BIOLOGICAL SELF-ASSEMBLED MONOLAYERS FOR PHOTOSYNTHETIC SOLAR
CELL AND SENSING APPLICATIONS
Kien B. Lam1,2, Elizabeth F. Irwin3, Kevin E. Healy3,4, and Liwei Lin1,2
1
2
Department of Mechanical Engineering, 2Berkeley Sensor and Actuator Center
Berkeley Sensor and Actuator Center, 4Department of Materials Science and Engineering
University of California at Berkeley, Berkeley, CA 94720
ABSTRACT
Photosynthetic sub-cellular plant structures called
thylakoid were immobilized onto a gold electrode surface
that had been functionalized by bioelectrocatalytic selfassembled monolayers (bio-SAMs) of cystamine and
pyrroloquinoline quinone (PQQ). The goal is to achieve
direct transfer of electrons from thylakoids to the
electrode via the bio-SAMs to increase the electrical
output of MEMS photosynthetic fuel cells.
The
immobilization technique could also be used in MEMS
bio-sensing and microbial fuel cell applications. Quartz
crystal microbalance with dissipation (QCM-D) was used
to monitor the deposition kinetics of cystamine, PQQ,
and thylakoids. Using QCM-D, the surface coverage of
these three layers was determined to be, respectively, 7.9
× 10-10 mol/cm2, 3.3 × 10-10 mol/cm2, and 1.5 × 106
thylakoids/cm2. The cystamine and PQQ monolayers
formed within 5 min, while the thylakoid layer required
over 1 h. Each layer was shown to be covalently linked
to the substrate or layer underneath and thus was able to
survive repeated rinsing in water or buffer.
Keywords: photosynthesis, self-assembled monolayer
(SAM), thylakoid, solar cell, fuel cell
SAMs via a surface technique known as quartz crystal
microbalance with dissipation (QCM-D). Thylakoid
immobilization via bio-SAMs has not yet been
demonstrated in the literature, and our supposition is that
such a scheme would promote direct transfer of electrons
from thylakoids to the electrode, thus enhancing electrical
output.
In addition to MEMS PSC applications,
thylakoids immobilized on electrode surfaces can be used
in MEMS bio-sensing applications, taking advantage of
the thylakoids’ ability to detect herbicides and other
environmental contaminants [4].
THEORY
Photosynthetic organisms unicellular bacteria to plants
capture solar energy to power the splitting of H2O,
releasing electrons that reduce CO2 to produce
carbohydrates (CH2O) and other complex nutrient
molecules [11]. In plant cells, photosynthesis comprises
complex reactions that are catalyzed by enzyme
networks—collectively called photosystems (PS)—
embedded in the membranes of sub-cellular plant
structures known as thylakoids (Fig. 1b). Thylakoids
Thylakoids
INTRODUCTION
Photosynthetic solar cells (PSC) are electrochemical
cells that harness biocatalysts like photosynthetic bacteria
[1, 2], sub-cellular plant organelles [3, 4], or
photosynthetic enzymes [5, 6] to convert light energy and
water into electrical power. We recently demonstrated
MEMS PSCs (μPSC) powered by live blue-green algae
[7] and by sub-cellular organelles known as thylakoids [8,
9]. The μPSCs generated about 500 mV open circuit
voltage and on the order of 1 μA/cm2 closed-circuit
current density, results that are comparable to many
biological electrochemical cells yet not powerful enough
for most practical applications [10]. We previously
showed that the primary issues limiting PSC electrical
output were (1) the “slow” transport of diffusional
biocatalysts and redox (electron) mediators used in the
anode and (2) the sensitivity of the biocatalysts and
mediators to oxidation by O2.
To address these two issues, in this paper, we propose
immobilizing photosynthetic thylakoids directly onto a
gold electrode surface by means of bioelectrocatalytic
self-assembled monolayers (bio-SAMs).
We then
monitor the deposition kinetics of bio-SAM formation on
the gold surface and thylakoid immobilization the bio-
NADP(H)
PSII
e-
PSI
H+
H2O O2
(a)
Chloroplast
Thylakoid
membrane
e-
(b)
ATP
ee-
O2
ee-
(c)
H2O
Thylakoid
⎯ PQQ ⎯HN⎯S
Thylakoid
⎯ PQQ ⎯HN⎯S
Thylakoid
⎯ PQQ ⎯HN⎯S
Au
Figure 1. (a) Chloroplast, a membrane-bound organelle inside
plant cells that contain smaller membrane-bound structures
called thylakoids. (b) Detail view of a thylakoid, which have
photosynthetic enzymes embedded on the outer membrane. (c)
Proposed function of bioelectrocatalytic self-assembled
monolayers (bio-SAMs): illumination of thylakoids releases
electrons that are transferred via the bio-SAMs to the gold
electrode surface.
Cr/Au (2000 Å) on
Si Substrate
PQQ, EDC
Cystamine
(a)
(b)
Thylakoid
Thylakoids, EDC
HEPES, pH 7.5
Cystamine =
(e)
(c)
PQQ =
(f)
HEPES, pH 7.5
Thylakoid
(g)
NH2 ⎯
NH2 ⎯
(d)
NH2 ⎯
Figure 2. Chemical reaction steps of synthesizing bioelectrocatalytic SAMs (cystamine and PQQ) on gold substrate and immobilizing
thylakoids on SAMs.
themselves reside in sub-cellular plant organelles called
chloroplasts (Fig. 1a). The electrons extracted from the
light-powered splitting of H2O are transported along the
photosystems enzyme network, from which the electrons
are “siphoned” off in PSCs as electrical current. In biosensing applications, agents that interrupt electron
transport in the photosystems can be “detected” by
thylakoids.
In our most recent μPSC, a reaction mixture consisting
of diffusional thylakoids and a redox mediator in solution
was used in the anode.
To address the two
aforementioned issues limiting electrical output, we
propose the thylakoid immobilization scheme in Fig. 1c.
The gold electrode surface is first functionalized with
cystamine, to which the redox mediator pyrroloquinoline
quinone (PQQ) is then linked. Finally, thylakoids
isolated from plant cells are immobilized on PQQ. Our
supposition is that, under illumination electrons and in the
presence of H2O, the thylakoid photosystems would
transfer electrons directly to PQQ to cystamine and,
finally, to the gold electrode surface.
Bioelectrocatalytic SAMs consisting of cystaminePQQ have previously been shown to support direct
electron transfer [12] and be insensitive to O2 oxidation
[10]. In addition, enzymes linked to similar bio-SAMs
have been demonstrated to increase the current densities
of enzymatic fuel cells from the order of μA/cm2 to
Thus, the thylakoid
hundreds of μA/cm2 [10].
immobilization scheme of Fig. 1c has the potential of
solving both current-limiting issues discussed earlier and,
thus, increasing electrical output.
EXPERIMENT
The gold electrode surface was first fabricated by
thermally evaporating Cr/Au 500/1500 Å onto a Si wafer,
which was then diced into 1 cm × 1 cm substrates and
cleaned in heated 5:1:1 ultra-pure water (UPW): NH4OH:
H2O2. Thylakoids were isolated from baby spinach
purchased from the grocery store using a common
fractionation procedure [13], yielding a chlorophyll
concentration of 0.14 mg chl/mL. The specific activity of
the
isolated
thylakoids
was
measured
spectrophotometrically in conjunction with the electron
acceptor 2,6-dichloroindophenol (DCPIP) and found to
be of 0.44 μmol DCPIP reduced/mg chl/min.
The chemical reaction steps of the deposition of
cystamine, PQQ, and thylakoids are illustrated in Fig. 2.
All chemicals were purchased from Sigma Aldrich and
used as received.
The Au substrate was first
functionalized by soaking in 0.02 M cystamine for 2 h,
followed by rinsing in UPW and HEPES buffer (pH 7.5)
to remove physisorbed (i.e. non-chemisorbed) cystamine
(Fig. 2a-b). Next, pyrroloquinoline quinone (PQQ, 3
mM) was chemisorbed to cystamine using EDC (10 mM)
to crosslink the carboxyl groups of PQQ to the amino
group of cystamine to form amide bonds (Fig. 2b-c).
EDC is the acronym for the carbodiimide N-(3dimethyaminopropyl)-N’-ethylcarbodiimide; it is a zerolength crosslinking agent because, in forming the amide
bond, it does not introduce additional chemical structure
between the conjugating molecules [14]. Rinsing in
HEPES buffer followed to remove non-chemisorbed
PQQ. Finally, the isolated thylakoids—which have
amino groups on their outer membranes—were
immobilized on the PQQ, also using EDC carbodiimide
chemistry.
We then monitored the deposition kinetics of
cystamine, PQQ, and thylakoids using the QCM-D
surface chemistry technique. QCM-D is a sensitive mass
sensor in which an AC voltage is pulsed across a
piezoelectric quartz crystal at the crystal’s resonant
frequency (5 MHz) and at several overtones (n = 3, 5, 7,
corresponding to 15, 25, 75 MHz) [15-17]. The pulses
cause the crystal to oscillate in shear mode at each
frequency. The shear wave at each frequency is then
allowed to dampen, which provides viscoelastic
information about a film adsorbed to the crystal. In our
experiments, we used the Q-Sense D300 QCM-D system,
which is sensitive to surface coverage as low as 0.18
ng/cm2 [15]. For the special case of thin, rigid films, the
Sauerbrey equation relates the change in the resonant or
H2O baseline Introduce cystamine
Rinse w/ H2O
Rinse w/ HEPES
Introduce PQQ &
EDC in HEPES
00
-5
-5
Δf/n
(Hz)
Δf/n(Hz)
overtone frequency, Δf, of the crystal with the change in
mass per unit area, Δm, of the film adsorbed to the crystal
surface, including bound water [15, 16]: Δm = (C/n)Δf,
where Δm is in units of ng/cm2, C is a constant that
depends on the physical properties of the crystal (= 17.7
ng/cm2/Hz for the quartz crystal in our case), and n is the
overtone number (= 1, 3, 5, 7…). Then the surface
coverage film could be calculated as Γ = Δm/(FW), where
Γ is in units of mol/cm2 and FW is the formula weight of
the adsorbed species. Cystamine, PQQ, and thylakoids
were deposited on custom Q-Sense gold-covered crystals
in the QCM-D reaction chamber using the procedure
previously described for the Si/Au substrates.
Rinse w/ HEPES
-10
-10
-15 Δf3/3, Δf5/5 Δf7/7
-15
Deltaf3/3
-20
-20
Deltaf5/5
Deltaf7/7
The QCM-D monitored in real-time the frequency
change, Δf, at the resonant frequency (5 MHz, overtone n
= 1) and several overtones (n = 3, 5, 7, or 15, 25, 75
MHz) as material was being deposited. Figure 3 plots
frequency change normalized by the respective overtone,
Δf/n; because Δf is typically noisy for n = 1, Δf/n is
shown only for n = 3, 5, 7. As shown in Fig. 3a, when
cystamine (FW = 152.27 g/mol) was introduced to the
crystal surface, Δf/n for n = 3, 5, 7 dropped drastically
within 5 min and, after rinsing with UPW and HEPES,
settled at Δf/n = 6.8 Hz (average of overtones, negative
sign ignored). The cystamine surface coverage was then
be calculated to be Γ = 7.9 × 10-10 mol/cm2 (including
bound water).
When PQQ and EDC in HEPES were introduced, Δf/n
again dropped sharply within 5 min; after rinsing with
HEPES, the net average decrease was Δf/n = 6.2 Hz (Fig.
2a). The PQQ (FW = 330.21 g/mol) surface coverage
was then determined to be Γ = 3.3 × 10-10 mol/cm2
(including bound water).
We note that cyclic
voltammetry had been used in previous work to deduce a
PQQ surface coverage of 1.0 × 10-10 mol/cm2 [12, 18];
however, because QCM-D directly monitored PQQ
adsorption onto the crystal, the QCM-D measurement
was more accurate.
We also note that the surface coverage of both
cystamine and PQQ lay in the range of ~ 10-10 mol/cm2,
which corresponds roughly to a monolayer [19] and
justifies the use of the term SAM. In addition, both bioSAMs were stable to rinsing, indicating chemisorption
rather than just simple physisorption. Finally, we observe
that surface coverage of cystamine was over twice that of
PQQ because the formula weight of cystamine is less
than half that of PQQ.
The kinetic monitoring of thylakoid immobilization
onto the cystamine-PQQ bio-SAMs is shown in Fig. 3b.
When thylakoids and EDC in HEPES were introduced to
the crystal surface, over about 70 min, Δf/n gradually
decreased a net average of 19.4 Hz, which meant that the
mass surface density of immobilized thylakoids was,
using Sauerbrey, Δm = 340 ng/cm2. We note in Fig. 3b
that thylakoids introduced without EDC merely
(a)
-25
-25
00
50
50
100
100
Introduce HEPES
150
200
150
200
Time (min)
Time (min)
250
250
300
300
350
Introduce thylakoids
& EDC in HEPES
350
00
Rinse w/ HEPES
-5
-5
Δf/n
(Hz)
Δf/n (Hz)
RESULTS AND DISCUSSION
-10
-10
Introduce thylakoids
w/o EDC
-15
-15
-20
-20
Δf3/3
Deltaf3/3
Deltaf5/5
Deltaf7/7
-25
-25
(b)
-30
-30
00
20
20
Δf5/5
Δf7/7
40
60
40
60
Time (min)
Time (min)
80
80
100
100
Figure 3. QCM-D monitoring of deposition kinetics of (a)
cystamine and PQQ monolayers onto Au surface and (b)
thylakoids onto cystamine-PQQ monolayers.
Normalized
changes in frequency, Δf/n, of QCM-D crystal for several
overtones (15, 25, 74 MHz) are used with the Sauerbrey
relationship to calculate surface coverage of deposited layers.
physisorbed to PQQ and were easily removed by HEPES
rinsing. If we model thylakoids as flat discs of diameter
1 μm, thickness 0.25 μm, and density ρ = 1.2 g/cm3
(average of densities of water and protein, 1.0 and 1.4
g/cm3, respectively), then the thylakoid number surface
density could be estimated as Γ’ = Δm/(ρ×Vol) = 1.5
× 106 thylakoids/cm2.
By modeling thylakoids as a 1 μm flat discs, an area of
1 cm × 1 cm = 104 μm × 104 μm would have 104 × 104 =
108 thylakoids/cm2 in a packed monolayer. Yet, the
estimated number surface density was only ∼106
thylakoids/cm2, about 100 times less dense than the
expected thylakoid monolayer. A possible reason could
be that an insufficient number of thylakoids was
introduced to the QCM-D crystal surface, which could be
remedied by introducing additional doses of thylakoids
with EDC in HEPES buffer after the initial dose.
Another possibility was that 70 min was not long enough
for the thylakoids—which are orders of magnitude bigger
than cystamine and PQQ molecules—to self-assemble
(i.e. arrange and pack) properly. Certainly, a longer time
period could be allocated for the thylakoid
immobilization step. A third possible reason could be
that because of the thylakoids’ size, multiple EDCcatalyzed amide bonds were likely needed to “tie” a
thylakoid down to the underlying PQQ. Thus, it is
probable that higher concentrations of EDC would be
needed.
CONCLUSION
We have successfully functionalized a gold electrode
surface with a cystamine monolayer, onto which we
covalently linked a monolayer of the redox mediator
PQQ. We subsequently isolated thylakoids from baby
spinach and immobilized them onto the cystamine-PQQ
bio-SAMs. Using QCM-D, we monitored the formation
of the cystamine-PQQ-thylakoids layers and verified that
cystamine and PQQ indeed formed SAMs whereas
thylakoids formed in sub-monolayer surface densities.
The next steps entail electrochemically verifying that
direct electron transfer from thylakoids via the bio-SAMs
to the electrode has been achieved. This thylakoid
immobilization scheme could potentially increase the
current densities of MEMS photosynthetic fuel cells two
orders of magnitude to hundreds of μA/cm2. The scheme
could also easily be extended to MEMS bio-sensing
applications utilizing immobilized thylakoids to detect
herbicides and various environmental pollutants. Finally,
the immobilization chemistry developed in this paper
could be applied to immobilizing other membrane-bound
cellular or sub-cellular structures—such as unicellular
microorganisms for MEMS microbial fuel cells [20].
ACKNOWLEDGEMENTS
This work was supported in part by an NSF grant (ECS0300542), a DARPA/MTO/BioFlips grant (F30602-00-20566), and an ITRI fellowship.
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