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Danielle Klaus CHM 530 9/22/2017

1

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

Danielle Klaus CHM 530 9/22/2017

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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.

Danielle Klaus CHM 530 9/22/2017

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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

Danielle Klaus CHM 530 9/22/2017

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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

Danielle Klaus CHM 530 9/22/2017

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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

Danielle Klaus CHM 530 9/22/2017

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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

Danielle Klaus CHM 530 9/22/2017

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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

Danielle Klaus CHM 530 9/22/2017

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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.

Danielle Klaus CHM 530 9/22/2017

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