j. vincent edwards , krystal r. fontenot, nicolette ... · 476 j. vincent edwards, krystal r....

In: Nanocellulose, Cellulose Nanofibers … ISBN: 978-1-63483-860-3

Editor: Md. Ibrahim H. Mondal © 2016 Nova Science Publishers, Inc.

Chapter 20

SYNTHESIS AND ASSESSMENT OF PEPTIDE-

NANOCELLULOSIC BIOSENSORS

J. Vincent Edwards, Krystal R. Fontenot,

Nicolette Prevost, Sunghyun Nam, Monica Concha

and Brian Condon Southern Regional Research Center,

USDA, New Orleans, LA, US

ABSTRACT

Nanocellulose is an ideal transducer surface for biosensors: it provides a high surface

area, easily derivatized with bioactive molecules, and abrogates binding of proteins

present in biological fluids where analytes and clinical biomarkers are of interest. Here

we consider approaches to biosensors of human neutrophil elastase (HNE), a biomarker

for chronic wounds and a therapeutic target for a variety of inflammatory diseases.

An unchecked influx of neutrophils degrades growth factors and collagen formation,

thereby indefinitely delaying the healing of chronic wounds. Over the years, the use of

cellulose and other polysaccharides as matrices for wound care has grown. Among these,

nanocellulose is suitable for both wound dressing and point of care diagnostic sensor

applications.

Synthetic approaches to nanocellulose derivatization include esterification of the

transducer surface for the covalent immobilization of peptides and enzymes, and

numerous pathways for annealing peptides and proteins. Nanocellulosic transducers,

which are considered here, include cotton cellulose nanocrystals (cCNC) and wood

cellulose nanocrystals (wCNC). The cCNC and wCNC provide transducer surfaces of

varying reactivity and specific surface area. The comparison of cCNC and wCNC

matrices illustrates the relative efficacy of putative transducer surfaces as colorimetric

and fluorescent peptide conjugate sensors of HNE. Here we discuss routes to their

synthesis and analysis. The resulting peptide-nanocellulose conjugates demonstrate

promise for application to diagnostic technologies with the potential to interface with

therapeutic paradigms in advanced wound dressings.

E-mail: [email protected].

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J. Vincent Edwards, Krystal R. Fontenot, Nicolette Prevost et al. 476

Keywords: sensors, peptides, cotton and wood cellulose nanocrystals, human neutrophil

elastase

INTRODUCTION

Point-of-care protease detection has been receiving increasing interest and adopted

clinically in some countries to enable chronic wound treatment decisions [1, 2]. The

combination of detection with wound dressing treatment of chronic wounds in a single

dressing is not available at this time. Thus, the ability to combine measurable protease

detection with a dressing motif that removes harmful proteases from chronic wounds is a

current goal of wound healing biomaterial design.

Biosensors at a molecular level are designed with a receptor-like recognition component

that can be activated by a cellular or biochemical event, which in turn prompts a signal that

can be detected electrically (evanescent field), optically (fluorescence and colorimetric), and

mechanically (quartz crystal microbalance) [3]. Of the different types of biosensors,

fluorescence based detection is one of the most sensitive.

A biosensor is comprised of a bio-molecule such as an enzyme or peptide, i.e., a ligand-

like molecule as appended to a matrix, and a transducer surface where the ligand is

immobilized [4]. The application of biosensors in the biomedical, industrial, or military field

is growing [4]. High sensitivity of detectors is required to improve the efficiency in complex

biological fluids [5].

Cellulose is an abundant polymer composed of repeat glucose units connected by beta-

glycosidic linkages to form long chains that typically aggregate into crystalline or amorphous

domains. The anhydroglucose unit of nanocellulose is covalently modifiable principally at the

hydroxymethyl position. An important benefit of cellulose when applied to sensors is its low

propensity to absorb proteins making it favorable in biological milieus [6]. An acid treatment

of cellulose produces nanocellulosic systems that have a greater propensity of hydroxymentyl

groups.

Natural sources of nanocellulose include plants, trees, and bacteria [7, 8]. Processing of

naturally occurring cellulosic sources into nanocellulose systems has given rise to a number

of beneficial materials that afford potential sensor transducer surfaces. These include

aerogels, cellulose nanofibrils, cellulose nanocrystals, microfibrillated cellulose, nanopaper,

and thin films from different sources of cellulose [9]. These nanocellulose systems are highly

versatile, abundant, biocompatible, and biodegradable [10] with unique utility including high

specific surface area.

NANOCELLULOSE TRANSDUCERS AS OPTICAL SENSORS

Nanocellulose systems functionalized with covalent or non-covalent attachment of

fluorescent molecules enables sensitive detection in potential biomedical applications

including optical bioimaging, biosensors, and photodynamic therapy [11]. Previously

reported fluorophores anchored to nanocellulose include fluorescein-5‘-isothiocyanate (FITC,

bioimaging) [12], terpyridine and terpyridine-modified perylene fluorophore (bioimaging)

Synthesis and Assessment of Peptide-Nanocellulosic Biosensors 477

[13], 5-(and-6)carboxyfluorescein succinimidyl ester (FAM-SE, pH sensing) and Oregon

Green 488 carboxylic acid – succinimidyl ester (OG-SE, pH sensing) [14], polyethylenimine-

chlorin p6 derivatives (PEI-chlorin p6, photodynamic therapy) [15], 5-(4, 6-dichlorotriazinyl)

aminofluorescein (DTAF, bioimaging) [16], and 7-amino-4-methylcoumarin (AMC,

bioimaging) [17].

The covalent attachment of biomolecular substrates to nanocellulose as a sensor

transducer surface [11], offers specificity for biological recognition by enzymes or receptor

proteins. For example, DNA oglionucleotides grafted onto nanocellulose that gives rise to a

biocompatible material, which behaves as a biosensor for enzyme/protein immobilization [11,

18]. Furthermore, nanocellulose transducers of peptide-cellulose conjugates can function in

concert with the peptide as a molecular recognition element of proteases by negative charge

and surface polarity interaction with positively charged HNE.

Human Neutrophil Elastase in Chronic Wounds and Methods of Detection

Chronic wounds are severe wounds that typically do not heal within four to six weeks (up

to three months) or alternatively the wound size does not reduce by 20 – 40% after two to

four weeks of treatment [19-21]. These wounds may occur due to a variety of pathologies

including diabetes, repeated damage to tissues, insufficient pressure relief treatment or lack

thereof in the initial stages of wounding. It has also been noted that the body‘s production of

abnormally low levels of proteases inhibitors are etiological to chronic wound development

[19, 22]. Wounds heal through four overlapping stages of hemostasis, inflammation,

proliferation, and maturation [19, 23]. However, chronic wounds remain in the inflammation

stage, and can secrete harmful proteases that have a deleterious effect on the non-healing

wounds [22].

Human neutrophil elastase (HNE) is an elevated serine protease released from

neutrophils, a type of white blood cell, which is present in the inflammation stage of a chronic

wound [22, 24]. The HNE and matrix metalloproteases (MMPs) are required for debridement

of wounds during the healing process, but when HNE and MMP levels are unchecked, they

breakdown growth factors and collagen inhibiting the formation of new tissue [25, 26].

The sensor-based detection of elastase has varied from a microchip [27], microdialysis

probe [28], fluorometric supramolecular pore sensor [29], immobilization of peptides on

quantum dots [30], immobilization of HNE on a biosensor chip for surface plasma resonance

[31], and the use of a colorimetric and fluorometric peptides coupled to cotton cellulose

nanocrystals [25, 32]. However, enhancing the detection limits of HNE released from chronic

wounds continues to be of importance.

Peptide Analogs That Detect HNE

The serine protease, HNE, has specificity for small-uncharged amino acids including

alanine (A) and valine (V) [33-36]. An example is seen in the protein elastin, which can be

degraded as a result of pathologies associated with the lungs and connective tissue. Elastin is

subject to breakdown by HNE especially where alpha-antitrypsin is depleted in some

inflammatory disease states. Elastin contains repeat amino acid motifs of alanine and valine


residues that are segmented by proline (P) which constitutes the substrate-recognition sites for

HNE binding and hydrolysis [34].

Synthetic peptide substrates containing these hydrophobic amino acids vary in size,

sequence, and have a detectable signal, which can be associated with a sensor, that is

mediated through release of a chromophore or fluorophore upon HNE hydrolysis between the

COOH-terminal valine or alanine and respective signal molecule. The HNE selective peptide

sequences have a minimum of 3 – 4 hydrophobic uncharged amino acids with sequences of

Ala-Ala-Ala, Ala-Pro-Ala, and Ala-Ala-Pro-Val. As shown in this study some of these

sequences AAA [37], APA [38], and AAPV [39] have a high propensity to form β-turn

motifs. The amino and carboxyl termini of these peptides have also been derivatized with a

succinic acid linker and a chromophore or fluorophore (the signal molecules), respectively.

Figure 1 show the minimized low β-turn conformational energy of the tetrapeptide (Suc-

AAPV) with the 4-paranitroaniline (pNA, chromophore) and 7-amino-4-methylcoumarin

(AMC, fluorophore) attached to the esterified cellotriose chain as a portrayal of the motif that

is assembled on the surface of the cellulose nanocrystal.

Figure 1. The minimization energy of the tetrapeptide (Suc-AAPV) with the pNA (A) or AMC

fluorophore (B).


Cotton and Wood Cellulose Nanocrystals

Cellulose nanocrystals (CNC), which are referred to in the literature as nanocrystalline

cellulose, cellulose whiskers, cellulose microcrystallites, and microcrystals [40] are typically

generated from acid hydrolysis, which gives rise to cellulose crystallites that are less

accessible to acid degradation, and can be isolated through repeated dialysis of the acid [41-

45].

Cotton CNC (cCNC) has beneficial properties including high surface area, mechanical

robustness, and is characterized by geometrical rod-like or whisker shape nanocrystals with

fibril diameters of 15 nm, a length of 159 nm, and a density of 1.5 gcm-3

[25, 46].

Nanocrystals made from wood (wCNC), are prepared in a similar way to cCNC but during

the wood pulping process, more of the amorphous regions remain intact. Cotton cellulose

nanocrystals contain cellulose I nanocrystals, and wCNC contains both cellulose I and II [47].

The wCNC has similar properties to cCNC and has rod-like particles that are comparable to

cCNC but it differs in the fibril diameter (10 nm), length (112 nm), and density (1.6 gcm-3

)

[48-50]. Applications of wCNC are currently being explored in the automotive, aerospace,

electronics, and medical industries [48, 51].

Elastase Biosensor In Situ Paradigm for Wound Dressing Motif

Commercial applications of wound dressings that utilize bacterial nanocellulose are

beginning to emerge [52]. It is notable that nanocrystalline silver has also been applied to

dressings (used to prevent and treat infection) in the late 1990s [53], the system contained

high-density polyethylene sandwiching a layer of rayon/polyester gauze where the outer layer

contains a nanocrystalline form of silver coating [53]. These types of dressings have been

found to both inhibit matrix metalloproteases and wound specific bacteria. Nanofiber-based

wound dressings (NFD) have been applied to burn wounds, and confer high filtration, liquid

absorption efficiency, semi-permeability, conformability, functional ability, and confer scar

free properties [21,53]. Recently, we have proposed a system for the use of peptide

derivatized cellulose nanocrystals as biosensors interfaced within the dressing material [25].

Cotton and Wood Nanocellulose Systems as HNE Biosensors

Here we contrast the design, synthesis, and characterization of cotton and wood

nanocellulosic sensors, which shed light on the relationship of contrasting material properties

including specific surface area, degree of substitution and protease detection sensitivity by

examining two nanocellulosic transducer surfaces. The cCNC and wCNC transducer surfaces

immobilized with colorimetric and fluorescent elastase peptide substrates, Suc-AAPV-pNA

and Suc-AAPV-AMC, are contrasted as HNE sensors. This is with a view to further

understanding surface chemistry properties necessary to interface sensors with dressings.


EXPERIMENTAL

Materials

Chemicals N‘N-dimethylformamide (DMF), dichloromethane (DCM), and filter paper

were purchased from VWR. All chemicals were used as received without further purification.

Ethylcyanoglyoxylate-2-oxime (Oxyma Pure), diisopropylcarbodiimide (DIC), N,N-

diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP), and N-(1-naphthyl)

ethylenediamine dihydrochloride (NEDH) were purchased from Sigma Aldrich. The 9-

fluorenylmethoxycarbonyl-glycine (Fmoc-Gly) was purchased from Peptides international,

Louisville, KY, USA. The peptide substrates succinyl-Ala-Ala-Pro-Val-para-nitroanilide

(Suc-AAPV-pNA) and succinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (Suc-AAPV-

AMC) was purchased from Bachem without further purification. Human neutrophil elastase

was purchased from Athens Research Technologies. Electrospray liquid chromatography

mass spectrometry (ESI-LC/MS) data was collected on an Agilent 6520 QTOF LC-MS/MS

instrument. The cotton cellulose nanocrystals (cCNC) were provided as a freeze-dried powder

supplied by Dr. Quiglin Wu from Louisiana State University, Baton Rouge, LA. A solution of

wCNC was provided as a solution supplied by David Haldane, Innovatech. The wCNC was

freeze-dried into a powder form.

Methods

Esterification of Transducers

In a 50 mL centrifuge tube Oxyma Pure (0.75 mmol), DIC (0.75 mmol), and DMAP

(0.075 mmol) in DMF were added to Fmoc-Gly-OH (0.75 mmol). The cCNC or wCNC (0.75

mmol) was added to the centrifuge tube and sonicated at 25 – 30˚C for 3 h. The esterified

nanocrystals were washed thrice with DMF by centrifuging and decanting with a pasture

pipette. Deprotection of the Fmoc protected glycine on nanocrystals was accomplished by

soaking in 20% piperidine/DMF deblock solution for 15 mins while sonicating, see loading

substitution section. The deprotected esterified nanocrystals were washed thrice with DMF by

centrifuging and decanting with a pasture pipette. After which, the deprotected surfaces were

prepared for peptide coupling or washed thrice with methanol, air dried, and stored for further

use.

Immobilization of the Peptide Substrate on the Transducers

To a 50 mL centrifuge tube Oxyma Pure (0.68 mmol), DIC (0.68 mmol), and DMAP

(0.068 mmol) in DMF were added with the respective peptide substrate (Suc-AAPV-pNA or

Suc-AAPV-pNA) (0.052 mmol) in minimal DMF. The esterified cCNC-Gly or wCNC-Gly

(0.67 mmol) were added to the solution and swirled. The centrifuge tube was sonicated at 25

– 30 ˚C for 3 h and then placed in the refrigerator overnight. The biosensors cCNC-Pep or

wCNC-Pep was purified by washing with thrice with DMF and methanol using vacuum

filtration or centrifuged and decanted using a pasture pipette. The biosensors were allowed to

air dry and stored at ~ 4 – 8 ˚C until further use. Note biosensor will be defined as the

transducers immobilized with glycine and the peptide.


Upon the completion of drying, 10 mg of each biosensor was subject to peptide removal

by adding a cleavage cocktail of TFA/water/triisopropylsilane (95/2.5/2.5) to the biosensor

for three hours. The solution for each biosensor were diluted with water (1:10) and submitted

for ESI-LC/MS. The generation of the biosensors was confirmed via its molecular weight.

Loading Substitution

The supernatant from the deblock solution was measured using ultraviolet visible

spectroscopy (UV-VIS) and the loading substitution of the different transducers was

calculated [54]. The supernatant of each matrix were transferred to a quartz cuvette and the

absorbance was measure at 301 nm. The loading substitution was calculated using Equation

1.

Loading (mmol/g) = (Abssample) / (mg of sample x 1.75) Equation 1

Elemental Analysis

All samples were submitted as solids to Midwest Microlabs for carbon (C), hydrogen

(H), and nitrogen (N) analysis. Nitrogen content calculations for peptide mass (μg) and

peptide (μg/mg) were performed in Excel 2007 using equations 2 and 3. The peptide mass

determines the amount of peptide on the surface of biosensor by multiplying the nitrogen

mass by the nitrogen factor of 6.25, Equation 2. The amount of peptide per μg/mg of support

was achieved by dividing the peptide mass by the weight of the biosensor, Equation 3.

Peptide mass (μg) = N mass*6.25 Equation 2

Peptide (μg/mg) = peptide mass/weight of biosensor Equation 3

Attenuated Total Reflectance Infrared Imaging (ATR-IR)

A Platinum Alpha ATR-IR Bruker spectrometer, A220/D01 was used to determine the

functional groups of the cCNC, and wCNC transducers and biosensors. The OPUS 6.5

software was used to collect; sixty-four scans at a resolution of 4 cm-1

between the 4000 –

500 cm-1

region and to analyzed the spectra. All spectra were graphed using Microsoft Excel.

Degree of Substitution

Degree of substitution (DS) of the biosensors was calculated using the Touzinsky and

Gordon method where the percent nitrogen was determined using elemental analysis.[55] The

application of DS herein is as follows: PC1 is the percent nitrogen, MWu is the molecular

weight of one cellulose unit, MW1 is the molecular weight of one nitrogen atom, N1

represents the number of nitrogens, and MWG1 is the molecular weight of the peptide with

the glycine linker.

dS1 = PC1(MWu) / [(MW1)(N1)(100) – (MWG1)] Equation 4


Specific Surface Area

The specific surface area of the cCNC was calculated with a density of 1.5 gcm-3

a width

of 15 nm and length of 159 nm and wCNC with a density of 1.6 gcm-3

using width of 10 nm

and a length of 112 nm [25]. The volume of cylinder (Equation 5), density formula (Equation

6), number of particles (Equation 7), the surface area of a cylinder (Equation 8), and the

specific surface area formula (Equation 9) were used to estimate the specific surface area

formula.

ν = π * r2 * h Equation 5

V = m/ρ Equation 6

N = V/ ν Equation 7

Sa = 2πrh + 2πr2 Equation 8

SA = N * sa Equation 9

Molecular Modeling

Computational studies were used to compute the minimization energy of the tetrapeptides

(Suc-AAPV-pNA and Suc-AAPV-AMC) anchored to cellotriose. The peptide substrate

models were built using GaussView 5.0.9 and optimized using a semiempirical Hamiltonian

method [Stewart 89, Steward89a] (PM3) contained in Gaussian 09 revision A.02 molecular

orbital software [56].

Microscopy

Hirox Optical Microscope equipped with a MGX lens was used to obtain high range

images with a 350x magnification, a resolution of 0.5 μm, and a scale of 200 μm of the

transducers. The iridescence of the transducers was observed with polarized filters. The

samples were placed onto the microscope stage and imaged without further processing.

The field emission scanning electron microscopy (FE-SEM) matrices were imaged using

a FEI Quanta 3D FEG FIB/SEM with magnifications of 65000, 80000, and 100000x with a

500nm scale. The samples were sputter coated with a thin 4 nm layer of gold-palladium using

a Leika EM ACE600. A thin layer of coating was used to ensure the surface morphology of

the matrices was not altered.

Spectroscopy

The phosphate buffer solution (PBS) consists of 0.1 M sodium dihydrogen phosphate

(NaH2PO4) and 0.5M sodium chloride (NaCl) in millipore water and the pH was adjusted to

7.4 with 1N sodium hydroxide (NaOH) or 1N hydrochloric acid (HCl). The PBS buffer was

filtered with a 0.45 μm filter for all assays.

The absorbance spectroscopy of the pNA peptide substrate with 2 U/mL of HNE and its

amplification with the NEDH chromogen was performed as previously reported [25]. The

emission spectrometry measurements of the AMC peptide substrate were obtained using

RF5301 spectrofluorometer. The peptide substrate solutions 0 – 0.5 μmol/mL were prepared


using the aforementioned PBS solution and 100 μL were added to the centrifuge tube. A 2 mg

sample of biosensor wCNC-Pep and a 4 mg sample of FP-Pep were placed into centrifuge

tube with 100 μL of PBS. A volume of 100 μL of the elastase enzyme 1 U/mL was added to

the wells containing the substrate and biosensors for a total volume of 200 μL. The centrifuge

was placed onto an Accu block digital dry bath heating element from Labnet International for

30 min at 37˚C. These samples were diluted (15x) with PBS and fluorescence measurements

were scanned from 405 – 625 nm with an excitation at 390 nm.

HNE Activity Assay

Stock solutions of the chromophore were prepared as previously reported [25] and the

fluorescent peptide substrate were prepared from which serial dilutions were made using a

PBS buffer solution. A standard curve of the substrate was prepared with a serial dilution

ranging from 1 to 0.0156 μmol/mL. The biosensor stock solutions were prepared by

suspending 20 mg of the cCNC-Pep or wCNC-Pep in 1 mL of PBS, which equals ~ 2 mg of

sample. To start the reaction, 50 μL of human neutrophil elastase at various concentrations

ranging from 2 - 0.015 U/mL was added to the standard curve and to biosensor to provide a

total well volume of 150 μL.

Measurement commenced immediately at 37˚C and continued for 1 h at 1-min intervals.

The 96 well plate was shaken before each measurement for 3 seconds. The fluorescent

substrate was excited at 360 nm and the emission was recorded at 460 nm to measure the

increase in fluorescence of the amidolytic activity.

RESULTS AND DISCUSSION

Synthesis and Characterization of Biosensors

Conjugation of the tetrapeptide containing either a chromophore (p-nitroaniline) or

fluorophore (7-amino-4-methylcoumarin) to the transducer surface occurs in two steps. The

esterification of the transducer surface with glycine via the primary hydroxyl group of

cellulose was accomplished using carbodiimide coupling (Figure 2).

The loading substitution of the first amino acid (glycine, three letter abbreviation Gly)

anchored on the transducer surface is measured by base-mediated deprotectiion of the Fmoc-

amino protecting group, which releases flourescent dibenzofulvene (through β-1 elimination).

The absorbance intensity of the chromophore is employed to calculate the loading substitution

of glycine, which averages ~ 0.16 mmol/g for both the cCNC-Gly and wCNC-Gly.

It is noteworthy that the biosensor is defined by the transducer immobilized with glycine-

linked tetrapeptide. Esterification of the cellulose nanocrystals with Fmoc-Gly provides an

anchor for the covalent immobilization of the tetrapeptide to the transducers where an amide

bond is formed between the N-terminus of the alpha amino glycine and the N-terminus of the

succinyl carboxylate of the peptide substrates (Suc-AAPV-PNA or Suc-AAPV-AMC). The

peptide nanocellulose conjugates were confirmed via its molecular weight using ESI-LC/MS,

the parent ion value (m/z) of the peptide adducts is reported in Table 1.


Figure 2. Esterification and immobilization of peptides on transducers.

Table 1. The ESI-LC/MS, average nitrogen percent and peptide content as determined

by elemental analysis, calculated concentration, and sensitivity concentration

of the biosensors

ESI-LC/MSb Average N% Peptide Calc. conc. Namea (m/z) (mg) (μg/mg) (μg/mL)

cCNC-Gly 1.06

cCNC-Pep [M] 633.6590 1.46 27.56 35.73c

wCNC-Gly 2.17 wCNC-Pep [M+H] 671.3005 2.54 88.13 134.86d

aThe abbreviation Pep indicates the peptide with glycine (G-Suc-AAPV-pNA or AMC).

bThe [M]

calculated for C32H42N6O10 m/z 670.72 and includes the glycidyl link attached to the peptide. cThe

absorbance concentration for cCNC-Pep and the dfluorescence calculated concentration for wCNC-

Pep are based on measurements of released chromophore and flourophore from peptide conjugate

in the HNE assay at 1-2U HNE/mL.

Elemental analysis of the biosensors identified the percent of nitrogen present on the

biosensor from which the level of peptide incorporation was calculated (Table 1). The wCNC

has a 2-fold increase in esterified glycine and peptide immobilized surfaces compared to the

cCNC. As a result, the level of peptide attachment in μg/mg for the wCNC is 88.13 μg/mg,

which is higher than the peptide attachment for the cCNC of 27.56 μg/mg. The higher-level

of percent nitrogen and peptide attachment for the wCNC may be due to the smaller size of

the wood nanocrystals, which in turn accounts for a higher specific surface area that

accommodates more peptide.


Spectroscopy

The calculated concentration of the absorbance and fluorescence intensity of the

biosensors with 1 – 2 U/mL of HNE is provided in Table 1. The pNA absorbs at 405 nm and

AMC is excited at 360 and emits at 460 nm.

Figure 3. HNE pathway of hydrolysis for A) the pNA biosensor and its amplification with NEDH and

B) the AMC biosensor.

The cCNC-peptide analog has an absorbance-calculated concentration of 35.73 μg/mL

and the emission calculated concentration of the wCNC-peptide is 134.86 μg/mL. All faces of

the cCNC-Pep and wCNC-Pep nanocrystal surfaces freely interact with HNE, thereby

promoting access of the substrate peptides to the HNE enzyme active site.

A

B


The higher concentration of the wCNC-peptide is due to increased peptide substitution on

the nanocrystals and the higher sensitivity of the fluorescent AMC. The detection signals of

the chromophore and fluorophore with human neutrophil elastase varies. Figure 3 shows the

pathway of A) pNA amplification with NEDH and B) the AMC fluorescence pathway. Figure

3A portrays HNE hydrolyzing the amide bond between valine and pNA, which is further

reacted with NEDH. An azide bond forms between pNA and NEDH, which results in a red

shift in absorbance to 545 nm. The low detection signal of the pNA chromophore is amplified

with NEDH, which increases the colorimetric response [25]. Figure 3B demonstrates the one-

step reaction that occurs upon HNE hydrolysis of the amide bond between valine and AMC

subsequently resulting in a strong fluorescence signal.

Surface Analysis

Attenuated total reflection infrared (ATR-IR) identified different characteristic bands

present on the surface of the cellulose nanocrystals, see Figure 4. The ATR-IR of bands of

cellulose has been well studied for its functional groups [57-59] and contains similar bands to

nanocrystalline cellulose (Table 2) [57-61].

Table 2. Characteristic ATR-IR bands of the nanocellulose transducers

Sample Wavenumber

(cm -1)

Literature

(cm -1)

Assignments

Transducers 3489 and 3439a 3486 and

3439

Intramolecular hydrogen bonding

cCNC and

wCNC

3335 -3200 3570 – 3200 ν(O―H) stretching vibration

2900 – 2893 3000 - 2800 ν(C―H, C―CH3, C―H2) stretching

1636 1633 ν(H2O) absorbed molecules

1428 1429 ν(C―H) wagging - in-plane bending

1370 – 1314 1372, 1336, ν(C―H) bending, ν(O―H) in-plane

1205 1204, 1320 bending, and ν(C―H) wagging

1055 – 1031 1042 ν(C―O) stretching vibration

899 – 895 898 ν(C―H) bending and (C―H2) stretching

666-664 700 – 600 ν(C―C) stretching vibration

Biosensors

cCNC-Pep

and

wCNC-Pepa

3489 and 3439a 3486 and

3439

Intramolecular hydrogen bonding

3334 - 3200 3570 – 3200 O―H stretching vibration

2898 – 2892 3000 - 2800 C―H, C―CH3, C―H2 stretching

1652 – 1650 1650 amide I band(C―O stretching)

weak 1568 1540 amide II band (C―N stretching and N―H bending)

1636 1633 ν(H2O) absorbed molecules

1428 1429 ν(C―H) wagging - in-plane bending

1360 – 1315 1372, 1336, ν(C―H) bending, ν(O―H) in-plane

1204 1204, 1320 bending, and ν(C―H) wagging

1055 – 1031 1042 ν(C―O) stretching vibration

899 – 895 898 ν(C―H) bending and (C―H2) stretching

662 700 – 600 ν(C―C) stretching vibration

The transducers represent the cCNC and wCNC without any modification and the biosensors represent

the cCNC-Pep and wCNC-Pep with that are functionalized with the peptide. aThe additional peaks

observed for the nanocellulose matrices.


a

b

Figure 4. ATR-IR spectra of the transducers (A) and biosensors (B) with cCNC (pink) and wCNC

(blue) without and with the peptide.

Differences in the ATR-IR spectrums between the cCNC and wCNC and the cCNC-Pep

and wCNC-Pep are notable, Table 2. Figure 4A shows the additional bands present for the

cCNC and wCNC including the 3489 cm-1

and 3439 cm-1

and these spectra have equal

intensities. The 3489 cm-1

and 3439 cm-1

bands are ascribed to intramolecular hydrogen

bonding [62-64].

The ATR-IR spectrum of the biosensors show an additional peak at 1652 – 1650 cm-1

,

which represents the amide I stretch from the peptide [65], see Figure 4B. The peak intensity

of the cCNC increases after peptide immobilization on the surface. In comparison to

previously reported cellulose spectra the bands for the nanocellulose transducers are sharper

and may indicate a higher density of hydroxyl groups present on the surface of the

nanocrystals [62]. Furthermore, the ATR-IR results support the ESI-MS findings that the

tetrapeptide is anchored on the surface of the biosensor.

60012001800240030003600

Wavelength (cm-1)

cCNC

wCNC

60012001800240030003600

Wavelength (cm-1)

cCNC-Pep

wCNC-PepA


Degree of Substitution and Specific Surface Area

Degree of substitution (DS) levels, which measure ratio of derivatized anhydroglucose

hydroxyls, were calculated for the esterified transducers cCNC-G and wCNC-G and

biosensors cCNC-Pep and wCNC-Pep based on the Touzinsky and Gordon method (Table 3)

[55]. The glycine-esterified transducers have DS levels of 0.128 (cCNC-Pep) and 0.276

(wCNC-Pep), respectively. The high DS level for glycine is expected since glycine is a small

molecule with limited steric hindrance. The peptide on the transducer surfaces has DS levels

of 0.025 (cCNC-Pep) and 0.061 (wCNC-Pep). This indicates that approximately 2 – 6

peptides are linked per 100 anhydroglucose units (AGU) for the biosensors. The wCNC-Pep

has a 2.3 fold increase in peptide substitution compared to the cCNC-Pep. These findings are

further supported by the elemental analysis (percent nitrogen and peptide incorporation) and

the absorbance and fluorescence intensity results. The low DS of the cCNC-Pep and wCNC-

Pep in comparison to cCNC-Gly and wCNC-Gly, as previously reported by Edwards et al.

[25] is partially due to the synthetic modification that occurs at the surface.

Table 3. Degree of substitution, specific surface area, and the sensitivity values for

cellulose nanocrystals

Name DSa Specific surface areab

(m2g-1)

Sensitivity conc.c

(U/mL)

cCNC 186.2

cCNC-Gly 0.128

cCNC-Pep 0.025 0.050

wCNC 261.2

wCNC-Gly 0.276

wCNC-Pep 0.061 0.015 \aDS is the degree of substitution as calculated by Touzinsky and Gordon method.

bThe specific surface

area was calculated using the Equations 5 – 9 in the experimental section. cThe activity of the

biosensors towards HNE.

The specific surface area (SSA) of the transducers was calculated based on the surface

area and volume formula of a cylindrical shape [25] Table 3. The transducer cCNC has a

specific surface area of 186.2 m2g

-1 when calculated with a density of 1.5 gcm

-3, a width of 15

nm, and a light of 159 nm while the wCNC has a specific surface area of 261.2 m2g

-1 with a

density of 1.6 gcm-3

, a width of 10 nm, and a length of 112 nm.

The SSA of wCNC has a 1.4 fold increase in the total surface area of the cCNC. The

higher surface area of the wCNC compared to the cCNC is due to the differences in diameter

and length (diameter of 5 – 10 nm and length of 50 – 60 nm) and (diameter of 15 nm and

length of 159 nm), respectively.

The elemental analysis and degree of substitution further supports that the wCNC has a

higher SSA and accounts for the higher percent nitrogen and peptide incorporation. Smaller

size nanocrystals lead to higher surface area and increased hydroxymethyl titer for peptide

attachment.


Microscopy

Images of the transducer were obtained with a polarized optical microscope and field

emission scanning electron microscope (FE-SEM), Figure 5. The polarized optical images

reveal the iridescent nature of the nanocellulose transducers, Figure 5A [9].

Figure 5. The microscopy images A) polarized optical images of cCNC and wCNC with polarizer and

magnification of 350X, resolution of 0.5 μm, and a scale of 200 μm and B) the FE-SEM images of

cCNC and wCNC at 65000 x with at 500 nm scale.

The images show the rod-like shapes of the crystals, which appear to emit a

purplish/bluish hue in both the cCNC and wCNC. This photonic property may represent

reflection bands with a shorter wavelength [66]. Nanocellulose is known to exhibit photonic

properties including selective reflection of left-handed circularly polarized light with the pitch

of the chiral nematic phase varying within the wavelengths of visible light [67]. The FE-SEM

images reveal whisker-like crystals of cCNC and wCNC with an uneven disbursement as

shown in Figure 5B.

HNE Activity with Cellulose Nanocrystals

As shown in Table 3, the activity of the peptide-cellulose nanocrystal conjugates in the

presence of various concentrations of HNE revealed that the wCNC-Pep had the greatest

sensitivity levels (0.015 U/ml) towards HNE whereas the cCNC-Pep had a lower sensitivity

wCNC

200 μm 200 μm

cCNC

500 nm500 nm

cCNC wCNC

A

B


level (0.05 U/ml) yet still within the range of that found for chronic wound exudate titer of

HNE. The higher sensitivity of the wCNC supports the findings from the higher peptide

loading, degree of substitution, the calculated concentration from fluorescence spectroscopy,

and the higher surface area.

CONCLUSION

The synthesis and application of peptide-nanocellulose from wood nanocellulose as a

biosensor for HNE is reported and contrasted with cotton nanocellulose. Esterification and

immobilization of the cotton and wood cellulose nanocrystals with glycine and the

tetrapeptide was confirmed using ESI-MS, elemental analysis, and spectroscopy. Surface

analysis using ATR-IR revealed sharp peaks present for the nanocrystals and additional peaks

representing the tetrapeptide-nanocellulose conjugate. The higher DS levels and specific

surface area observed for wCNC results in the high florescence levels and a high

concentration of the peptide, which provided enhanced sensitivity levels towards human

neutrophil elastase. Although the cCNC has a lower surface area in comparison to the wCNC

both nanocrystals show promise as transducer surfaces that can be functionalized with

peptides to serve as potential point of care diagnostic sensor to detect HNE in chronic wound

fluid.

ACKNOWLEDGMENTS

This project was funded by the U.S. Department of Agriculture. We thank Zhongqi He

and Dorselyn Chapital for allowing us to use their UV-VIS instrument and Dr. Casey Grimm

for collecting mass spectral analysis.

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