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Paper-Based Membraneless Co-Laminar Microfluidic Glucose Biofuel Cell With

MWCNT-Fed Bucky Paper Bioelectrodes

Article  in  IEEE Transactions on NanoBioscience · July 2018

DOI: 10.1109/TNB.2018.2857406


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2 authors:

Prakash Rewatkar Sanket Goel

BITS Pilani, Hyderabad BITS Pilani, Hyderabad


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Paper based Membraneless Co-Laminar

Microfluidic Glucose biofuel cell with MWCNT
fed Bucky Paper Bioelectrodes
Prakash Rewatkar and Sanket Goel

 materials to contribute extra mechanical support.

Abstract—The present work establishes a membraneless, co- Due to their unique properties, paper based enzymatic biofuel
laminar flow-based approach to develop a cost-effective cells (EBFCs) are increasing attention in the scientific
microfluidic paper-based analytical device for enzymatic biofuel community. An EBFC is a bioelectronics device that efficiently
cell (µPAD-EBFC). The developed µPAD-EBFC supporting the
self-capillary fluid transport action, consists of Y-shaped paper
converts the chemical energy into the electrical energy
microchannel with the fuel (glucose) and oxidant (O2) streaming harnessing naturally available enzymes as electrocatalyst [5-7].
in parallel over carbon nanotube (CNT) based bucky paper (BP) They are being employed in many application such as disease
electrodes modified with biocompatible electrocatalytic enzymes diagnosis, glucose sensing, securities, point-of-care monitoring
such as Glucose oxidase (GOx) and Laccase without any additional etc. [4],[8-11]. In addition, paper based batteries are enticing
redox cofactor. The electrochemical performance for the modified growing attention as new energy harvesting systems for small
bioelectrodes i.e. electrocatalytic oxidation and reduction reaction
was carried out using Linear Sweep Voltammetry (LSV), Cyclic
electrical and electronic devices. However, the current state-of-
Voltammetry (CV) and Open Circuit Potential (OCP). The overall the art manifests that such devices produce less power density
performance of µPAD-EBFC was evaluated using polarization in an unsustainable manner to operate most of the small
studies. Subsequently, the catalytic activity of enzymes on the electrical devices and sensors [3],[12-14]. Except the critical
electrode surface was validated by Scanning Electron Microscope factor of power performance, there is a necessity to addresses
(SEM). This simple and portable µPAD-EBFC can generate the several other important challenges, such as the geometry of cell,
maximum power density to the order of 100 µW/cm2 (600 µA/cm2)
at 0.505 V over prolonged durations of around 50 hours. Hence,
shorter lifespan, electrode materials properties (porosity,
the presented µPAD-EBFC shows good power density and conductivity and surface area) and complex enzymes
stability, leading to its strong potential to power miniaturized immobilization strategies before realizing the µPAD-EBFC in
microelectronics devices and sensors. practical applications [15-18].
To address such challenges, even though extensive research
Index Terms— Microfluidic paper based analytical device for work is being carried, however, most of the published work
enzymatic biofuel cell (µPAD-EBFC), Carbon nanotube (CNT), describes the non-co-laminar fluid-flow approach, and cell and
Bucky paper (BP), Linear Sweep Voltammetry (LSV), Cyclic
Voltammetry (CV) and Open Circuit Potential (OCP), Scanning electrode fabrication using wax printer and screen printing
Electron Microscope (SEM). respectively [19-21]. Further, the persistent requirement of
membrane has been reported and broad array type structure is
presented for enhancing the power performance [22-28]. In a
I. INTRODUCTION recent work, Y-shaped paper EBFC with membrane under co-
laminar flow produced 24 µW/cm2 power density [29]. To the
I N the last few years, there has been a growing interest in the
development of microfluidic paper based analytical devices
(µPADs),These micro and nano fluidics devices for energy
best of our knowledge, till now no work is reported with
membraneless microfluidics paper based analytical device for
conversion put more emphasis on miniaturization and offer enzymatic biofuel cell (µPAD-EBFC) under co-laminar flow.
many unique advantages over conventional devices [1]. Such Hence, there was a strong need and more extensive work on
device has precise control and manipulates fluidics at small co-laminar, membraneless, simple in structure and cost-
scale. The miniaturization of µPADs are not only a cost- effective electrochemistry to fabricate bioelectrodes and
effective, lightweight, flexible, biocompatible and easy to µPAD-EBFC. Table 1 summarizes the accomplished work on
disposable but also provide self-pumping fluid transport via µPAD-EBFC leveraging time-consuming, additional costly
capillary action and high surface to volume ratio (SVR) [2][3]. redox cofactor, an electrode material, and expensive equipment
Hence, the necessity of external pressure system and pump not for the fabrication of cell and complex immobilization
necessary for transporting fluid through paper based strategies for bioelectrode fabrication. Based on this, it can be
microfluidics devices. Moreover, paper based devices promote manifested that there is a strong need of simple in fabrication;
good coherence with a large number of chemical and membraneless and co-laminar flow based paper based fuel cell.
bioreagents and can be compatible with low-cost plastic Herein, a membraneless µPAD-EBFC is demonstrated with

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simple fabrication process, low-cost, under co-laminar, sustained power production up to 4 days. Here, the self- driven

References I. Shitanda et al., I. Shitanda et al., Maria Jose et al., F. Javier et al., I. Shitanda et al., I. Shitanda et al.,
2017 [22] 2017 [21] 2017 [27] 2016 [29] 2017 [28] 2013 [30]
Type of Paper Whatman #1 Whatman #1 Whatman #1 Whatman #1 Whatman 1 Whatman 1
Electrode Carbon Ink Carbon Ink Carbon paper Carbon paper Carbon ink Carbon ink
Fabrication Screen printer Screen printer Screen printer
Anolyte Glucose oxidase Glucose oxidase Glucose Glucose Glucose oxidase Glucose oxidase
(GOx) (GOx) dehydrogenase oxidase (GOx) (GOx)
(GDH) (GOx)
Mediator Tetrathiafulvalene Tetrathiafulvalen Os(dmobpy)PVI Tetrathiafulvalene Tetrathiafulvalene
(TTF) e Fc-C6-LPEI (TTF) (TTF)
Catholyte Bilirubin oxidase Bilirubin oxidase Bilirubin oxidase Laccase Bilirubin oxidase Bilirubin oxidase
Triton X-100 Os(bpy)PVI Triton X-100 Triton X-100
Mediator TBAB-Nafian
Single Cell
Device Size - - 35 x 25 mm2 45 × 5 mm2
CD - - 225±14 0.48 mA/cm2 0.45 mA/cm2
PD 46 µW/cm2 76±26 µW/cm2 90 µW/cm2 µW/cm2 80 µW/cm2 0.12 mW/cm2
OCV 0.57V 0.61 V 0.65±0.05 V 24±5 µW/cm2 0.5 V 0.55 V
0.55±0.04 V
Array type 5 series 4 series/4 parallel 2 series
- -
CD 350 µW 1 mW 0.48 mA/cm2
PD 2.65 V 2.5 V 180 µW/cm2
OCV 0.94 V
Stability Not defined Not defined Not defined Not defined Not defined Not defined

pumping eliminates the need of external pressure pumps and 1). Fuel cell design and fabrication
co-laminar flow mechanism eliminates the need of a membrane. µPAD-EBFC was constructed using Whatman #1 filter
Moreover, cost-effective, additional redox co-factor free and paper. The Y-shaped design was cut using a commercial cutter
time efficient electrochemistry was used to create highly and plotter (Graphtec, Japan – Graphtec CE-2000) with speed
conductive carbon nanotube composed bucky paper (BP) based and force of 4 cm/s and 10 g respectively. The cutting operation
bioelectrodes. This BP based bioelectrode was used to harvest was performed at the single pass to prevent the tearing of the
energy longer in the presence a redox mediator. paper. Graphtec studio software was used to design the Y-shape
paper based microchannel. The Y-shape microchannel
II. EXPERIMENTAL consisted of two inlets and one outlet. Out of these two inlets,
A Materials and Reagents: one carried glucose and other carried oxygen due to the
Whatman #1 filter paper was purchased from (Whatman), capillary action to allow prolonged energy harvesting.
Buckeye Composites Multiwall carbon nanotube (MWCNT) MWCNT based buckeye composite (BP) was used as
based Bucky paper (BP) were purchased from (NanoTechLabs, electrodes of the µPAD-EBFC. The BP was cut in a rectangular
Inc.), USA. Glucose oxidase (GOx) from Aspergillus niger, 15 mm x 8 mm size and positioned in parallel on either side of
Laccase from Trametes Versicolor and other supporting the microchannel 20 mm downwards from inlets and isolated 1
chemicals like D-(+)-glucose, Monosodium phosphate mm from each other. In this configuration, the active electrode
(NaH2PO4), Disodium phosphate (Na2HPO4), P-Benzoquinone, surface area of the microfluidic channel was 0.3 cm2. Fig. 1
2, 2- azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) shows stepwise fabrication process flow of Y shaped
(ABTS), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide microfluidics paper based enzymatic biofuel cell
(EDC), N-hydroxy succinimide (NHS), Hydrochloric acid 2). Microfluidics device setup
(HCl), Sodium Hydroxide (NaOH), Isopropyl alcohol (IPA) A small mini-platform to hold the µPAD-EBFC was
and Acetone was purchased from Sigma-Aldrich. For the fabricated using the flash forge creator pro (Flash forge, USA)
experimentation, ultra-pure 18.2 MΩ cm MilliQ water 3D printer using Polylactic acid (PLA) filament. This design
(Millipore) was used. 0.1M pH 7.0 and 5.0 phosphate buffer contained two reservoirs (for anolyte and catholyte) and one
solution (PBS) was used for bioanode and biocathode holder was used to keep the microscope glass slide to hold
electrochemical analysis. µPAD-EBFC. Double sided tissue based adhesive tape was
used to adhere the µPAD-EBFC cell on the microscope glass
B Microfluidics fuel cell fabrication side and electrode. Alligator clips were used for external

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Transactions on NanoBioscience

electrical connections of the device. In electrochemical analysis, various parameters, such as

3). Modification of Bioelectrode Linear sweep voltammetry (LSV), Cyclic voltammetry (CV)
The modification process for BP based Bioelectrodes is and Open circuit potential (OCP) were investigated for the
similar to the earlier published work by our group [31][32]. In fabricated bioanode and biocathode. Finally, polarization
brief, for fabrication of the bioanode, first BP was cut in the response of the Y shaped µPAD -EBFC was carried out. The
dimensions of 15 mm × 8 mm. This BP was cleaned with electrochemical and polarization performance of µPAD -EBFC
isopropyl alcohol (IPA) and cured at 90ºC for 2 hours. Then the are summarized in Table 2
BP was immersed in EDC/NHS (30 mM/90 mM) in 10 ml of
Milli-Q water for 2 hours at the room temperature for activating III. RESULT AND DISCUSSION
carboxylic group. Thereafter, enzyme solution was prepared to A FESEM analysis of BP
dissolve 5 mg GOx in 1 ml PBS (0.1 M pH 7.0) solution and
a b
BP (15mm × 8 mm) were immobilized in GOx solution for 2
hours at the room temperature.
In the case of biocathode, a similar process was followed till
the activation carboxylic group on BP surface. Thereafter, 5
mg of Laccase was dissolved in 1 ml PBS (0.1 M pH 5.0)
solution and immobilized BP was immersed in Laccase solution
for 2 hours at room temperature under proper ventilation.
Finally, modified bioanode and biocathode was assembled on
1 µm 100 nm
Y-shaped paper microchannel. The overall size of µPAD -
EBFC was (50 mm × 25 mm) with anodic surface area 1.5 cm
c d
× 0.2 cm.

100 nm 100 nm

e f

100 nm 100 nm

Fig. 2. Images of untreated (bare) and surface modified BPs. (a) and (b) SEM
images of bare BP (different magnification). (c) and (d) SEM images of GOx
immobilized BP (different magnification). (e) and (f) SEM images of Laccase
immobilized BP (different magnification).
Fig. 2 shows the nanoarchitecture of enzymes modified BP
over untreated (bare) BP. As can be seen from Fig. 2 (a) & (b),
thin and long fiber-like the structure of the agglomerated
Fig.1. Fabrication process flow of microfluidic paper based analytical MWCNTs uniformly dispersed on the surface were observed,
enzymatic biofuel cell
resulting in rough surface morphology under different
magnification of untreated (bare) BP. These highly porous
4). Electrochemical measurement
structures of BP lead to strong bonding between enzymes
The electrochemical analysis of bioanode and biocathode
during the enzymes solutions deposited on the electrode
was conducted independently using a computer controlled
surface. Fig. 2 (c) & (d) and Fig. 2 (e) & (f) show SEM images
Potentiostat/Galvanostat (Biologic SP-150) at 10 mV/s. scan
of GOx and Laccase immobilized on BP respectively, which
rate at the room temperature. Conventional 3 electrode cell (5
clearly confirm enzymes aggregation everywhere over the
ml), with Ag/AgCl (3 M NaCl), platinum and BP as a reference,
electrode surface
counter, and working electrodes respectively, was used.

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B Electrochemical analysis of bioelectrodes current peak was clearly noticed in presence of Laccase
In electrochemical characterization, linear sweep saturated with air (red line) and a small reduction current peak
voltammetry (LSV) and cyclic voltammetry (CV) was carried was observed in the absence of oxygen saturated with nitrogen
out to evaluate the electrochemical behavior of modified BP gas (blue line). The maximum current density 2.1 mA/cm2 at
based bioanode and biocathode using 3 electrode 0.3 V was observed and reveling direct electrocatalytic activity
configurations. First, LSV study of bioanode was performed to of Laccase enzymes on the surface of BP.
know the electrocatalytic oxidation current by varying glucose 12
concentration, i.e. 10, 20, 30, 40, 50 and 60 mM, in presence of Absence of Glucose
1 mM P-Benzoquinone as a redox mediator in PBS (0.1 M, pH Presence of Glucose
7.0). As shown in Fig. 3, the oxidation current increased 8

Current density, mA/cm2

linearly with gradation in glucose concentration up to 40 mM
after that gradual current drop and reached at steady state
[33][34]. This oxidation reaction accomplished the saturation 4
kinetics with no further change in current with continuous
increase in glucose concentration after 40 mM. The LSV
analysis manifests that the successful enzyme-catalyzed 0
oxidation of glucose occurred on the BP electrode modified by
10 -4
9 20mM -0.5 -0.3 -0.1 0.1 0.3 0.5 0.7 0.9
Voltage, V
8 Fig 4. CV of GOx modified BP based anode in the absence and presence of 40 mM
Current density mA/cm2

7 50mM glucose in PBS (0.1 M, pH 7.0) in presence of 1 mM P-Benzoquinone redox

60mM mediator with 10 mV/s. scan rate at room temperature
4 Saturated with Nitrogen
3 Saturated with Air
Current density, mA/cm2

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 -0.5
Voltage,V -1
Fig 3. LSV of GOx modified BP based bioanode in PBS (0.1 M, pH 7.0) -1.5
with various glucose concentration i.e.10, 20, 30, 40, 50 and 60
presence of 1mM P-Benzoquinone redox mediator.
After LSV analysis, the CV analysis of the same the bioanode -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
was investigated at 40 mM glucose concentration. During the
Fig. 5. CV of Laccase modified BP based biocathode in absence of oxygen
bioanode measurement, immobilized BP bioanode was (saturated with nitrogen) and presence of oxygen (saturated with air) in PBS
immersed in PBS (0.1 M, pH 7.0) containing 40 mM glucose (0.1 M, pH 5.0) in presence of 1mM ABTS redox mediator with 10mV/s scan
and 1 mM P-Benzoquinone saturated nitrogen gas. Fig 4 shows rate
the cyclic voltammograms of BP based bioanode with and
without 40 mM glucose concentrations at 10 mV/s. scan rate. A
catalytic oxidation redox peak clearly observed due to the
presence of glucose (red line) revealing the electrocatalytic
character of GOx enzyme towards the C6h12O6/C6H12O7 redox
couple. On the other hand, a small catalytic redox peak was
recorded on bioanode in the absence of glucose (blue line).
Based on this redox wave, the maximum current density 10.07
mA/cm2 at 0.36V was recorded.
Similarly, the CV of BP based biocathode was carried out
with presence (saturated with air) and absence (saturated with
nitrogen gas) of oxygen. During the biocathode CV analysis,
Laccase immobilized BP dipped in PBS (0.1 M, pH 5.0)
containing 1mM ABTS cathodic mediator saturated with air at
room temperature. As shown in Fig 5, catalytic reduction

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Fig 6. Complete experimental setup of Y shaped microfluidic paper based

analytical enzymatic biofuel cell 0.6 120
Subsequently, the Y-shaped µPAD-EBFC was assembled in
3D printed mini-platform as shown in Fig. 6. This mini- 0.5 100
platform has two reservoirs for transporting fuel and oxidant

Power density, µW/cm2

towards the electrode side. In anode side, 40 mM glucose and
0.4 80
1mM, P-Benzoquinone in PBS (0.1 M, pH 7.0) was used

whereas 1 mM ABTS in PBS (0.1 M, pH 5.0) was used as
catholyte. The polarization experiment of the complete fuel cell 0.3 60
with these solutions was performed by connecting anode to the
counter and reference terminal while biocathode to the working 0.2 40
terminal of the potentiostat.
0.1 20

0 0
0 200 400 600 800 1000 1200
Open circuit Potential, mV

Current density, µA/cm2

Fig 8 Polarization performance and power curves measured at 40mM
0.4 glucose, 1mM of P-Benzoquinone in PBS (0.1 M, pH 7.0) at the anode and
1mM of ABTS in PBS (0.1 M, pH 5.0) at the cathode. Standard deviation
0.3 based on n=3

0 20 40 60 80 100 120
Time, Hr. Electro Bio anode Bio cathode Fuel Cell

Fig 7 Operational stability of paper based enzymatic biofuel cell by chemical

measuring Open circuit potential (V) with hours. Standard deviation based Characterization
on n=3
LSV 40 mM Glucose - -

By assembling the Y-shaped µPAD-EBFC on to the 3D CV Current density Current density Current
printed mini-platform, the Open circuit potential (OCP) and (CD) (CD) density (CD)
polarization performance of µPAD-EBFC was evaluated. As 10.07 mA/cm2 at 2.1 mA/cm2 at 600 µA/cm2
shown in fig 7, the develop µPAD-EBFC records 0.540 V OCP 0.36 V 0..3 V Power
after 2 hours and achieve maximum OCP 0.570 V after 24 hours density (PD)
and remains sustained up to 50 hours. Till date no such single 100 µW/cm2
Y-shaped and other paper based EBFC design generate the
OCP 20 mV 485 mV 570 mV
power production OCP for up to the 50 hours and remains
sustained for more than 2 days. Thereafter it began to decrease
in OCP which may be attributed to the change in porosity of IV. CONCLUSION
Whatman filter paper. Subsequently, polarization performance Herein a simple in structure, portable and membraneless
of paper based fuel cell was measured shown in Fig. 8. The µPAD-EBFC has been demonstrated using BP bioelectrodes.
maximum power density 100 µW/cm2 (600 µA/cm2) at 0.505 V The unique advantage of the proposed µPAD-EBFC is that the
was observed which is higher than the reported two streams and co-laminar flow and capillary flow mechanism eliminates the
other paper based enzymatic biofuel cell as summarized in need for membrane and external pressure pump for transporting
Table 1. These values obtained by several repeats of same the electrolyte towards the electrode side. The combination of
experiments (n=3). additional redox co-factor free enzymes and redox mediator
producing more sustainable power output compared to
previously reported paper based laminar or non-laminar based
fuel cell. The simple µPAD-EBFC consisted of Y-shaped paper
microchannel which was imposed on a coverslip using a
double-sided tape. These enzymes modified BP electrode were
combined together in the Y-shaped microchannel and complete
µPAD-EBFC was realized in a 3D printed holder. The major
limitation of the proposed µPAD-EBFC is the shorten

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