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1. Background
1.1. Properties of Cellulose: Cellulose rich plant fibers have been harnessed by humans throughout humanity
due to its rich abundance and diverse yet distinctive structural properties.1-3 Cellulose is the main component of
the plant cell wall and is responsible for over half of the carbon in the biosphere. The plant cell wall is accountable
for the load bearing function and the mechanical stability is comparable to that of steel.5 The strength can be
attributed to the ordering of cellulose within the plant walls.5 The primary cell walls of plants is comprised of
crystalline cellulose microfibrils implanted in a hydrated, disordered matrix of hemicellulose, pectin, lignin, and
glycoproteins.1
Figure 1: Plant cell wall matrix i) linear polymer of cellulose ii) cartoon depiction of organized cellulose forming cellulose microfibrils (CMFs) (square boxes) vs
amorphorous regions (squiggle lines) iii) combination of amorphous hemicellulose with highly organized cellulose crystalline fibers (CCFs) iv) different layers of plant
cell wall, hemicellulose and CCFs located in between each layer v) CMFs stack upon each other to form highly organized CCFs vi) Xyloglucan, major component of
hemicellulose.
Cellulose is a linear polymer that is comprised of up to 15,000 D-anhydroglucose rings that are linked by β-(1® 4)
-glyosidic bonds at the anomeric carbon (Figure 1i).6 The successive glucose rings are flipped over 180° with
respect to each.6 This allows intramolecular hydrogen bonding between the C3-hydroyl group and the ring oxygen
(O5) on the neighboring sugar ring, which stabilizes the linkage and is responsible for the linear configuration of
the cellulose chain. Van der Waals and intermolecular hydrogen bonds between hydroxyl groups and oxygens of
O
O
O
HO
OH
O
O
O
HO
OH
O
OH
HO
O
O
O
OH
HO
O
OH
OH
O
O
O
OH
HO
OH
OH
HO
HO
O
O
O
HO
OH
HO
OH
OH
OH
O
OH
OH
OH
H
0 or 1
n
0 or 1
XG
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adjacent molecules promote parallel stacking of multiple, highly cohesive cellulosic chains forming simple fibrils
that further aggregate into larger microfibrils (Figure 1ii).6-7 The intra- and inter-chain hydrogen bonding network
makes cellulose a relatively stable polymer and gives the cellulose fibrils high axial stiffness.7
1.2. Dichotomy of Cellulose and Xyloglucans The interconnected cellulosic scaffold is also held together by
intra and inter-molecular hydrogen bonding from interactions with hemicellulosic polysaccharides called
xyloglucans (XG) (Figure 1vi).1, 7 It is the dynamic hydrogen bonding and Van Der Waal forces between XG and
cellulose that gives strength, rigidity to the wall, and makes them water insoluble despite its hydrophilistic nature.1,
8 XGs have an intrinsic affinity to develop strong, spontaneous binding interactions with cellulose via its backbone
and side chains (vide infra) which are independent of temperature, molecular weight, and precise sugar
composition as well as water and buffers with different pH and ionic strengths.7, 9 The binding is essentially
irreversible, except at high pH concentrations.4, 10 The interaction is so specific that XGs are used in
chromatography as a means of separating cellulose from other plant materials found in the plant cell wall matrix.2,
11 In industry and research groups, the most widely used XGs are readily obtained from the tamarind seed of the
tropical tamarind tree (Tamarindus indica). Aqueous or basic extraction is used to acquire “tamarind kernel
powder (TKP)”, which is 60% xyloglucan by mass.2 The sugar units of the TKP form a common linear backbone of
β-(1® 4) -glyosidic residues which are covalently bonded at the anomeric carbon, nearly identical to the cellulose
polymer. The exception is the xyloglucans are decorated with xylosyl residues that are substituted with galactosyl
sugar rings.2, 7 These repeats are combined in various proportions which interact to form a refined structure that
varies in conformations.7 Research by York et.al determined that the complex branching pattern is comprised of
oligosaccharide repeats XXXG, XXLG, XLXG, and XLLG in a molar ratio of 1.2:3:1:5.4. Figure 2 uses single
letters to denote the observed structures as well as the detailed structure of the tamarind xyloglucan.
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Figure 2: Left: Single letters to denote the observed structures Right: detailed structure of the tamarind xyloglucan
2. Applications of Enzymatic Biosensors
2.1. Enzymatic Biosensors: Biosensors are distinctive types of bioelectronics devices generally utilized in
bioanalysis.12 Enzymatic biosensors can be defined “as an analytical device having an enzyme as a bioreceptor
integrated or intimately associated with the physical transducer to produce a continuous digital electronic/optical
signal that is proportional to the concentration of analyte present in the sample”.13
In the biomedical field, advances in enzymatic biosensors have been revolutionizing healthcare due to the
capability to diagnose and measure the progression of disease onset and advancement.12 This is particularly true
in the area involving detection and monitoring of blood glucose levels, cancer, malaria, neurodegenerative
diseases, and wound healing.12-14 Nanomaterials, such as cellulose crystalline fibers (CCFs, Figure 1v), have high
surface area matrix for functionalizing and biocompatible properties that make ideal transducer surfaces for
optical biomarkers.14 Point of care devices (POCs) that utilize cellulose mircrofibrils (CMFs) as substrates in the
matrix, such as dip-stick enzyme tests, lateral flow assays (LFAs), and microfluidic paper analytical devices
(μPADs), are becoming more popular in the biomedical field due to their economic practicality and minimum
consumption of resources.3-5 Potential application of both fluorescent and colorimetric detections to markers like
endogenous enzymes would reduce the need for highly sophisticated and costly instruments in resource limited
areas.4, 15
O
O
O
HO
OH
O
O
O
HO
OH
O
OH
HO
O
O
O
OH
HO
O
OH
OH
O
O
O
OH
HO
OH
OH
HO
HO
O
O
O
HO
OH
HO
OH
OH
OH
O
OH
OH
OH
H
0 or 1
n
0 or 1
XG
Xyl
Glc Glc Glc Glc
Xyl
Xyl Xyl
Glc Glc Glc Glc
Xyl
Xyl
XXXG XLXG
Gal
Xyl
Glc Glc Glc Glc
Xyl
Xyl
Gal
Gal
Xyl
Glc Glc Glc Glc
Xyl
Xyl
Gal
XXLG XLLG
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2.2. HNE Biosensors: Bacterial nanocellulose (BNC) or microbial cellulose (MC), is a polymer that is produced
by certain types of bacteria that has unique biomedical applications in soft-tissue replacement, reconstruction
applications, and wound healing due to its biocompatibility, sterilizability, tensile strength, surface chemistry, and
pharmacological properties.4, 15-16 BNC-based wound dressings, such as XCell, Bioprocess, and Biofill, are
already commercially on the market for applications in wound healing and detection of pathogens or endogenous
enzymes.4, 16 Edwards et. al developed a simple method for detecting the overabundance of human neutrophil
elastase, a serine protease responsible for the degradation of extracellular matrix proteins found in chronic
wounds.16 A peptide substrate containing chromogenic p-nitroaniline or fluorogentic 4-amido-7-methyl coumarin
was conjugated to cotton cellulosic fibers. When the HNE enzyme came into contact with the terminal C amide
biomarker, the yellow p-nitroaniline chromophore, for example, was released, allowing direct visual detection
(Figure 3).16
Figure 3: Synthesis of peptide substrate for detection of human neutrophil elastase.
2.3. Limitations: While relatively simple in the approach, there are 3 general limitations to the above mentioned
techniques. First, small aromatic compounds can cause an inflammatory response due to the peptide being
cleaved and diffusing into the surrounding environment (Figure 4A).4, 17-18 The cleavage of the chromogenic
moiety also results in signal attenuation, which is a general problem with POC devices such as dip stick enzyme
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tests.4, 19 Lastly, chemical modification of the microfibril surfaces for different applied purposes is limited to the
hydroxyl groups decorated on CNFs and CMFs. The integrity of cellulose microfibrils is dependent on the
hydrogen bonds between the hydroxyl groups of neighboring glycosyl sugar rings. Organic solvents and strong
bases that are used to extensively modify the hydroxyl groups gradually destroy the integrity of the fiber structure
and disrupt intrinsic molecular interactions.4, 11, 20-21
3. Methods and Materials
3.1. Two Birds, One Stone: To resolve the first two for mentioned limitations, Brumer et. al developed a reverse
substrate approach by tethering the fluorophore, instead of the target biomolecule, to the cellulosic transducer
with a tetraethylene glycol azide linker (Figure 4B).4, 22 This approached allowed the fluorophore to stay tethered
after the substrate cleavage took place, which addressed the signal attenuation. The biomolecule would diffuse
into the surrounding environment to be metabolized in vivo, therefore preventing possible toxicity.
Figure 4: A. Surface tethering vai biomolecule; B. Surface tethering via chromogen or fluorgen4
Scheme 1. Approaches for Cellulose-Based Esterase Biosensors
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3.2. Pre-“Click” Functionalization: Due to the intrinsic chemical properties of cellulose mentioned in the
introduction, cellulosic microfibrils are generally incompatible with organic solvents, making the method of
attaching a bioactive moiety limited.4, 22-23 However, noncovalent approaches such as printing techniques and
surface-adsorbed polymers have been successful in attaching bioactive moieties to the CMFs.10, 24-26 Using the
intrinsic binding capabilities of native XGs, which spontaneously form with CMFs, Brumer et. al developed a
method to functionalize xyloglucans which could be appended via the facial copper (I)-catalyzed azide-alkyne
cycloaddition (CuAAC), a type of “click chemistry”, to expand a useful reaction to produce a sensitive cellulosebased
esterase biosensor.4, 22-23
Considering a modification of the C-6 hydroxyl position on the galactosyl sugar ring of the XG polymer is more
regioselective in terms of reactivity, a well-defined functionalization was a necessary preliminary step towards a
“click” modification. Previous reports called for equal amounts of galactose oxidase (GO)27, but yielded only 24%
of the oxidized galactosyl residues.9 During optimization, 5 times the amount of GO was used as a catalyst (see
“repeat ratio” in Introduction) to selectively oxidize 90% of the C-6 hydroxyl groups of the terminal galactose to
aldehydes.4, 27 The aldehydes were then reduced with sodium borohydride as a heavy isotopic label and the
former oxidized galactosyl residues were labeled with one deuterium atom and formed an alcohol.27 Digestion of
the polysaccharide by endoglucanase produced xyloglucan oligopolysaccharides (XGO), which were quantified
for analysis by Matrix Assisted Laser Desorption Ion-Time of Flight-Mass Spectroscopy (MALDI-TOF-MS).4
3.3. Synthesis of Fluorescein Esters: Three ester derivatives, diacetyl, dibutryl, and diheptonoyl, of 5(6)-
carboxyfluorescein-tetraethylene glycol (TEG)-azide (FTA) were created utilizing a scalable, two-step synthesis
from starting materials of the parent fluorphore and the hydrophilic, biocompatible linker amino-TEG-azide.4 The
substrates for the detection of esterase were produced in high yields, confirmed by NMR analysis.4 TEG-azide
was used as a linker to optimize the fluorescein moiety28-29 and diacylation has been shown to block fluorescence
of the fluorescein moiety for esterase substrates.28, 30 Despite high yields, diheptonoyl-FTA was poorly soluble in
water and diacetyl had a high spontaneous hydrolysis despite being conjugated to the cellulose surface.4, 29-31
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3.4. Clickable Xyloglucans by chemo-enzymatic modification: The oxidized XG were propargylated by reductive
amination under optimized conditions with 10 equivalents of the amine and 5 equivalents of sodium
cyanoborohydride per mole of galactose in the polysaccharide.9 The solvent composition was 7:3 water/acetonitrile
to slow the rate of hydrolysis during the reductive amination but also allow the fluorescein ester derivative to go into
solution.4, 9 The propargylated XGO was adsorbed onto Whatman No.1 paper. The polysaccharide sorption was not
altered by the chemo-enzymatic modification.9 Optimized CuAAC conditions to conjugate FTA dibutyrate onto
propargylated xyloglucan-activated cellulose substrate consisted of the ligand tris-hydroxylpropyl triazolylamine
(THPTA).9 Generation of CuI was formed by the reduction of CuSO4 by sodium ascorbate and the solvent remained at
a ratio of 7:3 water/acetonitrile for better solubility of FTA dibutryl and reduced the amount of spontaneous
hydrolysis during the CuAAC.9 Extinction coefficient of 61700 M-1cm-1 at 495nm was used to calculate total
concentrations. Previous experiments used Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass
Spectroscopy (MALDI-TOF-MS) in positive reflectron mode with delayed extract ion to confirm that the terminal
alkynes were transformed into the FTA-esters biosensors.22
Cellulose Substrate
Cellulose Substrate
Cellulose Substrate
O
HO
OH
O
OH
OH
n
O
HO
OH
O
O
OH
n
O
HO
OH
O
NH
OH
n
O
HO
OH
O
NH
OH
n
O
O
O
O
O
O
NH
O
O
O
N
O
O
N
N
O
HO
OH
O
NH
OH
n
O
HO
OH
O
NH
OH
n
O2C
OH
O
O
O
N
H
N
N
O N
3
O2, galactose oxidase,
catalase, peroxidase,
H2O, RT
Propargylamine, NaCHNBH3,
HOAc, H2O or
H2O/Methanol
Cellulose Substrate ,
aqueous
Porcine liver esterase (PLE),
tris buffer, pH 8
FTA-butylrate, CuSO4 5H2O,
Sodium ascorbate, tris( 3-hydroxypropyltriazolylmethyl)
amine (THPTA), H2O/acetonitrile (7:3)
native
xyloglucan
oxidated
xyloglucan
propargylated
xyloglucan
FTA diester biosensor
Figure 5: Reaction scheme of chemo-enzymatic synthesis. Reaction chemicals and conditions listed. Previous experiment MALDI-TOFMS
a) native XGO b) Propargylated XGO c) Cellulose substrate functionalized via CuAAC.
Scheme 2. Chemo-Enzymatic Preparation of Cellulose Based Esterase Sensors4
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Discussion of Results
4.1. Initial-rate enzyme kinetic analysis: Agilent Technologies Cary 60 UV-Vis spectrophotometer was utilized for
absorbance measurements.4 An extinction coefficient of 14200 M-1cm-1 400nm. The specific activity of porcine liver
esterase was assayed against p-nitrophenyl acetate (p-NPA) as a benchmark.4 Since the FTA-butyrate crashed out
before reaching complete enzyme saturation, 10% acetonitrile slowed the specific activity of p-NPA by 30%, which
assisted the FTA-butyrate into going into solution while slowing down the specific activity of p-NPA. Simple, complete
hydrolysis of diesters to free FTA was assumed for calculations.4 FTA diacetate had a calculated solubility limit of
~90μM and a specific activity of ~0.45μM min-1mg -1.4
4.2. Porcine-catalyzed cleavage of conjugated FTA dibutyrate on cellulose substrates: Varying concentrations of
porcine liver esterase (PLE) (0,1.19,5,95,23.8, and 119 μM PLE) in 20μL 20mM tris buffer at a pH 8, were loaded onto
a Whatman no.1 filter paper conjugated with FTA-dibutyrate (Figure 6A, left side).4 An unmodified blank Whatman
no.1 filter paper was used as a blank. The fluorescence intensity over time was measured using a Bio-Rad Pharos FX
Plus Molecular Imager with an external laser using excitation of 488nm laser and 530 nm emission filter (Figure B, left
side).4 The fluorescence intensity was quantified using Quantity One 1-D analysis software. 1mL of 0.01M NaOH was
added to completely hydrolyze the FTA dibutyrate ester.4 For direct visualization, a 385 UV LED Blacklight (Figure C,
right side) and an UltraSlim LED Illuminator (Figure A, right side) were used.4 Limit of detection was 0.36μg of FTA
dibutyrate in solution after 30 minutes, assuming the diacylfluorescein hydrolysis kinetics are not linear.4
To further demonstrate the broad applicability of bioactivation via CuAAC of functionalized xyloglucans, cotton gauze
(Figure 6D) and bacterial cellulose (Figure 6E) has the FTA dibutyrate clicked on the substrates. Figure 6 shows how
this method can be used to incorporate a wide variety of functional groups without compromising the integrity of the
fibers.4
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Figure 6: Left side A) Varying concentrations of porcine liver esterase (PLE) (0,1.19,5,95,23.8, and 119 μM PLE) in 20μL 20mM tris buffer at a pH 8, were
loaded onto a Whatman no.1 filter paper conjugated with FTA-dibutyrate.4 An unmodified blank Whatman no.1 filter paper was used as a blank. The
fluorescence intensity over time was measured using a Bio-Rad Pharos FX Plus Molecular Imager with an external laser using excitation of 488nm laser and
530 nm emission filter.4 1mL of 0.01M NaOH was added to completely hydrolyze the FTA dibutyrate ester.4 B) Relative quantitation of ester hydrolysis
assuming FTA completely hydrolyzes. Right side: For direct visualization, C) a 385 UV LED Blacklight and an A) UltraSlim LED Illuminator were used. B) Close
up of discs under Ultra Slim LED Illuminator. D)cotton gauze functionalized using click CuAAC FTA dibutyrate and E) bacterial cellulose being visualized by
esterase activity using clicked FTA dibutyrate.
5. Conclusion
Dr. Brumer et. al successfully developed a versatile chemo-enzymatic method to develop a fluorogenic esterase
biosensor using cellulose as a substrate and a propargly functionalized xyloglucan as an anchor.4 Surface
activation was successfully accomplished on a wide variety of substrates such as a cotton gauze and bacterial
cellulose without disturbing the intrinsic matrices and interactions.4 Another advantage of this method is that
the fluorophore is tethered to the propargylated xyloglucan, which prevents signal attenuation and possible
toxicity in vivo.4 This method could extend to a wide range of Point of Care devices.
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