 18 in an excited state.
106
The exact
mechanism of formation of the excited-state species is still
under debate. A few different mechanisms have been proposed
for the breakdown of FMN-4a-peroxyhemiacetal species 16,
including the proposed formation of a dioxirane intermediate
109
or a flavin-mediated intra-molecular electron transfer mechanism
initiating the collapse of 16. There is greater computational
110
and
experimental data
111
to support the intra-molecular flavin-mediated
electron transfer from the 5-N of the isoalloxazine ring to the distal
oxygen atom of FMN-4a-peroxyhemiacetal species 16.Light
emission occurs when the flavin mononucleotide species FMN-4a-
hydroxide 18 relaxes to its ground state 19 and emits a photon of
bluish-green light (l
max
490 nm) (Table 3). The long-chain aldehyde
is oxidised to the corresponding carboxylic acid as part of the cycle.
In essence, the entire light emission machinery including
luciferase production, luciferin biosynthesis and recycling
Table 2 Properties of common marine luciferases
Rluc
91
Gluc
92
Mluc2
89
Wavelength 480 nm 485 nm 488 nm
Optimum pH 7.4 7.5–8.4 7.5–8.4
Optimum temperature 32 1C
91
15–20 1C
92
51C
89
Optimum [NaCl] 0.5 M 1.6 M 1.0–1.5 M
Quantum yield 5.3 0.1%
93
Not determined Not determined
Fig. 3 The mechanism of coelenterazine bioluminescence.
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machinery is genetically encoded for together in a single operon,
so transgenic auto-luminescence is possible. Exogenous adminis-
tration of luciferin is not required in any imaging applications,
unlike what is observed in fireflybioluminescence imaging and
coelenterazine bioluminescence imaging. This is of great value in
synthetic biology, especially as the administration of expensive
and unstable luciferins in some hosts such as plants is
difficult.
112,113
However, considerable work and optimisation of
the genetic make-up was required to make bacterial operons
useful in eukaryotic cells and machinery. Although in early studies
only luxA and luxB were expressed in plant cells such as tobacco
and carrots to form functional luciferase in them, autolumines-
cent tobacco plants were later designed using the operons form
Photobacterium leighognathi lux operon.
114,115
Although previously,
Fig. 4 The mechanism of bacterial bioluminescence.
Table 3 Properties of the bacterial bioluminescent system
Bacterial luciferase
Wavelength 490 nm
122
Optimum pH 6.8
122
Optimum temperature 23 1C
123
Quantum yield 30%
124
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Table 4 Summary of the key properties of the remaining luciferin–luciferase pairs
Luciferin Luciferase size Wavelength
(l
max
)Required
components Key facts
B61 kDa
22
452 nm
22
Luciferin, luciferase
and O
2
Bioluminescence mechanism is very
similar to that of coelenterazine
It is a secreted luciferase
Several applications exist which will be
discussed in depth in later sections.
Quantum yield = 28%
125
B29 kDa
126
520 nm
126
Luciferin,
127
luciferase
and O
2
The only eukaryotic auto-luminescent
system.
So far used to create GM glowing
plants.
128
Quantum yield not determined.
B135 kDa
129
3 distinct
domains that are each
catalytically active 476 nm
129
Luciferin,
130
luciferase
and O
2
at pH 6.3
No chemical synthesis of the luciferin
reported to-date
Fewer than half a dozen known
applications of the luciferase exist.
Examples include use as a reporter
enzyme for in vitro gene expression,
131
measuring quantities of cell-surface
expressed membrane proteins,
132
and
pregnancy-specific glycoproteins in
HeLa cells to investigate cell
senescence.
133
Quantum yield not determined.
B600 kDa
134
468 nm
135
Luciferin,
138
luciferase
and O
2
No chemical synthesis of the luciferin
reported to-date and a pure sample of
luciferin not isolated either – structure
elucidated through crude luciferin
sample and oxyluciferin sample
Cross-reactivity is observed between
krill luciferin and dinoflagellate
luciferase and dinoflagellate luciferin
and krill luciferase
136
No known applications to-date
Quantum yield not determined.
B173 kDa
137
536 nm
138
Luciferin,
130
luciferase,
purple protein and O
2
The structure of the light-emitting
species is still unknown
a
Synthetic analogues of the luciferin
have been reported that are less active in
bioluminescent emission
139–141
Quantum yield = 17%
142
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the bacterial lux operon had been used to produce a weakly
autoluminescent human cell line (HEK293 cells), more recently
codon-optimised lux sequences have been developed that produce
bright, autonomous bioluminescence in mammalian cells.
116,117
Further details of the codon-optimisation and enzyme engineering
involved are presented in Section 3.3.
Bioluminescent bacteria are often used as biosensors in
ecotoxicological studies as these can often be tuned to detect
concentrations of a variety of different organic (alcohol, carboxylic
acids, aromatic compounds) and inorganic substances (heavy
metal ions).
118–120
Moreover, bioluminescent bacteria are also
being used to create unique glowing artwork and proposals
have been suggested to use them in indoor aquariums as part
of aesthetic architecture in skyscrapers.
121
As both bacterial luciferase and luciferin are encoded for in
the lux operon, this has led to limited mutations in the native
luciferase structure and no mutations to the luciferin, although
shorter-chain aldehydes are also tolerated and as expected
result in no change in wavelength of the light emitted.
105
2.4 Other known luciferins
There are over 30 known bioluminescent systems but the
luciferin–luciferase pairs of only 11 systems have been
characterised to-date with a handful of systems only partially
characterised.
10
Of these D-luciferin, coelenterazine and the
bacterial bioluminescent systems have found most utility in
biotechnological applications, by virtue of a more comprehensive
understanding of them. The table below (Table 4) details
succinctly the other known bioluminescence systems and some
of their applications.
The discovery and characterisation of yet-unknown luciferins
and luciferases is an area of active research and recent highlights
include reports on the luciferin–luciferase systems of the New
Zealand glow-worm Arachnocampa luminosa and the diptera
Orfelia fultoni from the United States.
151,152
Both systems emit
blue light and although structures of the luciferins have been
proposed based on NMR spectroscopy, mass spectrometry and
isotopic labelling studies although, the proposed structures need
to be validated through chemical synthesis.
3. Designer luciferins and their
luciferases
Advances in synthetic chemistry and protein engineering have
been crucial to take the known natural luciferin and luciferase
systems to unchartered territories and use them for a wide
Table 4 (continued)
Luciferin Luciferase size Wavelength
(l
max
)Required
components Key facts
B300 kDa
143
copper
dependent 500–530 nm
144
Luciferin,
144
luciferase
and H
2
O
2
Used in analytical tools to detect H
2
O
2
levels and peroxidase activity.
145
A major limitation on its use is that
the luciferase has not been cloned so
needs to be sourced directly from the
earthworms themselves.
146
Quantum yield = 3%
a146
B35 kDa
147
478 nm
147
Luciferin,
148,149
luci-
ferase, Mg
2+
ions, O
2
and ATP
A major limitation on its use is that
the luciferase has not been cloned so
needs to be sourced directly from the
earthworms themselves.
146
Quantum yield not determined.
B35 kDa
150
510 nm
150
Luciferin, luciferase
and O
2
Luciferin structure was reported in
late 2019.
11
Quantum yield not determined.
No known applications to-date.
a
This is the proposed structure for the luciferin although other proposals suggest that the light emitting species could be a flavin moiety as is the
case in the bacterial bioluminescent system.
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range of applications. Significant work has been done in
both fields to prepare optimum luciferin–luciferase pairs that
give access to previously inaccessible applications through
bioluminescence imaging (BLI). Although significant research
has been done in accessing ‘caged’ luciferins and ‘tagged’
luciferins as well as ‘split’ luciferases and ‘tagged’ luciferases
– this particular portion of the review will focus on the basic
structural modifications of both luciferins and luciferases that
have bestowed them with beneficial properties, whereas as
‘tagged’ and ‘caged’ luciferins and ‘split’ luciferases will be
discussed where relevant and have been discussed in greater
detail in Sections 4.5 and 4.7. This section will focus on some of
the key developments in the area of novel luciferins and
luciferases.
3.1 Novel D-luciferin analogues and luciferase mutants
As the D-luciferin/Fluc assay is the most red-shifted and bright
natural luciferin to date, it has emerged as the assay of choice
for in vivo imaging in small mammals. However, it does have
some drawbacks that limits quantitative deep-tissue imaging in
small mammals (Section 2.1). In order to address these, the
quest for bright, non-toxic, red-shifted luciferin analogues
that have optimum biochemical properties such as solubility,
biodistribution and the ability to be useful in quantitative,
multi-parametric imaging continues.
The chemical synthetic routes towards luciferin analogues
were succinctly compiled by Podsiadly et al. in 2019.
69
Herein,
we briefly highlight synthetic luciferin analogues, particularly
those that have found use in applications, or have useful and
interesting properties such as brightness that could potentially
make them useful in applications. As the primary use of
red-shifted D-luciferin analogues is in in vivo applications, we
particularly highlight analogues that emit in the near infra-red
region (4650 nm), and of those analogues, specifically those
that have been successfully tested in vivo (Table 5). It is
important to note that although the Fluc/D-luciferin combination
emits at l
max
=558nmin vitro, there is a spectral red-shift
observed in in vivo imaging, with l
max
B610 nm due to
attenuation through tissue.
153
Of all the synthetic luciferin analogues reported to-date,
BtLH
2
28 was reported to have the highest bioluminescence
quantum yield (70%) relative to that of D-luciferin 1with wild-
type Fluc. Although BtLH
2
28, had a bioluminescence l
max
of
523 nm which was around 20 nm blue-shifted, it was also
reported to have a longer-lasting and sustained bioluminescence
signal compared to that of D-luciferin.
154
This could possibly
make it a better candidate for applications that require blue-
shifted and longer-lasting light emission. One of the earliest
reported synthetic luciferins was the aminoluciferin 29 by White
et al. This synthetic luciferin was red-shifted (l
max
= 594 nm)
compared to D-luciferin (l
max
= 558 nm) but only about 10% as
bright in vitro.
61,155
Since then, several other synthetic amino-
luciferin analogues were reported, and although all of them are
dimmer than D-luciferin 1, with the wild-type Fluc, some have
useful properties for in vivo imaging. For example,aminoluciferin
analogues 30 and 31 have emission around l
max
B600 nm.
Both analogues 30 and 31 was reported to have better penetration
than D-luciferin through the blood–brain barrier in mice.
156,157
Moreover, analogue 31 CycLuc1 was reported to have brighter
bioluminescence output from cells at lower substrate concentrations
than D-luciferin 1, indicating that it had better cell-permeability
than D-luciferin 1.
62
Both analogues 30 and 31 are commercially
available. The Prescher group reported a brominated luciferin
analogue, that was red-shifted (l
max
= 625 nm) and had an
appreciable relative bioluminescence quantum yield of 46%
compared to D-luciferin 1at 100 mM substrate concentration
and 1 mg of Fluc.
158
At these concentrations they reported
aminoluciferin 29 to be 61% as bright as D-luciferin 1.
A handful of synthetic luciferin analogues truly emit in the
nrIR region (4650 nm). The analogues 32–34 were reported by
Maki et al. and have been successfully taken on to in vivo
detection of tumours in mice.
159–161
In particular, the hydro-
chloride salt of Akalumine 32 was used with the engineered
firefly luciferase Akaluc in single-cell bioluminescence imaging
(BLI) of deep-tissue in the lungs of live, freely-moving mice and
to image small numbers of neurones in the brains of live
marmosets.
64
The analogues Akalumine 32 and Sempei 34 are
now commercially available from Merck. The analogues 34 and
36 were also reported by Maki et al. as significantly dimmer
substrates than Akalumine 32 but whose emission was B700 nm.
162
Anderson et al. reported the analogue iLH
2
38 as a significantly
red-shifted luciferin that showed enhanced tumour burden in
deep-seated tumours such as liver metastasis in mice due to
less scattering and greater penetration of the near-infrared
(nrIR) light.
66
The luciferin iLH
2
38 was designed to emit
different colours of light with different Fluc mutants through
retention of the phenol group which is deemed necessary to
modulate the colour emission of D-luciferin analogues. This
ability of iLH
2
38 to emit different colours of light with different
Fluc mutants made it unique, and it was proposed that iLH
2
38
was a suitable analogue for multiparametric imaging and
tomography. Recently, the suitability of iLH
2
38 also demon-
strated in a report by Anderson and co-workers in which
racemic iLH
2
38 together with stabilised colour mutants of
firefly luciferase (Fluc_green B680 nm and Fluc_red B
720 nm) were shown to be a suitable system for nrIR dual
in vivo bioluminescence imaging in mouse models where they
simultaneously monitored both tumour burden and CAR T cell
therapy within a systemically induced mouse tumour model.
68
The Anderson lab also reported the analogue PBIiLH
2
37 as a
racemic compound, which was prepared as a conformationally
restrained infra-luciferin analogue.
163
Although PBIiLH
2
37 was
only tested in in vitro assays and found to be less bright than
racemic iLH
2
38, it did demonstrate an increased bimodal
emission with increasing pH. A primary bioluminescence peak
at 608 nm was observed with Fluc x11 and a secondary peak at
714 nm of increasing intensity. This emission pattern could
potentially be used to monitor pH, although the work does not
build on this possibility.
164
In 2018, Mezzanote et al. reported
the most red-shifted luciferin analogue 40 and sister compound
39 with mutant CBR2opt luciferases without the use of
resonance transfer.
165
Both analogues 39 and 40 were tested
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in HEK-293 cell lines expressing CBR2opt, and as the analogue
39 gave higher light output than the analogue 40, the analogue
39 was tested in in vivo mouse studies. The most useful output
from this study appeared to be the development of the mutant
Table 5 A selection of synthetic D-luciferin analogues that have useful properties organised in increasing wavelength
l
max
o550 nm Natural substrate l
max
B550–600 nm
l
max
B600–650 nm
l
max
B650–700 nm
l
max
4700 nm
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luciferase CBR2opt as the D-luciferin 1and CBR2opt
combination was demonstrated to be the brightest and most
useful in all the experiments reported in the work, whilst both
analogues 39 and 40 were dimmer than D-luciferin with
CBR2opt.
In the area of luciferase engineering, several firefly and
beetle luciferase mutants have been reported with improved
properties such as increased stability, increased substrate
affinity, and increased brightness over the years and these were
comprehensively reviewed in 2016 by Yampolsky et al.
10
Some
highlights since then include engineered luciferases for
improved light output of specific synthetic substrates such as
Akaluc for Akalumine 32 and CBR2opt for the analogue 39.
165,166
Other highlights include work by Miller et al. on mutants that
have a significantly higher K
m
for D-luciferin and ATP than for
cyclic amino-luciferins. The rate of reaction when the enzyme is
saturated with substrate is the maximum rate of reaction, V
max
.
For practical purposes, K
m
is the concentration of substrate
which permits the enzyme to achieve half V
max
. An enzyme with
a high K
m
for a particular substrate has a low affinity for that
substrate and requires a greater concentration of the substrate
to achieve V
max
. Hence the mutants developed by Miller et al.
are more selective for cyclic aminoluciferins than for D-luciferin
and this allowed substrate-selective BLI in mouse-brain.
167
The
Prescher group reported an elegant piece of work in which they
prepared a library of 159 mutant luciferases by mutations of
23 key residues near the active site of the enzyme.
168
These were
then screened against 12 synthetic luciferins to identify
orthogonal luciferin–luciferase pairs. Three of the ‘hit’ pairs
from this analysis were taken up for in vivo mouse studies of
mammary carcinoma.
169
Imaging conditions are sensitive to a
variety of factors, such as concentration of the imaging agent,
the type of cell line used and the type of mouse model and
tumour or infection studied. Although a number of synthetic
luciferin analogues and mutant luciferases have been reported,
there are very few studies reported that compare these against each
other to match the best luciferin analogue and its complementary
luciferase for a particular application in one study.
3.2 Coelenterazine analogues and luciferase mutants
Coelenterazine 7(CTZ) is utilised by both photoproteins and
luciferases. Coelenterazine utilising photoproteins such as
aequorin, are often activated by Ca
2+
ions and hence these
are routinely used to detect intracellular Ca
2+
ion concentration
in biological studies. These photoproteins and the synthetic
coelenterazine analogues with improved properties have been
reviewed elsewhere.
8,10
A host of luciferases including Renilla luciferase (Rluc) from
the sea pansy, Gaussia luciferase (Gluc) from the marine
copepod and Oplophorus gracilirostris luciferase (Oluc), from
the deep-sea shrimp use coelenterazine 7as their
substrate.
24,25,79–84
A number of key developments using protein engineering
were carried out on these coelenterazine utilising luciferases,
which resulted in useful imaging and visualisation tools. For
example, Nagai and co-workers developed the Nano-lantern,
which was the brightest luminescent protein reported at the
time in 2012. This was a chimera of Rluc8 (a brighter and more
stable Rluc mutant)
93
and a fluorescent protein called Venus
which has high BRET efficiency.
170
They then further developed
this work by using different fluorescent proteins as BRET
acceptors of Rluc8 and other Rluc mutants to develop a suite
of Nano-lanterns that emit light of different colours, including
the most red-shifted Nano-lantern ReNL (l
max
585 nm).
171–173
The Nano-lantern series was shown to have broad applicability
in both in vitro and in vivo imaging, as well as in the detection
of Ca
2+
ions.
In another ground-breaking development, the catalytically
active portion of Oluc was identified and mutated using a
combination of both rational mutagenesis and random muta-
genesis for enhanced thermal stability and light output by
Promega.
174
This small 19 kDa mutant enzyme was called
Nanoluc and it was optimised to perform best with the syn-
thetic substrate furimazine (Fz) 42 Table 6.
175
Both Nanoluc
and furimazine are now commercially available. However, the
Nanoluc-furimazine combination emits blue light (l
max
B
456 nm) which makes it unsuitable for in vivo applications,
although the fact that this system has ATP-independent emis-
sion has led to possible advantages in some applications over
the firefly bioluminescence system.
176
Following the development of Nluc, there were reports of
chimeric proteins that use Nluc as the BRET donor together
with a fluorescent protein as the BRET acceptor. For example,
the LumiFluor series was developed by creating chimeras of
Nluc with bright, fluorescent proteins such as eGFP to get
emission of around B460–508 nm or with an orange light
emitting GFP variant LSSmOrange for emission B572 nm.
177
The LumiFluor series was shown to be useful in the in vivo
imaging of tumours as well. In a complimentary approach to the
development of Nano-lanterns, small-molecule fluorophores
could also be appended to Nluc through the development of
Nluc-Halotag fusion proteins.
178,179
BRET then occurs from the
furimazine oxyluciferin to the fluorophore.
A number of coelenterazine analogues including 43 and 44
were reported by Shimomura et al. and these were tested
against the wild-type luciferases that naturally utilise
coelenterazine.
180
The analogue e-CTZ 43 was reported to be
B1.4 times brighter than CTZ 7with Rluc and had a 7.5 times
higher initial peak intensity than CTZ 7. This could potentially
be due to the fact that this analogue is a conformationally
restrained analogue of CTZ 7. The analogue v-CTZ 45 was
reported to be 0.73 times brighter than CTZ 7with Rluc but
had a 6.4 times higher initial peak intensity than CTZ 7. It was
also unsurprisingly more red-shifted than CTZ 7, possibly due
to extended conjugation of the p-electon system. This led to an
emission of l
max
B512 nm. Both these analogues have been
used with modified and optimised Rluc luciferase systems in
the in vivo imaging of small mammals and are commercially
available.
181,182
Recently, some furimazine analogues such as 45 and 47
have been reported which have red-shifted emission with
Nanoluc in the absence of any resonance transfer fluorophore.
183
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However, these analogues were about 10
2
–10
4
times dimmer in
in vitro assays than the Fz-Nanoluc combination. Consequently,
Nanoluc was mutated further in attempts towards red-shifted
emission. These attempts had limited success, with light
emission being red-shifted only up to 509 nm which is still
blueish-green light.
184
In an attempt to create bright and red-
shifted reporters, Nanoluc was fused with CyOFP1, a bright,
engineered, orange-red fluorescent protein that is excitable by
cyan light (497–523 nm), to develop a BRET-based genetically
encoded reporter called Antares. The Fz-Antares combination
was reported to be the brightest in vitro and in vivo when
compared with D-luciferin-Fluc, Fz-Nanoluc and Fz-Orange
Nanolantern combinations.
185
Building on from this work, Ai et al. created another fusion
protein Antares2 in which random mutations were introduced
in NanoLuc across the gene using error-prone PCR.
184
From this,
Table 6 Representative examples of synthetic coelenterazine analogues that have useful properties organised in increasing wavelength
l
max
o460 nm Natural substrate l
max
B460–550 nm
l
max
B550–600 nm
l
max
B600–650 nm
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they identified a Nanoluc mutant (Nanoluc-D19S/D85N/C164H)
with a 5.7-fold enhancement of DTZ bioluminescence and
named it teLuc as it gave teal coloured emission (l
max
B
502 nm). The teLuc fusion with CyOFP1 was named Antares2.
This BRET based reporter utilised DTZ 46 rather than Fz 42 and
emitted 3.8 times more photons above 600 nm than Antares.
Although this analogue is reported to be brighter than D-luciferin
1and Akalumine 33 in vitro, it suffers from poor solubility in
aqueous media and poor stability as like all coelenterazine
analogues it is prone to auto-oxidation. This makes in vivo
studies particularly challenging. In order to address these
challenges, Ai et al. reported a number of pyridyl analogues of
DTZ 46 including 8-pyDTZ 48 that had B13 times better aqueous
solubility and bioavailability than DTZ 46.
186
In their work they
reported the development of a teLuc mutant called LumiLuc
which was through a series of error-prone PCR experiments on
teLuc resulting in a total of 12 mutations, The emission from
8-pyDTZ/LumiLuc was B5 times brighter than 8-pyDTZ/teLuc
and had emission around l
max
B525 nm. LumiLuc was then
fused to a fluorescent protein mSCarlet-I to form LumiScarlet
which was useful in BRET based BLI and had emission around
l
max
B600 nm. The emission from 8-pyDTZ/LumiScarlet in
in vivo imaging was reported to be comparable to that by
Akalumine/Akaluc.
Two new substrates hydrofurimazine 49 (HFz) and fluoro-
furimazine 50 (FFz) were also reported recently to address the
challenges of solubility and bioavailability in coelenterazine
analogues. The analogue HFz 49 exhibited similar brightness to
AkaLuc with its substrate Akalumine 33, whilst a second
substrate, FFz 50 with even higher brightness in vivo. The
FFz-Antares combination was used to track tumour size
in vivo whilst Akalumine-AkaLuc combination was used to
visualise CAR-T cells within the same mice.
187
3.3 Bacterial bioluminescence
Several site-directed mutagenesis studies and some random-
mutagenesis studies have been carried out on the bacterial
luciferase system. However, most of these mutations have
resulted in reduced activity, poorer quantum yield and have
primarily served the purpose of improving our understanding of
the nature of and the key residues in bacterial luciferase.
188–192
A select few of these studies have resulted in altered properties of
the bioluminescent system that could potentially be of use in
applications. For example, an E175G mutation to the a-subunit
of X. luminescens luciferase using random mutagenesis, resulted
in faster kinetics and a faster decline in peak height, which could
be useful in certain applications.
193
Moreover, a number of
red-shifted, mutant bacterial luciferases from Vibrio harveyi were
reported.
194
The mutant aA75G/C106V/V173A was reported to
emit at 505 nm whilst the mutant aA75G/C106V/V173S emitted
at 510 nm. However, both these systems were 80–90% dimmer
than the wildtype system. Hence, they are too dim to be useful in
in vivo imaging and would need substantial optimisation to
make them more useful.
A breakthrough in this area was reported by Gregor, Hell and
co-workers, who engineered the ilux operon.
195
Prior to their
work, bacterial bioluminescence resulted in a weakly auto-
luminescent mammalian cell line.
116
Hell and co-workers
chose the luxCDABE operon from P. luminescens due to its
thermostability and systematically carried out studies to
identify the cause of poor luminescence in mammalian cells.
This led to codon optimisation and enzyme engineering,
including supplementing the FMN reductase in P. luminescens
with that from V. campbellii followed by error-prone PCR to
select brighter mutants. The final result ilux contains a total of
at least 15 mutations and around 8 times brighter than the
original construct allowing single-cell imaging of bacterial cells
for extended periods of time in vitro.
195
This work was then
further developed by codon optimisation of ilux for mammalian
cells, which led to brighter emission by around 3 orders of
magnitude compared to previous approaches. The light output
wasalsoreportedtobecomparabletothatofaD-luciferin/Fluc
system in HeLa cells.
4. Applications
4.1 ATP sensing
ATP is the energy currency in living cells and found in virtually
all prokaryotic and eukaryotic cells. As Mg-ATP is a necessary
co-factor in the mechanism of firefly bioluminescence, the
firefly luciferase and D-luciferin system has been adapted into
a variety of assays for ATP detection with different detection
limit levels for ATP. Luciferase reagent preparations and their
delivery devices for ATP detection vary from supplier to supplier
and are optimised for each system. Each reagent system is a
balanced cocktail of enzyme, co-factors, buffer and extractants.
196
Once the reagents are mixed at 25 1C, there is a 0.25 ms time
lag until light emission is observed after which light output
peaks at around 300 ms. This is followed by a rapid decay in
light output and then finally slow, sustained light emission.
197
This phenomenon is known as ‘burst kinetics’ and the decay in
light output is thought to be due to inactivation of the enzyme
or if a significant proportion of the substrates D-luciferin and
ATP are consumed per minute in the reaction, when their
concentration is low compared to that of the luciferase.
42
Inactivation of the luciferase can occur if the luciferase is
bound to surfaces or there is a significant concentration of
an inhibitor such as oxyluciferin – the product of the reaction,
or contaminants in the D-luciferin preparation such as L-luciferin and
dehydroluciferin. Inactivation of the enzyme can be counteracted
by using a highly pure D-luciferin sample to avoid contaminants,
and by the addition of stabilising substances such as bovine
serum albumin (BSA), neutral detergents and osmolytes for the
protein, so that stable light output is obtained.
198
In most ATP
sensing assays, a fixed amount of D-luciferin is added to the
assay mixture, which is in excess of ATP levels. At high luciferase
concentrations, the peak light output is proportional to the
amount of luciferase, as ATP is depleted in a first-order reaction.
When luciferase concentration is low, ATP is slowly depleted
and so light emission is stable and proportional to the ATP
concentration, when the ATP concentration is significantly
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below the K
m
of the enzyme i.e. ATP o0.1 mM L
1
, as the rate of
reaction and hence light output are proportional to the ATP
concentration below K
m
.
48
This is useful to monitor ATP forming
and ATP degrading reactions including kinetic and end-point
assays of enzymes and metabolites.
42,48,199
Through the careful manipulation of all of these factors,
a series of ATP-bioluminescence reagents have been made
commercially available as simple-to-use kits which include
the luciferin and luciferase preparations. These reagents are
of two main types based on the intensity and duration of light-
emission. Constant light emitting reagents have moderate
sensitivity towards ATP (working range: 10
6
to 10
11
M ATP).
The constant light signal is useful for kinetic studies of
enzymes and metabolic studies, or if coupled enzymatic assays
are applied. Such assays have been used to determine the
amount of ATP in various diseased and healthy cell lines using
both lysed human HeLa cells, mouse MEF cells and in worms
such as the round worm, as well as intact cells or isolated
mitochondria.
200–203
This type of reagent can also be used to
determine the activity of enzymes such as the activity of H
+
-ATP
synthase from live isolated mitochondria.
204
The second type of ATP-bioluminescence reagents are
high sensitivity light emitting reagents. These have a higher
concentration of luciferase and exploit the ‘burst kinetics’
phenomenon where the peak height is proportional to the amount
of ATP in the sample and dependent on the concentration of
luciferase. These reagent combinations have higher sensitivity
towards ATP (working range: 10
5
to 10
12
M ATP), although
reagents that report even lower detection limits are available
in the market. These reagents are often sold packaged with
cell lysis reagents, and are suitable for use in luminometers
where automatic injection of the reagents is possible such as in
tube luminometers and microplate luminometers (Table S1,
ESI†).
48
It is important to note that different types of cells have
varying levels of ATP. For example, bacteria have lower levels of
ATP compared to fungi or mammalian cells.
48
It is important to
pre-treat the sample effectively, and to use aseptic conditions to
ensure that ATP levels from the correct desired source are
detected. For example, a clinical urine sample may contain
3 different pools of ATP – extracellular ATP, ATP in mammalian
cells, and ATP in bacterial cells and the purpose of an ATP
bioluminescence measurement might be to estimate bacterial
levels. The level of ATP from all 3 sources can be detected by
using an appropriate kit containing ATP-degrading enzyme,
neutral detergent, strong extractant, ATP reagent, and ATP
standard (Fig. 5 and Table S1, ESI†).
48
Recently, there have been developments in luciferase
engineering that allow ATP bioluminescence technology to
reach unchartered territories. For example, Branchini et al.
reported a red-emitting chimeric firefly luciferase that has a
low K
m
for ATP and D-luciferin and would reach half of the
maximum rate of the bioluminescence reaction at lower levels of
ATP and D-luciferin than the wild-type enzyme making it suitable
for in vivo imaging in low ATP cellular environments.
205
Moreover
Pinton et al reported protocols for the in cellulo and in vivo the
use of chimeric luciferases that ensure the specific cellular
localisation of the luciferase in a cell i.e. in the mitochondrial
matrix and the outer surface of the plasma membrane to
determine the ATP concentration in those areas.
206
Viviani
and co-workers also reported a blue-shifted luciferase that
has the lowest reported K
m
for ATP, highest catalytic efficiency,
and thermal stability among beetle luciferases that was suitable
for ratiometric ATP, metal and pH biosensing assays.
207
Fig. 5 Determination of ATP levels in a sample containing extracellular ATP, mammalian cell ATP and bacterial ATP. Table S1 in the ESI†contains a list of
commercially available ATP reagents and their details.
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Metal sensing bioluminescence assays are covered in greater
detail in Section 4.3, while pH sensing assays are covered in
Section 4.5. There have also been advances in the development
of more sensitive and portable luminometers and sensing
devices.
208,209
More recently, Roda and co-workers reported a
low-cost wax-printed nitrocellulose paper biosensor that
immobilised luciferase/luciferin reagents, and whose light
signal could be detected and analysed by a smart phone to
detect E. coli levels in urine samples.
210
This low-cost, readily
available technology would be ideal for use in developing
countries where access to a luminometer and other specialist
kit would be limited.
4.2 Hygiene control
Bioluminescence based sensing technology has been of great
use in hygiene control for several decades now. In particular the
ATP-bioluminescence assay based on the firefly bioluminescence
system is routinely used to monitor the cleanliness of surfaces in
healthcare facilities such as hospitals and clinics and in the dairy
and meat processing industries.
211,212
This is the technique of
choice when the speed and ease of analysis are of vital importance
as alternative methods such as culturing or microorganisms
usually take days to offer results, whilst techniques based on
fluorescence need an external light source for excitation and do
not discriminate between living and dead cells.
213
Bacterial
bioluminescence is also used in the food industry to monitor
the behaviour of Lux-tagged bacteria in situ in complex food
systems, for problem-solving and for the development of modified
andimprovedprocessingandstorage purposes as discussed
further below (Fig. 6).
214
Several studies have been reported on the use of swab taking
and ATP bioluminescence as a quick and objective way of
monitoring the cleanliness of hospital surfaces, including
those of large objects such as tables and benches and small
pieces of equipment such as tweezers and other kit. Swabs that
are impregnated with buffer are often commercially available as
part of ATP-bioluminescence kits. These are used to sample
surfaces, and then processed with the ATP-bioluminescence
reagent in a portable luminometer. The swabs and portable
luminometer are often sold from the same supplier and
complement each other. However, this technique is still poorly
standardised at an international level and the difference in kit
and reagents used in different studies is one reason why
significant differences in ATP levels are reported.
215,216
Despite
these limitations over the comparability of results, ATP
bioluminescence remains a quick and cost-effective measure
for surface cleanliness and hygiene control in hospitals and
routinely informs the cleaning practices of housekeeping and
healthcare staff.
217
More recently these assays have been used
to monitor the cleanliness of not just surfaces but surgical
instruments and dentures as well.
218,219
ATP bioluminescence measurements are routinely used
to monitor quality control and hygiene in the food industry.
212
In particular, fish processing plants have used it for decades to
determine contamination levels.
220
Recently a study was
reported to determine the contamination levels in various fish
processing environments i.e. different lines of production
including different fish types such as trout and cod, different
types of meat such as protein-rich loin meat or fat-rich belly
meat and different levels of processing such as slaughtered or
cooked products, and the results were compared against
conventional culture and plating techniques.
221
It was
established that it is essential to set up critical limits after a
period of validation and calibration that are specific to each
processing plant, type of ATP-bioluminescence kit used, specific
areas, types of fish and fish meat and different hygiene zones,
to obtain more robust, consistent and meaningful results.
The dairy industry also benefits greatly from ATP bioluminescence
assays as these are used to determine the quality of milk by
selectively measuring the ATP from somatic cells and milk
spoilage by determining ATP levels from bacteria and other
microorganisms both before and after UHT treatment to
estimate shelf life.
222
The lux operon which is responsible for bacterial bioluminescence
has also found great utility in the food industry.
214
The lux
genes responsible for bioluminescence can be genetically
encoded onto bacteria that are not naturally bioluminescent,
and the localisation, population size and environment of these
bacteria can be monitored in real time. As all known bioluminescent
bacteria are Gram-negative, there were initial challenges in obtaining
a good level of light output and gene expression in Gram positive
bacteria. However, this was overcome by introducing translational
Fig. 6 ATP-bioluminescence assays are used for hygiene control in several settings and industries including healthcare and food processing plants,
whereas Lux-tagged bacterial bioluminescence is used to develop improved hygiene practices in the food industry.
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signals optimized for Gram positive bacteria in front of luxA,
luxC and luxE genes.
223,224
To date bioluminescent E. coli and
Listeria have been used to monitor the survival of these bacteria
post-processing in yoghurt and cheese.
225
A number of
lux-tagged bacteria such as Campylobacter jejuni and Salmonella
enteritidis have also been used to determine egg-shell
penetration and colonisation.
225,226
Lux-tagged bacteria have
also been used to monitor the development of bacterial
infection in plant seedlings so interventions can be made at
the appropriate time.
227
The lux-based gene expression system
has also been fused to genes of bacterial toxin production such
as the promoter of the cereulide toxin gene ces in B. cereus to
determine the ability of various foods to support toxin
formation.
228
Lux-tagged bacteria have been administered to
mice for in vivo imaging of the resultant developing bacterial
infection – for example lux-tagged L. monocytogenes were shown
to grow and localise in the gallbladder of mice and cause
re-infection inthe intestines when bile was released.
229
However,
as bacterial bioluminescence emits predominantly blue light
which is strongly absorbed by blood and tissue, it is important
do ex vivo analysis of the organs as well to ensure bacterial
colonies are not missed. Another use of lux-tagged bacteria has
also been to detect biofilms and to develop cleaning methods
against them, as well as to test the efficacy of hand sanitisers
and disinfectants. As well as monitoring the growth and
development of pathogenic bacteria, lux-tagged probiotic
bacteria can also be monitored in foods that contain them as
well as tracking the bacteria using in vivo imaging to under-
stand their lifecycle and environment.
230,231
4.3 Mapping pollution in ecosystems
The most widely used bioluminescence sensors in the toxicology
monitoring of ecosystems are whole-cell bacterial bioluminescence
sensors.
232
Like most bioluminescence-based assays, these
sensors provide a quick result to help assess toxicity levels.
Other protocols for measuring environmental toxicity often
involve exposing test organisms, such as fish, crustaceans,
plants or bacteria to environmental samples and to monitor
survivorship. The benefit of bioluminescent bacteria is that
their light output can be used as a quick measure of survival.
Moreover, their bioluminescence is directly linked to their
respiratory chain and so any toxin that interferes with their
respiratory chain, interferes with the light output.
233
These
sensors have been used to monitor a wide variety of contaminants
including both heavy metal contaminants and organic
compounds such as toluene and naphthalene.
If the lux gene is expressed continuously, luciferase and luciferin
will be formed continuously and the baseline light intensity would
change on addition of the target analyte, depending on how well
the bacterial cell survives. Alternatively, the lux gene can be
controlled in an inducible manner wherein it would be fused to
a promoter that is regulated by the compound of interest. In this
case, the concentration of the compound can be quantitatively
detected by measuring the bioluminescence intensity.
118
Previously
bacterial bioluminescence sensors were reported to analyse a
variety of analytes including zinc, bioavailable toluene and
uranium.
234–236
Some recent examples in the development of
bacterial bioluminescence biosensors to detect various analytes
of ecotoxicology interest can be seen below (Table 7). Like most
assays, careful pre-treatment of the sample to eliminate inter-
ference causing agents is essential to get meaningful results.
The other common application of bioluminescence in ecotox-
icology and pollutant monitoring is ATP quantification using the
firefly bioluminescence ATP assayinbothaquaticenvironments
and bioaerosols in the atmosphere. ATP bioluminescence-based
sensors have been used to determine the total ATP in water bodies
including ocean environmentsanddrinkingwateruptoa
detection limit of 1.1 10
11
M.
244,245
This would include ATP
from not just bacteria but also fungal cells and any parasitic
protozoa. ATP bioluminescence-based sensors have also been
used to detect the location and density of several air-borne
bacteria in both artificially created and natural bioaerosols in
indoor environments.
246
The biosensors reported have either used
fabricated paper disks immobilised with luciferase/D-luciferin or
sensors microfluidic chips.
247,248
Air was vented into and bubbled
into a bio-sampler bottle containing 20 mL of deionised water to
capture any cells found in the air. This solution was then
concentrated and heated to lyse the cells. This lysate was then
dripped on the fabricated paper disks immobilised with luciferase/
D-luciferin. The fabricated paper disks with immobilised with
luciferase/D-luciferin were reported to have up to 10 times
longer shelf-life compared to the liquid assay reagents when
stored at room temperature.
247
Although this is a quick method
to identify air-borne bacteria and their levels in studies where
the identity of the bacterium is known i.e. artificially created
bioaerosols, it is important to calibrate the assay effectively
with known samples and use the ATP bioluminescence assay
together with another assay to validate the results.
249–251
4.4 Culture and heritage – preservation of art work
The ATP bioluminescence assay from the firefly has also been
adapted to take surface measurements of ATP from old artwork
Table 7 Recent examples of the use of bacterial bioluminescence biosensors to detect various analytes in ecotoxicology
Target Microorganisms Detection limit Ref.
Common antibiotics Bacillus WT and E. coli FhuAT 0.043–324 mg L
1
Jonkers et al.
237
Mercury P. leiognathi 9.87 mg L
1
Kassim et al.
238
Chlorine E. coli mutants 1 mg L
1
Borisover et al.
239
PyC
12
Phe (ionic liquid) Vibrio fischeri 4.17 mg L
1
Kahru et al.
240
Sucralose (sweetener) E. coli mutants 1gL
1
Harpaz et al.
241
Terbutryn (herbicide) Aliivibrio fischeri 81 mg L
1
Conrad et al.
242
Arsenite E. coli 39.6 mg L
1
Ginet et al.
243
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to estimate bacterial, fungal, yeast, algae and lichen levels in
antique work for the purposes of cultural and historic
preservation.
252
A bioluminescence low-light imaging technique
was reported that was used on artwork consisting of paper,
stone, fibre and wood.
253,254
All reagents were applied directly
to the samples and the conditions were optimised for sample
geometry and surface conditions. More recently, the level of the
microbial contamination of the seventeenth-century wall paintings
in the nave of the old Church of the Holy Ascension (Veliki
Krcimir, Serbia) was evaluated using the ATP bioluminescence
method, and traditional cultivation-based method, using dip
slides that were commercially available.
255
It was established that
ATP bioluminescence measurements can be a quick way to
determine ‘hot spots’ of contamination on the art-work, allowing
a quick assessment of areas that require greater concern.
4.5 Sensing of pH, metal ions, reactive oxygen species (ROS),
enzymes, drug molecules, and membrane potential including
in cellulo applications
As the light output in bioluminescent reactions is dependent
on the conditions of the assay in vitro or in vivo, it has been
modified and adapted to be able sense various parameters such
as pH, concentrations of metal ions, glucose, reactive oxygen
species, enzymes and drug molecules. These sensing applications
can use either modified luciferins such as caged luciferin
structures or modified luciferases that are conjugated with
sensing domains in a form of activity-based sensing (ABS) where
the optical signal output is dependent on the intrinsic chemical
activity of the bio-analyte in question with either the sensing
domain of the luciferase or the caged-luciferin.
Luciferin based sensors. In the area of luciferin based
sensors for specific sensing applications, caged luciferins are
the most common and well-known.
256
In a caged-luciferin, one
of the key functional groups, is masked in some way and then it
is modified or revealed by external conditions that are then
detected by bioluminescence. The masked key functional group
is often the electron-donating –NHR or OH at the 6-position of
the benzothiazole, but in some cases the carboxylic acid in
D-luciferin was the group that was masked. These are often
designed and used as turn-on probes in which the bioanalyte in
question uncages the luciferin and the light output is triggered
in the presence of the bioanalyte (Fig. 7). There are fewer
examples of caged-coelenterazine probes, probably due to the
fact that the auto-oxidation of coelenterazine analogues make
them more difficult to manipulate and handle synthetically.
The reported caged-CTZ probes usually have the C-3 carbonyl of
coelenterazine caged by the analyte of interest. Such probes
have been reported to measure b-galactosidase activity,
257
for
the detection of thiophenols,
258
and for the targeting and
detection of biothiols such as cysteine, glutathione and
Fig. 7 Caged luciferins – left hand side – a general scheme showing the 2 common sites of caging in red. Uncaging of the luciferin occurs in the
presence of the bioanalyte of interest and the uncaged luciferin reacts with luciferase to emit light. A turn-on response is obtained in the presence of the
analyte of interest. Right hand side – specific examples of caged-luciferin probes from the literature. Probe 51 was reported to measure b-galactosidase
activity
261
and probe 52 was reported to measure the activity of Fatty acid amide hydrolase.
262
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homocysteine.
259
A number of caged-CTZ probes were also
included as glycosidase inhibitors in a patent by the Promega
corporation.
260
Caged D-luciferin probes have been used as sensors for
enzyme activity,
262–269
small molecule sensors for molecules
such as glycans,
270
hydrogen sulphide,
271
hypochlorous acid,
272
carbon monoxide
273,274
and hydrogen peroxide,
275
and sensors
for metal ions such as copper,
276
iron,
277,278
and cobalt.
279
The
benefit of bioluminescence is that no incident light is needed and
so the signal to noise ratio is higher and therefore more accurate.
Although a potential drawback of these bioluminescence-based
probes compared to similar fluorescent probes is that the
bioluminescent probes are administered in much higher
concentrations in cell-based assays than fluorescent probes,
and this might affect the physiological conditions of the cells.
Nonetheless, several caged-luciferins have been successfully
used for in vivo imaging of mice and there has been an excellent
recent review covering the advances in this area.
280
Moreover,
the first example of a bioluminescent probe that can measure
mitochondrial membrane potential in a non-invasive manner
in vivo has just been reported to be a caged luciferin probe
that called a ‘mitochondria-activatable luciferin’ (MAL probe)
(Fig. 8). The MAL probe is uncaged by a bioorthogonal
Staudinger reaction with an organic azide (Azido-TPP1), to
release a functional luciferin, which will emit light in the
presence of luciferase. The triphenylphosphonium (TPP)
groups on both the organic azide and the caged luciferin directs
both reagents to the mitochondria. The rate of uncaging and
hence rate of formation of active luciferin is proportional to the
combined changes in mitochondrial membrane potential and
plasma membrane potential.
281
The other type of luciferin probes that have been reported
are designed to use the Bioluminescent Enzyme-Induced
Electron Transfer (BioLeT) process to modify the light output
generated. Bioluminescent Enzyme-Induced Electron Transfer
(BioLeT) is analogous to photoinduced electron transfer (PeT)
which has often been incorporated in the design of fluorescent
probes.
282,283
The design concept is that the singlet excited-
state oxyluciferin species can be quenched by the electron
transfer from the highest energy molecular orbital (HOMO) of
an electron rich benzene moiety in close proximity. This was
first reported in the design of a sensor for nitric oxide (NO),
which is very dim in the absence of NO, due to BioLeT, but
significantly brighter in the presence of NO, due to the absence
of the electron-donating moiety (Fig. 9).
284
This work also
reported the successful use of this probe in vivo mice models.
The authors have also reported another BioLet probe with turn-on
luminescence that detect highly reactive oxygen species.
285
Luciferase based sensors. A smaller number of modified
firefly luciferases have also been reported for the ratiometric
analysis and detection of pH, metal ions and reactive oxygen
species (ROS). Viviani and co-workers reported a technique to
estimate the intracellular pH in E. coli using mutant firefly
luciferases,
164
as well as a mutant firefly luciferase for ratio-
metric pH sensing and the selective detection of cadmium.
207
A genetically encoded pH sensitive reporter has also been
recently reported that consists of a pH-sensitive GFP (super-
ecliptic pHluorin) that emits at 510 nm, and a pH-stable lucifer-
ase Antares that emits at 580 nm. On the addition of
furimazine, a ratiometric readout R
580/510
was indicative of
pH and this system was shown to be functional both in vitro
and in vivo using xenograft murine tumours to detect
Fig. 8 The design and functioning workflow of the mitochondria-activatable luciferin probe.
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acidosis.
286
A genetically encoded calcium ion indicator (GECI)
was also reported in which Nanoluc was fused on the
N terminus of the Ca
2+
sensitive GCaMP6s protein. The blue
light from the reaction of Nanoluc with furimazine was used as
an excitation source for the fluorescent GCaMP6s protein. In the
presence of Ca
2+
, the GCaMP6s was in the correct conformation
to receive this blue excitation light and emit yellow-green light as
asignofCa
2+
ion signalling.
287
The Nano-lantern developed by Nagai et al. (Section 3.2)
were also developed further in the same piece of work to detect
ATP concentration.
170
A chimeric fusion protein of the Nano-
lantern with a subunit of bacterial F
o
F
1
-ATP synthase led to the
development of Nano-lantern (ATP1) which exhibited an
increase in light output on the addition of ATP with a K
d
of
0.3 mM. This was then used to visualise ATP formation in
chloroplasts.
170
A number of Nano-lantern based GECIs were
also developed by genetically engineering the Nano-lantern
probe with a calcium-sensing domain from an established
fluorescent Ca
2+
sensor to form Nano-lantern (Ca
2+
) which gave
comparable output and sensitivity to the fluorescent, genetically
encoded Ca
2+
sensor it was developed from.
170
Another important class of luciferase-based sensors are the
luciferase-based indicators of drugs (LUCIDs) developed by
Johnsson and co-workers.
288
These are semisynthetic biolumi-
nescent sensor proteins that consist of three components: a
receptor protein for the drug of interest covalently linked to a
luciferase (Nluc), which is linked to a self-labelling protein such
as SNAP-tag (Fig. 10A). The self-labelling protein was further
linked to a synthetic molecule that consists of a fluorophore
that can accept BRET from the luciferin–luciferase reaction and
a ligand for the receptor protein. Binding of the protein with
the ligand, lead to close proximity of the Nluc with the
fluorophore and red-shifted emission due to BRET. In the
presence of a drug molecule, this interaction is perturbed
and hence a measure of the ratio of blue light/red light leads
to a measure of drug concentration. LUCIDs were shown
capable of detecting both small-molecule drugs and larger
peptidic and macrocyclic drugs as well. The LUCIB and analytes
were spotted on filter paper and the light output measured
using a digital camera making them useful candidates for
point-of-care diagnostics. Later Johnsson and co-workers
reported the use of antibodies in place of the receptor protein
to make the technology more easily accessible.
289
A number of BRET-based antibody sensors have also been
reported. Merkx and co-workers reported a luminescent antibody
sensing (LUMABS) technology to detect antibodies in blood
plasma.
290
In these single protein sensors Nluc is connected to
a green fluorescent protein mNeonGreen via asemiflexible
linker and two antibody binding epitopes. A helper domain is
found on each protein that keep both them in close contact to
allow efficient BRET in the absence of an antibody (Fig. 10B).
When an antibody binds to the sensing domain, the close
proximity of the two light emitting proteins is disrupted leading
to loss of BRET. A measure of this signal allows a ratiometric
measure of antibody concentration. Initially this assay was
optimised to a 384 well plate and the light output measured
using a mobile phone. This technology has been further
developed to enable identification of non-peptide epitopes,
291
optimised to use as a microfluidic paper-based analytical
device,
292
and optimised further to require very small volumes
Fig. 9 The use of a BioLeT probe to detect the presence of nitric oxide.
285
The BioLeT donor has a high energy HOMO that is capable of donating an
electron to the singlet excited state oxyluciferin.
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of blood (B5mL) by depositing the biological machinery on
cotton threads.
293
As discussed earlier, whole cell bioluminescent bacterial
biosensors are widely used to detect heavy metal concentrations
such as mercury, zinc and chromium in ecotoxicology
studies.
294–296
For more details on this please refer to Section 4.3.
4.6 Gene assays
A number of gene and DNA based assays have been reported
that use a bioluminescence-based readout to detect various
analyte levels. For example, Christopoulos and co-workers
reported some of the earliest work in this area, utilising either
Aequorin as the photoprotein,
297
or firefly luciferase.
298
This
expression immunoassay used a Fluc coding DNA fragment as a
label, and reported the limit of detection of prostate specific
antigen (PSA) as low as 1 pM ((Fig. 11).
298
The antigen was
isolated on polystyrene plates pre-coated with a suitable
capture antibody. The captured antigen was then reacted with
a biotinylated antibody. The biotin tag was then able to capture a
streptavidin bound Fluc DNA tag. Transcription and translation
of the Fluc DNA template resulted in 12–14 Fluc luciferase
molecules per DNA tag bound to the plate. On addition of
D-luciferin, the light output was proportional to the number of
antigens bound. Recently, this limit of detection was improved
to 0.007 pM by improving the number of enzyme molecules
produced per DNA template by using a highly productive E. coli
extract-based cell-free protein synthesis system.
299
ADNA
hybridization assay based on similar principles, but to detect
DNA instead using Fluc bioluminescence was also reported by
Christopoulos and co-workers.
300
One of the most common applications of Fluc and Rluc
bioluminescence is their use as reporter genes for the study of
Fig. 10 (A) LUCID developed by Johnsson and co-workers. BRET between NLuc and the fluorophore is disrupted in the presence of a drug molecule. (B)
LUMABS developed by Merkx and co-workers. BRET between NLuc and mNeonGreen is disrupted in the presence of an antibody.
Fig. 11 The gene expression immunoassay for detection of prostate specific antigen (PSA) reported by Christopoulos et al.
264
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gene expression in prokaryotic and eukaryotic cells and
systems.
301–304
A notable recent and relevant example is the
use of luciferase based assays to determine the infectivity of
viruses such as the various coronaviruses in different host cell
types.
305,306
Such an assay was also used to establish that
SARS-Cov-2 coronavirus and a closely related RaTG13 corona-
virus that was found in bats can both successful infect human
cells to produce daughter viruses (Fig. 12).
307
The interaction
and binding of the spike glycoprotein in coronaviruses to the
cell receptor Angiotensin-converting enzyme 2 (ACE2) in
human cells is considered key in the entry of the viruses into
human host cells.
308
Plasmids containing the genes for Fluc
and the genes for the spike protein from the coronavirus strain
of interest were co-transfected into host cells and incubated for
72 h. The pseudoviruses formed after this period were collected
and these pseudoviruses would have the desired spike-protein
on their surface and the genetic material encoding for Fluc
inside of them. These pseudoviruses were then incubated with
ACE-2 expressing human cells for 60 h. If the viruses are
successfully able to infect the human cells, daughter viruses
and Fluc would be produced. On addition of D-luciferin, the
light output would be a measure of infectivity.
There has also been a report of the blue light from a
Nanoluc-furimazine reaction being used to activate a photo-
active LOV protein that in-turn uncages a transcription factor –
so in essence the light is used to regulate gene expression,
although this assay can also be used as a protein–protein
interaction assay.
309
In their assay, protein A is linked to a
light-activated LOV protein as well as a transcription factor
through a protease cleavage site and protein B is linked to
Nanoluc as well as a protease (tobacco etch virus protease
TEVp). When proteins A and B come into contace with each
other, and blue light is emitted from Nanoluc in the presence of
Furimazine, this light activates the LOV protein, which changes
conformation to present the protease cleavage site to the TEVp
protease. The protease works on the protease cleavage site and
releases the transcription factor, which then heads towards the
nucleus for transcription. In the work, the transcription factor
induces the transcription of the red fluorescent protein
mCherry. The readout of the assay is the result of this
transcription and hence the expression and fluorescence of
mCherry (Fig. 13). This is one of the few examples of light from
the bioluminescent reaction being used to control an effector
function.
4.7 Protein–protein interactions (PPIs)
One of the most established uses of bioluminescence-based
assays is in the interrogation, analysis and determination of
interactions between proteins. The two main types of technology
used in this area are the bioluminescence resonance energy
transfer assays (BRET) using the NanoBRET technology,
175,310,311
and split-luciferase systems.
181,312
BRET is based on the concept of Fo
¨rster resonance energy
transfer, which is a non-radiative energy transfer between two
luminescent molecules, an excited state donor that transfers its
energy to an acceptor which then emits light. The efficiency of
the energy transfer is dependent on the distance between the
donor and acceptor and their respective dipoles, which means
that for efficient energy transfer to occur the molecules must be
in close proximity to each other (1–10 nm),
313
and have the
correct orientation.
314
In BRET the donor is a photoprotein
such as Aequorin or a luciferin molecule such as coelenterazine
or furimazine, while the acceptor is often the green fluorescent
protein GFP, which emits green light. This circumvents the
problems with Fo
¨rster resonance energy transfer (FRET) with
fluorophores, such as the need for an external light source,
Fig. 12 A Fluc/D-luciferin based assay used to determine the infectivity of coronaviruses in different host cells – image shows process used for
SARS-CoV-2 in ref. 273.
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photobleaching of the donor fluorescent protein, simultaneous
excitation of both the donor and acceptor molecules and
autofluorescence in cells or animal models (Fig. 14).
Previously, BRET based assays were well established to study
various protein–protein interactions of interest such as oncol-
ogy based targets including p53/hDM2 as well as G-coupled
protein receptors both in vitro and in cellulo.
315,316
More
recently the inhibition or stabitisation of other PPIs of interest
has also been successfully detected using the NanoBRET
technology in which BRET from the bright Nanoluc to an
acceptor chromophore allows the determination of the proximity
and orientation of two proteins of interest. Examples of such
analysis include the localisation and conformation of viral HCV
NS5A protein,
317
analysis of the interaction between the PRAS40
and hippo pathway,
318
analysis of the CD26-ADA-A
2A
Rtrimeric
complex in cells,
319
the use of miniG proteins as probes for
GPCRs,
320
and analysis of the interaction between the H2 relaxin
protein and the RXFP1 protein.
321
With the development of
BRET acceptors that emit red light, NanoBRET technology has
also been used to measure protein–ligand interactions in vivo
mouse models of breast cancer to determine target
engagement.
322
For a more detailed review on the developments
in NanoBRET technology, please refer to the recently published
review on the topic.
311
The vast majority of BRET systems use the
Fig. 13 The use of bioluminescence to control gene expression – (i) When proteins A and B are distant, the LOV protein is deactivated and nothing
happens. (ii) When A and B are in close proximity and interacting in the correct orientation AND Nanoluc reacts with furimazine to release light, the LOV
protein gets activated and changes conformation to present the protease cleavage sit to the protease. (iii) After cleavage the transcription factor is free to
move to the nucleus and initiate transcription of the fluorescent protein mCherry.
Fig. 14 (A) When both protein partners are away from each other only blue luminescence is observed due to the NanoLuc reaction with furimazine. (B)
When both protein partners are in close proximity, resonance energy transfer from NanoLuc to the acceptor GFP chromophore, results in the emission of
yellow-green light from GFP. (C) In the presence of an inhibitor of the protein–protein interaction, both proteins are again at a distance and hence no
BRET is observed.
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Nanoluc/Furimazine combination as the energy donor as it is
significantly brighter than D-luciferin/Fluc and is also much a
smaller enzyme than Fluc, which makes tagging it on to proteins
of interest easier and more useful as it is unlikely to disturb the
protein’s natural state. However, this is of limited use for in vivo
imaging studies. Consequently, some efforts have been made
towards red-shifted BRET systems. In this regard, Fluc enzyme
mutants such as Ppy RE10 (l
max
617 nm with D-luciferin) has
been covalently labelled with nrIR fluorescent dyes such as
Alexa-Fluor680. This resulted in BRET emission of l
max
705 nm with an acceptor to donor emission ratio of 34.0
(Fig. 15).
323
Another interesting avenue has been BRET from a
luciferase/luciferin combination to an appropriate quantum dot.
Quantum dots are nanoparticles (diameters B2–10 nm)
composed of a semiconducting material with diameters in the
range of 2–10 nm. Due to their high surface-to-volume ratios they
demonstrate a number of interesting properties such as fluores-
cence. For example, NanoLuc was covalently linked to a polymer-
coated CdSe/ZnS core–shell quantum dot QD705 that emits at
l
max
705 nm. BRET from the Nanoluc/Furimazine reaction to the
quantum dot led to red-shifted emission at 705 nm and this was
used to image a tumour in mouse.
324
Nonetheless, the toxicity of
quantum dots is a cause of concern for many, particularly for
in vivo applications, leading to research into more biocompatible
quantum dots.
325
As brighter and red-shifted BRET systems are being
engineered,
184
and tagging technology is improving as well,
326
it can be reasonably expected that increasing numbers of in vivo
monitoring of protein–protein interactions and protein–ligand
interactions will emerge in the near future.
Split luciferase assays are also widely used to detect and
evaluate PPIs.
327
These have been based on various luciferases
including the firefly luciferase, click-beetle luciferase, Gaussia
luciferase and Renilla luciferases, which have all been used to
sensitively monitor dynamic PPIs with close to real-time
kinetics both in vitro and in vivo.
328,329
The luciferase is split
into 2 portions with one consisting of the N-terminal and the
other of the C-terminal domain. Each of these portions is
tagged to proteins of interest. On addition of a ligand, the
proteins of interest are brought together and both termini of
the luciferase come in close proximity to each other, hence
reconstituting the luciferase reporter function (Fig. 16).
Before the development of Nanoluc, Gaussia luciferase was
arguably the most suited for protein-fragment complementation
assays (PCA) as it is ATP independent, can be located in the extra-
cellular space, is shown to be brighter than Rluc as part of these
assays and the N-terminal and C-terminal enzyme fragments are
small in size. The first split Gaussia luciferase assay was reported
by Michnick et al. In their work, they interrogated the FRB/FKBP
protein–protein interaction, using rapamycin as a ligand to
induce complex formation, and FK506 as a competitive inhibitor.
The PPI dynamics were visualised both in vitro and in vivo.
330
Tao et al. reported the development of a split Gluc template
which was adapted to visualise the protein–protein interaction
of three different PPIs namely CaM/M13 with Ca
2+
ions behaving
as the ligand and the interactions of the ligand binding domains
of (LBD) of representative steroid hormone receptors such as
androgen receptor (AR), glucocorticoid receptor (GR), and
oestrogen receptor (ER) with various petide or small-molecule
ligands.
331
Others have reported PCAs based on Gluc for
visualisation of PPIs both in vitro and in vivo mouse models.
332
Recently, split Nano luciferase has been used to determine
protein–protein interactions in plant cells, wherein the PPI
between receptor kinase flagellin-sensitive 2 (FLS2) and plant
receptor kinase BAK1 (BRI1-associated receptor kinase 1) was
found to be induced by bacterial flg22 peptide through a split
Nanoluc system.
333
Split luciferase assays have also been used to
study viruses,
334,335
as well as to detect protein–protein aggregation
in human cells.
336
4.8 High-throughput screening
Bioluminescence based assays are routinely used in drug-
discovery programmes as part of high-throughput screens
particularly against infectious pathogens such as bacteria,
viruses or parasites. This is due to the ease in converting the
assays to the commonly used 96 well or 384 well plate format
and a quick, sensitive and straightforward readout of light
output, that is often a measure of living cells. The gene, protein,
enzyme or pathogen of interest are genetically encoded
together with the luciferase gene and then pathogen replication
along with simultaneous luciferase expression can be determined
inthepresenceofluciferinthrough the light output of the sample
both in the presence and absence of drug targets.
For example, Tan and co-workers reported the development
of high-throughput screening assay to identify inhibitors of
coronaviruses.337 In particular, they replaced the ns2 accessory
gene in the human coronanavirus strain HCoV-OC43 with the
gene for Renilla luciferase (Rluc) to form a functional reporter
virus strain rOC43-ns2DelRluc whose pathogenicity was unaltered.
This mutant virus strain was then used to infect cells, and Rluc
was expressed in the infected cells during viral replication.
On addition of coelenterazine, the light output was proportional
Fig. 15 BRET from the oxyluciferin in the reaction of D-luciferin/PpyRE10
Fluc mutant to the covalently linked Alexa-Fluor680 resulted in emission at
705 nm with an excellent BRET ratio.
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to the Rluc levels in the cells, which are a measure of viral
replication. On addition, of small-molecule compounds that
inhibit viral replication, a significant reduction in light-output
was observed.
338
This assay could be readily adapted in a 96-well
format (Fig. 17).
More recently, similar high-throughput assays have been
developed for screening for antibiotics in aquatic samples,
237
screening of antibacterial dental adhesives against mutants of
streptococcus,
339
and monitoring and inhibiting kinase
activity.
340,341
Both Fluc and Rluc have been extensively used in high-
throughput screening campaigns against large compound
libraries in both biochemical assays and cell-based assays.
Whilst these assays are extremely useful and still widely used,
it is important to note that they have some limitations. For
example, both Fluc and Rluc can be inhibited by various small
molecules. For example, Fluc in particular suffers from
competitive inhibition from compound classes that have
similar chemical structures to that of its substrate D-luciferin
including benzothiazoles, benzimidazoles, benoxazoles and
biaryl oxadiazoles. This can lead to false-positives in inhibitory
assays for these compounds. Moreover, some compounds can
also lead to increased trasnscription or translation of the
reporter enzyme leading to a false-negative result. A critical
discussion on the use of bioluminesce based assays is covered
in seminal reviews written by Inglese and co-workers.
342,343
4.9 In vivo imaging
The exceptional sensitivity and specificity afforded by bioluminescence
imaging at the molecular level has rendered it particularly
useful for in vivo imaging in small mammals.
344
Whilst fluorescent
probes suffer from photobleaching and background auto-
luminescence and radioactive tracers have a short shelf-life
and need a synchrotron source to be produced, bioluminescent
probes do not suffer these disadvantages. Moreover, developments
in charge couple devices allow the detection of low levels of light
output. With the advent of engineered, brighter, substrate specific
luciferases and red-shifted, synthetic luciferins, significant
Fig. 16 General principle behind split luciferases – on the addition of a ligand (L), the N and C terminals of the luciferase come together to give
functional reporter luciferase and a glow and readout on administering of luciferin.
Fig. 17 The high-throughput screening assay for coronavirus inhibitors
that uses the coronavirus mutant strain rOC43-ns2DelRluc developed by
Tan and co-workers.
302
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advances have been made in in vivo imaging, most of which have
been discussed in Section 3. Most often, these new, engineered,
bioluminesce systems are first trialled and used in the imaging of
developing tumours in cell studies and in vivo in mice. Although
initial studies described the monitoring of tumours and gene
expression subcutaneously, more recent work has focused on the
imaging of more-deep seated tumours and challenging targets such
as imaging in the brain of moving animals with novel red-shifted
and bright luciferin–luciferase pairs.
345
For bioluminescence in vivo imaging the cells of interest
are genetically modified to include the gene for luciferase
production. These cells can then be injected and tracked in
the body of the small mammal, when the respective luciferin is
added. Whilst, eukaryotic cells are often genetically tagged with
Fluc, Rluc or Nluc mutants, bacterial cells are genetically
modified to incorporate the lux codon responsible for bacterial
bioluminescence. The light output is then recorded using a
cooled charge-coupled device camera (Fig. 18). This allows the
monitoring of in vivo processes in real time, without the need to
sacrifice the animal.
Bioluminescence imaging has also been effectively used in
imaging the development of infectious diseases both in vitro
and in vivo.
347
Genetically modified bioluminescent pathogens,
such as bacteria, parasites, viruses and fungi have been
designed and monitored both in vivo and in vitro, in the
presence and absence of therapies to test their effectiveness.
The bioluminescent light output has been shown to correlate
with the infection load.
Some notable examples include the first real time visualisation
of the influenza virus in ferrets infected with A/California/04/2009
H1N1 virus (CA/09) encoding Nanoluc (Nluc) luciferase.
348
The replication and development of human coronavirus
strain HCoV-OC43 in the central nervous system of live mice
was achieved using an Rluc reporter and coelenterazine,
349
while another study reported the entry sites of encephalitis
viruses in the central nervous system of mice, using the Fluc/
D-luciferin system.
350
An engineered firefly luciferase was
also used with D-luciferin to monitor the development of a
Candida albicans fungal infection real time in mice using in vivo
imaging.
351
In vivo BLI is also the modality of choice when monitoring
the development of parasitic infections that cause neglected
tropical diseases such as those from Toxoplasma gondii,
352
Trypanosoma cruzi,
353,354
and Leishmania amazonesis.
355,356
The gene for firefly luciferase can be readily encoded into these
parasites and as bioluminescent output from the D-luciferin/
Fluc system is ATP dependent, only living parasites are
selectively imaged, which would not necessarily be the case in
fluorescence imaging. Moreover, as the imaging technique is
non-invasive, the diseased mice can be kept alive and
monitored over time whilst being administered different
treatments.
Bacterial infections such as Klebsiella pneumoniae,Citrobacter
rodentium and antibiotic resistance to them is also monitored
using BLI; however the bacterial lux operon is often used for
this.
357,358
The bacteria of interest are genetically encoded with
the lux operon for bacterial bioluminescence and then injected
into the mouse. The mouse is then treated with antibiotics and
imaged over time to visualise the development of a disease. This
technique has often been used to visualise the effectiveness of
photodynamic therapy (PDT) on the treatment of bacterial
infections such as surgical wounds, burns and lacerations as
an alternative to treatment with antibiotics to combat antibiotic
resistance. For example, dermal abrasions on mice infected with
bioluminescent methicillin-resistant S. aureus (MRSA) were
monitored while a treatment of PDT using a phthalocyanine
derivative and toluidine blue with red light was administered by
Hamblin and co-workers.
359
Although now BLI reporters can emit light up to a wave-
length of 750 nm, there is still much to be desired in terms of
brightness to achieve desirable outcomes in in vivo imaging.
Moreover, research into fluorescent probes has demonstrated
that light output in the NIR-II window (1000–1700 nm) has
significantly better penetration through blood and tissue than
light in the NIR-I window.
360
This is the next frontier in
bioluminescence in vivo imaging.
Fig. 18 The process of in vivo imaging – (a) the cells of interest are genetically encoded with genes for expressing the luciferase of interest. (b) The
luciferase expressing cells are injected in tot the mouse and incubated for a set period of time. (c) The respective luciferin is added, and the cells are
tracked in real-time in vivo. Image acquisition is carried out using a camera with a cooled charged-coupled device. (d) An image of the tracked cells is
obtained. This exemplar image is of the fungal cells of C. albicans, tagged with Fluc and imaged after the administration of D-luciferin. Image reused with
permission from Oxford University Press on behalf of Brock et al.
346
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4.10 Disease therapy using the light from bioluminescence
The light from bioluminescence has been used as a localised
and controlled light source for the excitation of photosensitisers
in photodynamic therapy. Photodynamic therapy makes use of
photosensitiser molecules that are activated by light and
generate reactive oxygen species (ROS) radicals, such as singlet
oxygen that can destroy protein and tissue. This cytotoxicity can
be advantageous in cases where cell death is required as a form
of therapy such as in infection or cancer.
Both the Fluc and Rluc systems have been used as genetically
encoded sources of light for photosensitisers. For example,
Theodossiou et al. reported that increased cell death was
observed when both the photosensitiser Rose Bengal and D-luciferin
were administered to a Fluc expressing cancer cell line, in the
absence of ambient light.
361
This work was contested by a later
report that reported that the increase in cell death was
insignificant when non-toxic levels of photosensitiser and
luciferin were used, and that the quantum yield of the light
output from the D-luciferin/Fluc reaction was the limiting
factor.
362
Another study by Lai et al. reported Rluc-
immobilized quantum dots-655 (QD-Rluc8) for bioluminescence
resonance energy transfer (BRET)-mediated PDT using the
photosensitiser Foscan
s
to target cancer cells both in vitro and
in vivo in mice.
363
Although the PDT did not completely eradicate
the tumour, it significantly delayed tumour growth, and it was
proposed that this method of low-light dosage, compared to
the use of external light, may cause lower inflammation and
unnecessary death of healthy tissue.
Yun and co-workers reported Rluc and rose-bengal conjugates
that generate singlet oxygen by bioluminescence resonance
energy transfer (BRET). In their work, they used bovine serum
albumin (BSA) as a central backbone and conjugated Rluc and
rose-bengal to the BSA to achieve the desired distance between
Rluc and Rose Bengal which allowed both moieties to be
functional, without quenching the emission from either of them
(Fig. 19). When coelenterazine was administered to this system,
evidence of cytotoxicity and oxidative stress was observed in
cells.
14,364
Unsurprisingly, the uptake of the large and bulky
conjugate into cells was poor and optimisation is required on
that front.
It is to be noted, that an improvement in the light output
efficiency of the bioluminescent reaction, as well as the BRET
efficiency between the coelenteramide and the photosensitiser
would improve the generation of singlet oxygen.
An alternative approach that circumvents the need for
cellular uptake of large and bulky biological conjugates could
be to use a nanoparticle to bring the luciferase and photo-
sensitiser in close proximity. This approach was taken by Wu et al.
and in their work they encapsulated Rose-Bengal in biodegradable
poly(lactic-co-glycolic acid) (PLGA) nanoparticles, which were then
conjugated with Fluc. In the presence of D-luciferin, effective PDT
and cancer cell death was observed both in vitro and in vivo in
mice suffering from cancer of the liver.
365
Although increasing numbers of studies with PDT activated
by BRET from bioluminescence are being reported, the scope
of the work is somewhat limited by amongst other factors,
the brightness of the luciferin/luciferase reaction. As brighter
luciferin/luciferase pairs are developed, it is hoped that this
area will also expand into new territories.
4.11 Effector applications
Whilst traditionally the light from bioluminescence assays was
primarily used for sensing applications such as the ones
discussed previously, more recently bioluminescence-based
technology has found avenues in effector technologies, wherein
the light from the bioluminescent reaction can be used to affect
or control other processes.
366
This work was initially inspired
by the relatively well-established field of optogenetics, where
external light sources are used to control photoactive proteins
and ion channels and hence cells such as neurones.
367,368
Light from a luciferin–luciferase reaction provides an attractive
possibility of localised and controlled light output of a wave-
length of choice, compared to external light sources that are
often harsh and can indiscriminately excite other endogenous
fluorophores causing damage and autoluminescence.
The earliest targets to be affected by bioluminescent light
output were channelrhodopsins. Channelrhodopsins are light-
sensitive ion channels that are naturally found in algae and are
responsible for their movement in response to light i.e.
phototaxis.
369,370
When expressed in neurons, rhodopsins
allow light to control the state of the ion-channel by either
opening it or closing it and hence the ability of the neuron to
fire action potentials.
371
The channelrhodopsins absorb blue
light (480 nm), so are ideal targets for Rluc,Gluc or Nluc.
372
The gene for channelrhodopsins can be encoded together with
Fig. 19 Figure showing the Rose-Bengal–BSA–Rluc conjugate.
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the gene for luciferase leading to a fusion protein consisting
of a channelrhodopsins with a luciferase. This led to the
development of ‘luminopsins’ which are now a class of opto-
genetic reporters, in which the addition of coelenterazine
luciferin and subsequent light output controls the state of the
ion-channel, and hence the ability of neurone cells to fire
action potentials.
17,373–376
The use of bioluminescent light to
control gene expression has been discussed in Section 4.6.
Bioluminescent light has also been used for novel photo-
uncaging reactions by Winssinger and co-workers. In 2018, they
reported a photo-uncaging using BRET from Nluc to a
ruthenium photocatalyst to release pyridinium species.
Further developments by this group in 2019 saw the first true
‘bioluminolysis’ wherein no photocatalyst was needed to
uncage drugs and molecules of interest using BRET from
Nanoluc-Halotag chimera protein (Hluc) to a coumarin
photocage (Fig. 20).
15,16
Photopharmacology including photodynamic therapy,
photouncaging and photoisomerism is an area of growing
interest in medicinal chemistry to develop and herness new
therapies.
377
Bioluminescence has potential to be of great use
here as a light source in vivo in close proximity to species of
interest. The fact that luciferases are usually genetically
encoded makes this a challenging endeavour in humans and
complex on several fronts. However, with the advent of luciferase
conjugated nanoparticles, this challenge in the delivery of the
enzyme to its desired site of action might soon be getting
addressed.
5. Blue-sky research and new horizons
5.1 Glowing plants
Bioluminescence imaging has found limited use in the imaging
of plant metabolites, proteins and physiology. The fact that
plants have chlorophyll rather than haemoglobin means that
they have a different and distinct optical window compared to
small mammals.
378
The four types of chlorophyll each absorb
light at two distinct wavelengths; the first lB450 nm and the
second lB650 nm.
379
Moreover, the circulation of water and
nutrients in plants is not as rapid as in small mammals, and so
the delivery of luciferin to the cells of interest is a challenge.
This limited the use of BLI to small seedlings or cell cultures
that could be grown in Petri dishes in the lab. Nonetheless,
despite these limitations, noteworthy work was done to develop
our understanding of the circadian clock using a fused,
genetically encoded Fluc reporter in a pioneering study by
Kay et al.
380,381
In their seminal work, Kay et al. fused the Fluc
gene with a gene for the promoter of the chlorophyll a-b
binding protein (cab2), which is a membrane protein in plant
cells and plays an important role in photosynthesis. The
Arabidopsis plants were grown on sterile culture plates and
sprayed with a solution of luciferin. Bioluminescence imaging
was used to visualise the organ localisation of cab2 during the
light and dark. In this study, based on the cyclical changes in
cab2 expression levels, the length of the circadian clock cycle of
the plants could be determined.
380
After this work, the authors
reported another study in which they used this methodology to
identify Arabidopsis mutants which have aberrant circadian
clock cycles, and tried to investigate which genes were responsible
for these changes.
381
To circumvent the problem of delivery of D-luciferin to the
cells of interest, it was thought that an autoluminescent plant
would be of more use. To this end, genetically modified plants
were produced, in which the bacterial lux operon would be
expressed in the plastids, but this failed to produce sufficient
light, partly due to the fact that bioluminescent bacteria emit
blue light, whilst chlorophyll absorbs strongly in that region.
Moreover, the expression of the bacterial bioluminescent system
was found to be toxic to the plant.
115
Since the recent discovery
of the fully genetically encoded bioluminesce pathway of fungal
bioluminescence in 2018,
126
this year two different groups
reported successfully creating genetically modified plants with
the fungal bioluminescence pathway genetically encoded into
them, making them autoluminescent.
128,382
This opens up a new
avenue for BLI in plant research as it has moved BLI in plants
from the laboratory Petri-dish to more real life examples grown
in soil. Moreover, it also opens the exciting avenue of using such
plants for ‘green’ lighting purposes in the future. Notably, in
their work Yampolsky and Sarkisyan et al. also reported an
autoluminescent mammalian cell line using the genes respon-
sible for fungal bioluminescence.
128
However, no attempt was
made to compare the brightness of the light output with that
obtained from the bacterial ilux operon.
Apart from genetically encoded autoluminescent systems, a
nanobionic light emitting plant has also been reported in the
Fig. 20 The use of bioluminolysis for the release of the photocaged small-molecule kinase inhibitor ibrutinib by BRET.
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literature. In this system, Fluc was conjugated into silica
nanoparticles (SNP-Luc) and D-luciferin was conjugated into
poly(lactic-co-glycolic acid) (PLGA-LH
2
) nanoparticles. Both
types of nanoparticles were slowly able to release their cargo
inside plant cells to allow the bioluminescence reaction to take
place and to give the plant a yellow-green glow.
18
However, a
pressurized bath infusion of nanoparticles was used to
administer the mixture of nanoparticles to the plant, which
makes this sort of a light emitting plant unsustainable.
5.2 Lifestyle
The natural phenomenon of bioluminescence has enthralled
people for centuries and the advent of modern technology has
allowed us to bring this natural beauty, from the wilderness to
the lab and finally to our homes and places of recreation.
Indeed, bioluminescent systems are now being considered as
green alternatives to outdoor lighting in public spaces in
architecture.
383
Moreover, a simple search online would lead
one to lamps based on bioluminescent dinoflagellate to
decorate the home. The French start-up company Glowee was
founded in 2014 and has used bioluminescent bacteria to
develop lamps as a form of sustainable lighting. Since it’s
initiation, the company has managed to increase the lifespan
of the bacteria, and hence the lamps from 3 days up to 1 month.
As the use of bioluminescent systems become more wide-
spread, they have inspired both scientists and artists alike
towards innovation and novel applications. There are a number
of examples of artists that are inspired by and actively use
bioluminescence in their work. Novel and rare art work known
as ‘living art’ has been produced using some bioluminescent
systems – namely bioluminescent bacteria and the bioluminescence
from single-celled dinoflagellate.
384
The bacterial bioluminescence
art work is done on Petri dishes and lasts up to 2 weeks, gradually
dying off and depicting different aspects of the art as the light fades.
Thus, these bioluminescent systems serve as tools for the artist’s
expression as well as starting points for science communication
(Fig. 21).
5.3 Limitations of bioluminescent systems
Although the light from bioluminescence has quickly reached
several unchartered territories, it is still limited from use in a
wider-array of applications by the fact that most of the probes
have poor quantum yields, with the quantum yield of D-luci-
ferin/Fluc at 0.41, being one the highest photon outputs from a
natural wild-type system. Although, engineered systems such as
Nluc/coelenterazine have higher photon outputs, the wave-
length of emission of these systems is not ideal for all potential
applications and the more-redshifted variants of Nluc are not
commercially available. Some of the natural and synthetic
luciferins also suffer from issues arising due to poor stability,
cell-compatibility and bioavailability. The development of
complementary luciferin and luciferase pairs with improved
properties is an active area of research, and with further
advances, including the cloning and understanding of auto-
luminescent systems such as that of the fungi, will assuredly
lead to more novel applications.
6. Conclusions
Research in bioluminescence and bioluminescent organisms
has made phenomenal progress over time. From early discovery
research into undiscovered luciferins and luciferases, to new
Fig. 21 Art from bioluminescent creatures – (A) ‘flow visualisation’ by
Iyvone Khoo made using bioluminescent dinoflagellate, www.iyvonekhoo.
co.uk. (B) ‘Rabbit: stage 1’ photograph of drawing created with
bioluminescent bacteria, Hunter Cole, www.HunterCole.org.
This journal is The Royal Society of Chemistry 2021 Chem. Soc. Rev.
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Open Access Article. Published on 18 March 2021. Downloaded on 3/24/2021 9:09:19 PM.
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applications for known bioluminescent reporters, designer
luciferins and luciferases, and other developing technology
that assists these systems, a lot of great work has helped
develop and progress the field. Moreover, enzyme engineering,
biophotonics, computational chemistry and synthetic chemistry
are rapidly evolving fields as well and it can be reasonably
expected that developments in these areas will feed into the
research and applications of bioluminescence in biotechnology.
As bioluminescence has found utility in various fields including
medicine, biology, physics and engineering and led to exciting
multidisciplinary science across all of them, we hope this review,
that briefly details the origins and mechanisms of bioluminescence,
the currently available luciferin and luciferase systems and
covers extensively the key and recent applications of
bioluminescence in biotechnology across various fields and
makes them accessible to chemists in particular will stimulate
further exciting research and foster collaborations in the
community.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors would like to thank the artists Iyvonne Khoo and
Hunter Cole for sharing their work with us.
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T. A. Karataeva, K. A. Palkina, E. S. Shakhova,
Chem.Soc.Rev. This journal is The Royal Society of Chemistry 2021
Review Article Chem Soc Rev
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