1
Hydrogel e
ncapsulation of a
designed
fluorescent
protein
biosensor
for
continuous measurements
of
sub
-
100 nanomolar
nicotine
Aaron L. Nichols
1†
, Christopher B. Marotta
2†
, Daniel
A.
Wagenaar
1
, Stephen L. Mayo
1
, Dennis A. Dougherty
2
, Henry A.
Lester
1*
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91106
.
2
Division of
Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91106
Keywords:
iDrugSnFR, iNicSnFR12, nicotine, hydrogel, biosensor, continuous monitoring
ABSTRACT
:
The reinforcing and addictive properties of nicotine result from concentration-
and time
-dependent
activation
,
desensitization,
and upregulation of nicotinic acetylcholine receptors. However,
time
-resolved
[nicotine
] measurement
in peo-
ple who consume nicotine is challenging, as current approaches are expensive, invasive, tedious, and discontinuous.
To address
the challenge of continuous nicotine monitoring in human biofluids, we report the encapsulation of a purified
, previously
developed
fluorescent biosen
sor
protein
, iNicSnFR12
, into acrylamide hydrogels
and
polyethylene
glycol
diacrylate (
PEGDA
)
hydrogels.
We optimized
the
hydrogels
for
optical clarity and straightforward
slicing
. With fluorescence
photometry
of the
hydrogels in a
microscope and a
n integrated
miniscope,
[nicotine
] is detected
within a few min at
the smoking-
and vaping-
relevant level of
10
- 100 nM
(1.62
– 16.2 ng/ml), even in a 250 μ
m thick hydrogel at the end of 400 μ
m dia multimode fiber
optic
. Concentration-
response relations are consistent with previous measurements on isolated iNicSnFR12. Leaching of iN-
icSnFR12 from the hydrogel and inactivation of iNicSnFR
12
are
minimal for several days, and
nicotine
can be detected
for at
least
10 months
after casting
. This
work provides the
molecular, photophysical, and mechanical
bases for
personal, wearable
continuous [nicotine] monitoring,
with
straightforward extensions
to
existing, homologous “iDrugSnFR” proteins for other
abused and prescribed drugs
.
A
billion
persons
smoke nicotine
daily
, and an estimated 60% would like to quit. It has long been appreciated that the time
course of [nicotine] near nicotine acetylcholine receptors (nAChRs), which we term
[nicotine]
t
, partially
determines
both nic-
otine addiction and successful nicotine replacement therapy
.
1-5
A wearable continuous nicotine monitor would serve in research on four topics
that involve measur
ing
[nicotine]
t
. (1) Re-
searchers wish to “know the enemy”, the ways in which
[nicotine]
t
varies among individuals
who smoke, vape, and use oral
nicotine products
. (2) It will also be necessary to “know the therapy”. FDA
-approved nicotine replacement therapy (NRT) may
soon be enhanced by trials of prescription mesh nebulizers; and it is necessary to study how
[nicotine]
t
correlates with success
in smoking cessation. (3) The research community would like to “know the physiology”: by measuring
[nicotine]
t
, researchers
can corr
elate the contributions of activation, desensitization, and upregulation
of nAChRs
to reinforcement, withdrawal, and
addiction. (4) A wearable continuous nicotine monitor will produce data during
ad libitum
nicotine ingestion
.
Genetically encoded fluorescent
biosensors
pre
sent
one potential strategy
for the generation of a continuous nicotine
monitor
in animals and re
duc
ed systems.
To date, biosensors have been used to study endogenous molecules
such as serotonin, GABA,
glutamate,
and
dopamine
, as well as
abused and prescribed drugs –
including nicotinic agonists, opioids, rapidly acting anti-
depressants, and selective-
serotonin reuptake inhibitors (SSRIs).
6-16
G
enetically encoded biosensors
have been utilized in
mammalian cell culture, primary neuronal culture, and model organisms,
via transfection
and
viral transduction
.
Related techniques for gene transfer cannot
be used in humans for pharmacokinetic monitoring of drugs
. Instead, an
approach-
able
tactic
may be to build
a continuous fluorescent
monitor
by
entrap
ping
a soluble fluorescent
biosensor protein
into a trans-
parent
hydrogel
. Hydrogels have been
extensively studied for tissue engineering, biomimetics, biocatalytic scaffolds
, and even
a continuous glucose monitor (
CGM
).
17-19
Non
-degrading hydrogels provide excellent parameters for a medical device –
providing
an aqueous environment that allows small molecule diffusion into a
network, while simultaneously excluding
larger
biomolecule diffusion.
A successful hydrogel network restricts off-
target
binding,
limits chemical interfer
ence
, and reduces
protein degradation, while also slowing
diffus
ion of an
introduced protein out of the hydrogel
.
20
Encapsulating
target proteins
.
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2
during hydrogel formation allows for the design of needed structures through mold casting. In addition, hydrogel synthesis
utilizing photopolymerization
allow
s 3D printing of the desired structures for optimal device designs
.
21
This flexibility allows
device
construction
around the excitation and emission
needed for fluorescence detection.
Dermal i
nterstitial fluid (ISF)
can be probed with minimally invasive techniques. ISF
[nicotine]
t
, is expected to closely resem-
ble
[nicotine]
t
, in blood and in cerebrospinal fluid
3, 22
. The highest [
nicotine
] in the
se
three compartments
occurs during the
bolus of nicotine from puff
ing
on a cigarette/vape, ~ 100 nM
. This phase largely governs the reinforcing effects
of nicotine
.
23
When the smoking / vaping session
ends,
[nicotine]
t
partially declines within a few minutes Then a
prolonged, declining phase
begins, with a time constant of 2 –
4 h. Th
is taper
ing
[nicotine]
t
suppresses withdrawal symptoms and maintains the cellular
biological aspects of addiction
.
24
While we know the average
[nicotine]
t
for various modes of nicotine ingestion, individual
variability in nicotine processing is considerable
.
23, 25, 26
Two methods currently provide “
gold
standard” measurements of
[nicotine]
t
. 1)
Intravenous (IV)
blood draws provide instan-
taneous
time point measurements of nicotine concentration in the blood,
but
each IV sample
is costly and tedious to execute
and process.
27
2)
Positron emission tomography
has better time resolution (a few s) but also comes with a
high reagent and
machine cost.
28
While some i
ndirect, proxy measures for estimating nicotine
concentration
in
biofluids exist, such as the nicotine metabolite
ratio
(NMR)
, these measurements cannot directly
capture
[nicotine]
t
.
29, 30
Electrochemical nicotine sensors based on nicotine
-
oxidizing enzymes lack the molecular amplification of fluorescence and are, therefore, 20
- 100
-fold less sensitive than fluo-
rescence.
31-33
To date, electrochemical measurements have been tested only on sweat
.
31-33
which is less desirable than intersti-
tial fluid because of inherent time delays and acid trapping of nicotine
.
34
We
report
in vitro
continuous
fluorescence measurements
of
[nicotine]
t
with the
most evolved nicotine biosensor, iN-
icSnFR12
,
35
encapsulated in a hydrogel. We adapted techniques from measurements using genetically encode
d fluorescent
biosensors
on intact rodents (a miniature integrated fluorescence microscope, fiber photometry) and
brain slices (vibratome
slicing
, immobilization by a harp
). This work provides a first step toward the employment
of
designed fluorescent biosensors
for personalized monitoring of
[nicotine]
t
in human
ISF.
Experimental
Hydrogel formation
Acrylamide hydrogels
A 500 mM, pH 7.5 solution of TEMED was generated for acrylamide hydrogel reactions
. 4 mmol N,N dimethylacrylamide
monomer was crosslinked with 1 mmol tetra(ethylene glycol) diacrylate using a 0.025 molar ratio of TEMED as a co
-initiator
and 0.0025 molar ratio ammonium persulfate (APS) as a radical initiator, in the presence of 0.2x PBS, pH 7.4 and 0.5–
10 μM
final concentration of biosensor protein.
To ensure proper mixing and stability of biosensor protein stepwise addition of the components to a glass vial was as follows
:
Deionized (DI) water, monomer, crosslinker, TEMED, 1x PBS, pH 7.4, and biosensor protein. The vial was sealed with a
rubber stopper and the solution was sparged with argon
for ~30 s. The vial was cooled in an ice bath for 5 min, the rubber
stopper was removed, the APS was rapidly added, the rubber stopper was replaced, the vial was gently swirled and placed
immediately back in the ice bath for 1
-2 h. The vial was then held at
4 ⁰C for 1
-2 h. Next,
the vial was uncapped,
and the
formed hydrogel was rinsed with DI water. The vial was cracked to remove the hydrogel
puck. The puck was washed twice
using 50 mL of 1X PBS, pH 7.4 in a 50 mL conical tube
for 1
-2 h. The
puck was then
washed overnight in 50 mL of 1X PBS,
pH 7.4.
PEGDA
/ Irgacure
hydrogel
s
A 100 mg/mL stock of Irgacure 2959 (2-
hydroxy-
4′
-(2-hydroxyethoxy)
-2-
methylpropiophenone)) was solvated in ethanol/wa-
ter as a 70% v/v mix. Poly(ethylene glycol) diacrylate (PEGDA) (M
n
700) and Irgacure 2959 stock were mixed in 1X PBS,
pH 7.4 at 46% w/w and 4.4% w/w. The resulting mixture was combined in a 1:1
ratio with 20 μM iNicSnFR12 solution to a
final concentration of 23% PEGDA, 2.2% Irgacure 2959, 10 μM iNicSnFR12 in 0.97X PBS, pH 7.4, 3% ethanol.
21
The mixture
was irradiated on ice at 100% power using a 365 nm, 1350 mW LED, 1700 mA for 10 mins. The hydrogel puck was washed
twice in
50 mL of 1X PBS, pH 7.4 in a 50 mL conical tube
for 1
-2 h. T
he puck was then
washed overnight in 50 mL of 1X
PBS, pH 7.4
.
The addition of laponite (2.5%)
21
, rat collagen type I (50%), hyaluronic acid (4%), or
bovine gelatin (5%)
were tested
as
thickening agents for PEGDIrgacure formation. These agents gave sub
optimal results, including mottled translucence
and
insufficiently hard hydrogels
, and were consequently abandoned.
Hydrogel leaching tests
To test leaching of the biosensor from acrylamide and PEGDA
/Irgacure hydrogels, the 50 mL wash was concentrated to ~300
μ
L and measured on a Spark
M20
96-
well fluorescence plate reader (Tecan), with excitation at 485 nm and emission at 535
nm. Only background fluorescence was observed
Epifluorescence
imaging of hydrogel
s
.
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Acrylamide hydrogels could be manually chipped to uneven
slice
s at 0.5
-
1.0 μ
m
thick. PEGDA
-Irgacure hydrogels were sliced
at
250 μ
m
using
a Leica VT 1000S Vibratome
with a razor blade. Hydrogels were immobilized using manually placed weighted
wires and/or “
harps
” usually intended to immobilize brain slices.
Time
-resolved concentration–
response imaging was performed as described
36
on a modified Olympus IX
-81 microscope
(wide
-field epifluorescence mode using a 10× lens
,
λ
ex
= 470 nm λ
em
= 535
). Images were acquired at 1 frame/s with a back
-
illuminated
electron
multi
plier
CCD camera (iXon DU
-897, Andor Technology) controlled by Andor IQ3 software.
Solutions were delivered from elevated reservoirs
36, 37
by gravity flow via solenoid valves, providing solution changes in the
imaging chamber with a time constant < 10 s. The vehicle was 1x PBS, pH 7.4. Data analysis procedures included subtraction
of blank (
non
-hydrogel
) areas and corrections for baseline drifts using OriginPro 2018 software.
Displayed
time series data
were smoothed by moving averages over 30 s.
Miniscope imaging of hydrogel
s
Using the open-
source
instructions
, a v4.4 UCLA fluorescent miniscope
kit
was assembled.
38, 39
The elevated reservoirs
and
gravity flow setup described above were adapted so
that the miniscope
functioned as an
inverted
fluorescent
fiber photometer
.
We
3- D printed
an adapter to
hold both the miniscope and
the ferrule of a Thorlabs fiber optic (400
μ
m
dia
, 0.39 NA, 10 mm
length).
The adapter was held by a micromanipulator rod that usually holds an electrophysiology headstage. The instrument
end of the fiber optic was placed at the focus of the miniscope objective lens, and
the entire image of the fiber optic was
averaged
for the data shown.
The near
-tissue end of the fiber optic was
flush with the bottom of a
160
μ
m
cover slip
. Atop the
cover
slip, we placed a vibr
atome
-sliced hydrogel immobilized with a harp.
Images were collected at the slowest available rate
for the miniscope, 10
/s. Except for Figure 3B
below
, displayed time series data were smoothed by moving averages over 30
s.
Results and Discussion
Generation of hydrogels compatible with bacterially expressed and purified
fluorescent proteins
Our first generation of hydrogels was
composed of iNicSnFR12 Q368C
( a nicotine biosensor with a point mutation as a chem-
ical handle of iNicSnFR12)
35
encased in polymerized acrylamide at a final concentration of 2 μM. These hydrogels were brittle
and
fragile
, requiring manual chipping to produce samples for use in wide
-field epifluorescence experiments. The acrylamide
hydrogel showed an even distribution of biosensor throughout the hydrogel, with no detectable puncta. (
Figure 1A
).
We performed time
-r
esolved
concentration
-response relations with nicotine on the
hydrogel slices. We observed a robust flu-
orescent response to nicotine across a range of [nicotine]
> 100 nM (
Figure1B
-1C)
. The observed wash-
in and washout kinetics
characteristics were slower than the solution changes and were several fold slower than previous results with iNicSnFRs
in
mammalian cell culture and primary mouse hippocampal neurons.
13, 35, 37
Repeated applications of nicotine to the acrylamide
hydrogel showed minimal rundown, with a fluorescent decrease of 7-
8% over the course of a 75 mi
n experiment.(
Figure 1D
).
For all dose response relations
, we observed slower wash
-in and washout kinetics for the interior of the hydrogel versus more
solvent
- exposed
regions, suggesting that diffusion limited the access of the nicotine to the sensor protein. (
Supporting
Figure 1
.
iNicSnFR12
-Q368C in acryla-
mide hydrogels. (A)
Widefield
image of acrylamide hydrogel.
The dark line is a weighted wire
.
Scale bar, 50
μ
m.
(B, C
). Time
-re-
solved concentration
-response
relations over a [nicotine] range
from 100 nM to 100
μM in two
exemplar hydrogels. Wash-in
and washout kinetics are slower
than solution changes; see text
for analysis in terms of diffusion.
(D) In another gel, repeated ap-
plications of the same dose of
nicotine show a robust response
over 75 min.
.
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Information:
Analysis of solvent proximity to fluorescent response of iNicSnFR12 acrylamide hydrogel
) The fluorescent re-
sponse of iNcSnFR12 Q368C in concentration
-response relations with nicotine indicated
functional protein
, which demon-
strated that a chemical handle would not be n
eeded to stably encase iNicSnFR12 in the
hydrogel. Consequently, subsequent
hydrogel experiments used iN
icSnFR12
itself.
PEGDA/Irgacure hydrogels
While
the initial data for polyacrylamide gels show
ed that
iNicSnFR12
biosensor was functional in a hydrogel, it seemed
possible to improve on the
formulation
in
three
ways: 1) A
less toxic hydrogel matrix than acrylamide would be better suited
for use in biological systems
; 2) I mproved mechanical properties would
allow
more robust
manipulation and handling;
and
3)
Gel
-like consistency would allow systemat
ic shaping and slicing.
To accomplish these goals
, we
investigated
a PEGDA/Irgacure hydrogel formulation.
21
After experimenting with the addition
of thickening agents (including laponite, rat collagen Type 1, hyaluronic acid, and bovine gelatin), we determined that a
PEGDA/Irgacure iNicSnFR12
-containing hydrogel with no thickening agents provided the best
clarity
and flexibility
.
W
e cast PEGDA/Irgacure hydrogels at concentrations between 0.1 μM and 50 μM iNicSnFR12. Hydrogels with iNicSnFR12
concentration <10 μM provide
d less fluorescen
ce
than desired for accurate nicotine detection, while increasing [
iNicSnFR12
]
above 10 μM did not markedly increase
the
detectable signal. Additionally, increasing the biosensor concentration to 50 μM
resulted in fluorescent puncta, rather than a
homogeneous
distribution of fluorescence. (
Supporting Information: Widefield
imaging of varied PEGDA hydr
ogel preparations
).
Therefore, subsequent
experiments used
10 μM iNicSnFR12 in the
PEGDA/Irgacure hydrogels
.
Vibratome-
sliced PEGDA hydrogels
To obtain reproducible hydrogel thickness, we
adapted brain slicing techniques to the
PEGDA/Irgacure hydrogel
: we
obtained
250 μm thick slices using a
vibrating microtome (“vibratome”)
(
Figure 2A)
. Slices generated in this fashion had minimal
opacity and exhibited homogeneous
distribution of
fluorescence (
Figure 2B
). Concentration
-
response relations with 250 μm
thick slices showed a robust fluorescent response of iNicSnFR12 to nicotine across a range of concentrations
.
Concentration
dependence of nicotine
-induced fluorescence
In each of
four
slices tested (
examples in
Figure 2C, D
), we observed
responses at [nicotine] as low as10 nM
. The 10 nM
waveforms were noisy and distorted by mechanical artifacts, small changes in local pH, and other idiosyncrasies of the iN-
icSnFR series noted in previous papers
.
13, 15
Qualitatively, the 10
-fold scaled 10 nM waveforms in Figure 3
C,D
are comparable
in size
to the 100 nM waveforms, consistent
with the observed Hill coefficient of 1 for the iNicSnFR series
.
13, 15, 35
The 1
μ
M responses in
Figures 2
C,D
are
7.1
-fold
larger
than the 100 nM responses, markedly
less than the 10-
fold of a linear
relationship. This suggests
that 1
μ
M is an appreciable fraction of the EC
50
for the i
Nic
FR12
-nicotine dose
-response relation.
In a previous study, the EC
50
was 6 –
9
μ
M; this parameter may vary by several fold with small changes in pH, ionic strength,
and temperature
,
15
and perhaps the hydrogel environment.
Figure 2
.
Experiments with iNicSnFR12
PEGDA hydrogels, 250
μ
m
thick. (A) Photograph of hy-
drogels in a 35 mm dish. Hy-
drogel slices have optical clar-
ity. (B) Widefield fluorescence
image. Scale bar, 50
μ
m. (C)
Time
-resolved concentration
-
response relations to nicotine,
10 nM, 100 nM, and 1
μ
M.
Wash
-in and washout kinetics
are slower than solution
changes; see text for analysis.
(D) Ten months after casting;
storage at 4
°
C. The iN-
icSnFR12 PEGDA hydrogel
shows a reduced, but still
measurable response, In C and
D, the blue trace shows the 10
nM response, magnified 10
-
fold vertically.
.
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Semiquantitative
Analysis of diffusion
The rise times (10% -
90%) of the responses at 1
μ
M nicotine were 5 –
7 min
(Figure 2
C, D
). The experimental chamber
changed solutions within
10 s, much faster than the
se rise and fall times. In stopped
-flow measurements, the iNicSnFR series
responds to jumps of [nicotine] within 1 s
.
13, 15
The
refore, we present a semiquantitative analysis of the PEGDA hydrogel
waveforms based on diffusion properties
.
40
As
expected
for a diffusion
process
, the rise and decay waveforms
were the sum
of several exponential terms.
We assumed that nicotine undergoes diffusion within the hy-
drogel
plane of uniform thickness
x
= 250
μ
m.
We assume
d
that
[nicotine] at the upper surface of the hydrogel
is jumped
instantaneously to the level in the perfusate,
and the lower
surface, at the cover slip,
is closed to access from the solu-
tion.
We also assume
d that the free solution diffusion con-
stant
for nicotine
,
D,
equals
0.5
μ
m
2
/ ms
, typical of low
-
MW
alkaloids
.
41
We assumed that
the
effective diffusion
constant
D
eff
=
D/ab
, where
a
= 1.5
for diffusion within the
restricted
space of the hydrogel
. The factor
b
represents
re-
binding to the iNicSnFR
molecules and equals K
d
/ ([iN-
icSnFR12] +
K
d)
). Assuming that the K
d
equals the previ-
ously measured
35
EC
50
,
b
=
2.5
. The
refore,
D
eff
equals
0.13
μ
m
2
/ ms. The average diffusion time
t
is, there-
fore
,
t = x
2
/2D
eff
=
6
min, in rough agreement with the ob-
servations.
This estimate should be considered approximate
in view of the uncertain
param
eters
; nonetheless, diffusion
within the hydrogel seems to
dominate the waveform of the
fluorescence responses.
Minimal fluorescence from the hydrogel
In addition to advantages of the hydrogel strategy stated in
the Introduction, the optical isolation from surrounding tis-
sues and proteins leads to the hope that tissue contributions
to F
0
will be minimal. In measurements not shown, we found
that hydrogel fluorescence without introduced iNicSnFR12
was roughly 1/10 the F
0
value of a hydrogel cast with 10
μ
M
iNicSnFR12. This may allow absolute calibration of
[nico-
tine]
t
from measurements of
∆
F/F
0
.
Measurements after storage
To test the rate of biosensor release from
the
hydrogel
, we
incubated freshly crosslinked iNicSnFR12
PEGDA/Irgacure hydrogel in 50 mL of 1X PBS, pH 7.4,
for
three d
ays
while shaking. After the incubation, we concen-
trated the 50 mL of 1X PBS, pH 7.4 to ~300 μL and we de-
tected no significant fluorescence
above background (Data
not shown).
As an extreme test
of
durability
, we
stored
250 μm slices in
1X PBS, pH7.4
at 4
°
C
for ~10 months
(312 d)
. Values o
f
F
0
were 2
-3 fold lower than for the fresh hydrogel, and nic-
otine
sensitivity
(
∆
F/F
0
) was
~ 2- fold
lower than one
day af-
ter slicing (Figure 2D
). We
performed further experiments
on the storage solution
with a fluorescence plate reader. The
F
0
measurements
indicated that ~ 50% of the iNicSnFR12
had diffused from the gel to the storage
fluid
. In further
measurement on the iNicSnFR in the storage fluid, w
e as-
sessed
function by measuring fluorescence excitation spec-
tra
11
. The ratio between fluorescence excited at 405 nm a
nd
485 nM
was
~1 for fresh solutions of iNicSnFR12 and ~
0. 75
for the s
amples stored at 4
°
C, also suggest
ing
a modest de-
crease in iNicSnFR12 function
during 10 m
onths of storage
(Supporting Information: Excitation scans of iNicSnFR12
Figure 3
.
Fiber photometry of iNicSnFR12 PEGDA hydrogel using an inte-
grated miniature fluorescence microscope (miniscope). (A)
Schematic. The miniscope is inverted from its usual position on
a rodent head. Nicotine solutions are applied through the inlet
tube and re
moved by suction through the outlet tube. A harp im-
mobilizes a 250
μ
m PEGDA hydrogel slice. (B) iNicSnFR12
PEGDA hydrogel detects [nicotine] as low as 10 nM. BC, zero
-
nicotine buffer control, showing artifact due to solution
changes. (C) Smoothed miniscope
data using adjacent averaging
of 300 frames (30 s).
.
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;
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6
and 250 um iNicSnFR12 PEGDA hydrogel storage fluid).
However, the research uses stated in the
Introduction may require
only 1
-2 days per subject.
Measurements with an integrated miniature fluores
cence microscope
We tested whether
the
iNicSnFR12 PEGDA/Irgacure hydrogel could be measured with
the v4.4 version of the UCLA inte-
grated
miniaturized
fluorescence
microscope
(Figure 3
). The test rig operated
the miniscope as an inverted fiber photometer
while a
n iNicSnFR12 PEGDA/Irgacure hydrogel
slice was perfus
ed with various [nicotine]
. We
observed dose
-dependent
nicotine
-induced fluorescence
at [nicotine]
as low as 10 nM.
The 10 nM responses may be distorted by slight movements of
the hydrogel.
The miniscope plus fiber optic costs ~ $2500 US as a kit, roughly 1% of
the original cost of the fluorescent microscope that
provided the data in Figures 1 and 2.
The miniscope provided additional, inexpensive, off
-the
-shelf proof of concept for the
methods.
However,
its cellphone camera has poorly characterized linearity of responses, which vitiates systematic analysis of
the concentration dependence
; and as designed,
its image rate cannot be slowed
below
the
wasteful 10 Hz. Also, t
o avoid
obvious artifacts from overheating, we decreased the LED power to 20% of maximum. Importantly
, the miniscope’s LED,
excitation filter, dichroic mirror, and emission filter use the same epifluorescence technology as the fluorescent microscope;
and these sh
ould be retained in a future, more appropriate, and even
less costly specialized miniature fiber photometer.
Biofluid inter
actions
with iNicSnFR
12
In previous reports
using
fluorescent plate readers
, iNicSnFR12 and its predecessors responded to acetylcholine, varenicline,
and choline (the latter 100-
fold less strongly).
35
We explained how these low
-MW compounds are unlikely to interfere with
[nicotine]
t
measurements.
35
Dermal
ISF cannot yet be isolated in sufficient quantities to serve in a systematic characterization of iNicSnFR12 hydrogels
42
; therefore, we continue
d experiments with fluorescent plate readers.
Previously, t
he presence of 25% human serum
itself
increased background fluorescence
35
, which we term
ed F
0’
’. Human serum also apparently contained
then
-unidentified
com-
pound(s) that activated iNicSnFR12.
35
With added human serum, the lower limit of quantification increased by 2-
3 fold. It
was previously not possible to distinguish whether this altered sensitivity arose primarily from the increase
d F
0
’ or from the
background activation of iNicSnFR12 by the endogenous compound(s).
To probe
these interactions
further, we have tested individual component
s of
human biofluids. Because we evolved the binding
moiety of all iDrugSnFRs including iNicSnFR from a choline
/betaine
-binding binding, and based on our experience with
ligands for this series,
11-15, 44
, we selected 9 additional
biogenic amines or alkaloids present at > 1 μ
M in CSF, ISF, or plasma
3, 44
as candidates to activate iNicSnFR12. ISF concentrations are only partially characterized
45
; therefore, we cite
plasma
concentrations
3, 44
in
μM: tryptamine
(16.5),
putrescine
(8.4), , cadaverine
(2.0),
tyramine
(1.8),
spermidine
(3.1), u
rea
4.0,
L-
tryptophan
(65),
sarcosine
(1.4), and carnitine
(50). Of these, only carnitine consistently gave a detectable
∆
F/F
0
at the plasma
concentration
(S
upp
orting inf
ormation:
Concentration
-response relations for biogenic amines and alkaloids with iNicSnF12)
.
Referring these fluorescent plate reader data
to comparable previous data
35
, this carnitine
response would equal
the response
to
~ 15 nM nicotine, well within the linear range
and
unlikely to affect sensitivity by itself
. Nonetheless, we are considering
protective layers that might exclude carnitine. On the one hand, Nafion™
, an exemplar tetrafluoroethylene
based
fluoropoly-
mer
copolymer
, excludes nicotine and would not serve as a protective layer
33
. On the other hand, PDMS is quite permeant to
nicotine
46, 47
and would likely exclude carnitine, choline, and
acetylcholine
--all quaternary amines.
The
minimal effects of endogenous biogenic amines and alkaloids imply
that
the
more important challenge
is to
suppress
the
increased F
0
’ of biofluids. We previously suggested that the
elevation arises mostly from bilirubin
35
. Because bilirubin is
usually conjugated to albumin
,
43
w e hypothesize that
the hydrogel tactic will
decrease
F
0
’ by excluding albumin. A decisive
test o
f this
hypothesis
will
require
redesigning the optics to record fluorescence from only
the hydrogel, rather than including
surrounding test fluids or tissues. In summary, it is likely that a combination of the hydrogel strategy and protective layers can
optimize an iNicSnFR12
-based continuous nicotine monitor.
Conclusions
An
engineered, bacterially expressed, purified, fluorescent nicotine biosensor protein can be encapsulated in several hydrogel
formulations, while retaining the ability to continuously
detect dose
-dependent signals over several log units of [nicotine]
. The
most successful iteration, utilizing PEGDA/Irgacure
, was
readily
sliced by a vibratome,
and
showed minimal leaching
of iN-
icSnFR protein on
a time scale of days
a fter
preparation.
After nicotine appears at the hydrogel s
urface,
diffusion
slows access to the iNicSnF
R molecules.
We explain how diffusion
is slowed slightly by the gel and markedly by
rebinding to iNicSnFR12 itself. But iNicSnFR12 must b
e concentrated suffi-
ciently to give a good signal
-to-noise
(S/N)
ratio. This three
-effect
optimization
--buffering, diffusion, S/N
—also appears im-
portantly in the neuroscience
literature on genetically encoded fluorescent Ca
2+
sensor proteins. F
or measurements that resolve
the ~ 5 min bolus
of nicotine from smoking
or vaping,
we conclude that iNicSnFR
molecules mus
t be present at
10 μ M and
within
~ 200
μ
m of the biofluid surface.
The
approximately
linear dependence of induced fluorescence on [nicotine] and the minimal fluorescence of the gel itself lead
to the conclusion that absolute calibration of
∆
F/F
0
in terms of
[nicotine]
t
may be
possible
in tissue
. We conclude that, given
.
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7
additional resources,
a minimally invasive fiber
-optic based continuous monitor measuring
[nicotine]
t
in interstitial fluid can
eventually
be developed with
a form
factor
and cost comparable to
a modern amperometric continuous glucose monitor,
for
research on persons who ingest nicotine.
Further
engineering
48
would improve the sensitivity of the iNicSnFR series, further
integrate the electronics, optimize the optics including light shielding, adhere or attach the hydrogel to the fiber optic, test
protective coatings, and develop
an
insertion device.
Finally,
although we did not test the immobilization of
previously reported biosensors for other drug classes (iDrugSnFRs)
,
11-
14, 16, 44
photophysics and stability among iDrugSnFRs is nearly indistinguishable, which should result in comparable results if
they were similarly encased. This would expand the range of therapeutic and abused drugs that could be monitored continu-
ously
in a hydrogel
.
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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10
ACKNOWLEDGEMENTS
We thank Neal Benowitz, Bruce Cohen, Mario Danek, Sophie Dalfonso, Sujit Datta,
Ryan Drenan, Nick Friesenhahn,
Heather Lukas, Anand
Muthusamy
, and Koji Sode
for advice. We thank Carlos Lois for use of a vibratome.
Funding was provided by the Caltech
M
erkin Insitute for Translational Research, the Caltech
Sensing to Innovation
(S2I)
Fund, the Caltech
Rosen Bioengineering Center, the Caltech Rothenberg Innovation Initiative (RI
2
), and the Caltech
Carver Mead New Ventures Fund.
Insert Table of Contents artwork here
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if we receive a fa
vorable review
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