1

Enhancing the biocompatibility of Sodium Membrane Electrodes by Using Chitosan-

CuO and Chitosan-CuO-Heparin Composite Membranes

Ibrahim H. A. Badr1, A. Salim

1, A. M. Salem

1, M. Gouda

2, Hossam E. M. Sayour

3.

1 Chemistry Department, Faculty of Science, Ain-Shams University, Cairo, Egypt

2 Pretreatment and Finishing department, Textile Research Division, National Research

Center, Cairo, Egypt

3 Chemistry Department, Animal Health Research Institute, Cairo, Egypt

Abstract

We investigate the effect of introducing chitosan CuO-nanoparticle and heparin

loading chitosan CuO nanoparticle composite membranes to PVC based sodium selective

membrane electrode to enhance its biocompatibility and reduce its highly thrombogenicity.

The potentiometric responses of three sets of electrodes were studied: (1) conventional PVC

membrane electrode, (2) conventional PVC membrane electrode sandwiched with chitosan-

CuO membrane (CH-CuO-PVC), and (3) conventional PVC membrane electrode sandwiched

with chitosan-CuO-heparin membrane (CH-CuO-H-PVC). Potentiometric response

characteristics of the CH-CuO-PVC and CH-CuO-H-PVC membrane electrodes (e.g.,

detection limit, linear range, response slope, and selectivity coefficients) were found to be

comparable to that of the conventional PVC-based sodium electrodes. Biocompatibility was

assessed for all membranes (PVC and CH-CuO-PVC and CH-CuO-H-PVC) through platelet

adhesion protocol. SEM and platelet counts have shown that CH-CuO and CH-CuO-H have

improved biocompatibility compared to the classical PVC membranes.

Keywords: Membrane electrodes; Heparin; Biocompatibility;

Chitosan; CuO nanoparticles. * Corresponding author: Ibrahim H. A. Badr

Chemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt

E-mail: ibadr1@gmail.com Tel: +201153344232

1- Introduction

The technology of potentiometric sensors based on liquid or polymer membrane materials

is well established technology that spearheaded the integration of sensing devices into the

clinical laboratory for the automated testing of physiological samples for key electrolytes such

as potassium, sodium, calcium, and chloride, polyions, as well as for measuring the pH

[Young, (1997)]. This success came from the need of simple, direct, low cost, sensitive,

reliable, selective and rapid process that introduced by potentiometric sensors [Bakker, et

al.(1999); Bühlmann, et al.( (1998); Johnson & Bachas, ( 2003)]

Implantable electrochemical sensors have a great interest and development in biomedical

and clinical application especially for real- time monitoring of important physiological species

in blood, especially when such compounds are subject to a rapid change inside the body.

Measurements of electrolytes like Ca2+

, Na+, Cl−,

HCO3

−, and K+, blood gases, metabolits and

pH play a critical role in healthcare for diagnosis, monitoring, treatment or control of diseases.

Continuous monitoring of electrolyte or blood gases or pH are badly need in emergency cases

, intensive care , surgical procedures and for critically ill hospitalized patient (e.g., liver,

kidney or coronary pulmonary or in coma). Because this represents the physiological case of

body on time to help physicians to make right decisions regarding a patient's diagnosis and

treatment. Implantable sensors offer real time measurements which is critical in such cases.

Introducing any foreign materials such as chemical sensors in the blood stream have the

potential to activate platelets and also the potential to lead to thrombus formation that can

occlude the blood vessel. Chemical sensors even in the absence of full thrombus formation,

by activating platelets often lead to cellular adhesion on the surface sensors. These adherent

2

cells consume oxygen and glucose and produce CO2 as they are metabolically active.

Consequently, can cause inaccurate results for critical care analysis. The presence of platelet

adhesion and cellular activation and adhesion on sensors surface cause differences in the local

levels of PO2, PCO2, glucose, pH, etc., and on implanted sensor surface. As the platelet

adhesion and cellular activation and adhesion is dynamic process [Frost and Meyerhoff

(2015)]. This changing of the outer layer is one of the biggest practical concerns with using

intravascular sensors.

Many efforts have been made to develop materials that will resist or eliminate protein

adsorption on blood contacting polymers and utilized to prepare implantable sensors [Chen,

et al (2008); Chen, et al. (2010); Kim , et al. ( 2005); Li , et al. (2008 ); Zhang , et al.

(2008)]. Poly(ethylene glycol) (PEG)-based materials have shown the ability to decrease

protein adsorption [ Zhao , et al. (2010); Kim , et al. (2005)]. Zwitterionic surfaces have also

demonstrated a reduction in protein adsorption [Kim, et al. (2005); Li, ,et al. (2008); Zhang,

et al. (2008)]. Also studies to reduce protein adsorption create polymer brushes with tethered

pCBMA on surfaces [Zhang, Chen, Jiang (2006)]. Oligo (ethylene glycol) polymer brushes

tuned to thicknesses of 5–50 nm [Ma, Hyun, et al. (2004)], and tethered pSBMA on the

surface [Zhang, et al.( (2006)]. Those represent promising success at reducing protein

adsorption in bench top experiments. But, it still remains to be seen whether these approaches

can significantly improve in vivo performance of intravascular sensors. Approach of nitric

oxide (NO) release coatings that cause inhibition of platelet activation and adhesion has also

been explored [Frost, et. Al. (2002)]. Also combination of nitric oxide (NO) release coatings

with immobilized heparin improved blood compatibility by two means [Zhou, Meyerhoff

(2005); Wu et al.(2007)]. NO release coatings has also been applied recently to prepare

intravascular glucose sensors that yielded improved analytical performance and reduced

surface thrombosis when placed in the veins of rabbits [YanQ, et al. (2011)], also

immobilized heparin (to prevent bulk thrombosis/clot formation), and immobilized CD47 has

been proven successful [Finley, Rauova (2012); Finley, et al. (2013)].

Implanted devices suffer from biofilm formation and biofouling. Bacterial

colonization in the form of biofilms on surfaces causes persistent infections and is an issue of

considerable concern to healthcare providers [Mohankandhasamy and Jintae (2016)].

Biofouling is the accumulation of unwanted proteins and other analytes or organisms (cells

and bacteria) on surface of host materials [Banerjee, et al.(2011)]. The performance of

biomedical implants and devices can be changed by the adsorption of contaminating matter

and lead to patient infection, shortened durability, and increased healthcare cost by

replacement of devices. As a result there is an urgent clinical need to develop long-lasting

biomedical materials or devices with antibacterial and antibiofilm surfaces

[Mohankandhasamy and Jintae (2016)].

Different methodologies have been used to can enhance device biocompatibility and

functionality and material properties and surfaces can be modified to reduce microbial

contamination and prevent biofilm infections by different used include : antifouling coatings

[Banerjee, et al (2011)], antiadhesive surface modifications [Neoh and Kang (2011)],

addition of antimicrobials to the surfaces of medical devices [Shintani (2004); Kwok, et al.(

(1999); Sodhi, et al.( (2001)], Coating devices with polymer products [ Li, et al.( (2013)]

coating, lamination, adsorption, or immobilization of biomolecules [Khoo, Hamilton,

Grinstaff, , et al (2009), Lee, Wang, Yang,, et al. (2013); Arciola, , et al. (2012)] surface

engineering with chemical moieties [ Antoci ,et al. (2008); Gottenbos, et al. (2002); Hume,

Baveja, Muir et al.(2004); Pavlukhina, et al.(2012)]. Many approaches of Nanotechnology

provided a promising advancement to prevent drug-resistant biofilm infections of medical

devices and biomaterials. Copper, silver, gold, titanium, and zinc are known to have

antibacterial and antibiofilm properties that offer alternatives to antibiotics without increasing

3

the risk of resistance development. It has been established that metal-based nanoparticles have

much better antimicrobial activities than their micro-sized counterparts [Colon, Ward,

Webster (2006); Jones, et al.( (2008)].

CuO NPs exhibit effective antimicrobial activity against various bacteria, CuO

nanoparticles have effective antimicrobial action against a wide range of pathogens and also

drug resistant bacteria [ Alexandru et al. (2016), Kelly et al.( (2003); Raffi , et al. (2010).].

Chitosan is linear, semi-crystalline poly(aminosaccharide) cationic polymer. It consists of

two repeating units, N-acetyl-d-glucosamine and d-glucosamine linked by (1-4)-β-glycosidic

linkage with pKa of 6.5. Chitosan is obtained by alkaline deacetylation of chitin [Azuma et

al.( (2015)]. Chitin is the second important naturally occurring polysaccharide .It is found in

exoskeleton of arthropods mostly in crustaceans (shrimps ,lobster and crabs), insects, radulae

of molluscs and in beaks and internal shells of cephalopods (e.g. octopus and squid), cell wall

of fungi. Chitosan gained enormous importance in many filed specially in pharmaceutical and

biomedical applications (e.g. drug delivery, tissue engineering, gene delivery) chitosan is also

used in treating major burns, preparation of artificial skin and bones, surgical sutures, contact

lenses, blood dialysis membranes and artificial blood vessels etc. Chitosan has unique

properties due to its biological properties include biocompatibility, biodegradability, and non-

toxicity, mucoadhesive and antimicrobial activity. Besides this, it is fungistatic, bacteriostatic,

spermicidal, anticholestermic and anticarcinogenic. antitumor, blood anticoagulant,

antigastritis, hypochlesterolaemic and antithrombogenic agents [Pillai et al.( (2009), Saikia

et al. (2015); Croisier & Jérôme (2013)]. Chitosan is considered as promising polymer in

formulation of drug delivery system due to its unique chemical, physical and biological

properties like non- toxicity and ease of modification. The cationic nature of chitosan due to

primary amino groups impart a lot of properties such as controlled drug release, transfection,

mucoadhesion, and permeation enhancement [Felt , et al.( (1998); Park, et al.( (2010)].

Recently, metal oxides have been investigated in drug delivery applications because they have

the ability to improve drug loading, Control the drug release rate and targeting to specific

location in the body. For example ZnO nanoparticles stabilized with cationic chitosan coating

have been applied for preparation of doxorubicin loaded ZnO quantam dots [AbdElhady

(2012)]. Copper oxide (CuO) has attracted attention because of its antimicrobial and biocide

properties and they can be used in many biomedical applications [Kelly, Coronado, Zhao,

Schatz (2003); Raffi, et al. (2010)]. Therefore, CuO nanoparticles could be used to enhance

loading heparin into chitosan film and it also could be used to improve release of heparin

from the chitosan film.

In this study, we investigated the utility of chitosan-CuO nanoparticle membrane to

improve the blood compatibility of ion selective electrodes based on highly thrombogenic

polymers (e.g., PVC). Chitosan-CuO nanoparticle membranes used to the sensing PVC film

to prevent the contact between the highly thrombogenic PVC and the sample solution, and

therefore enhance the biocompatibility of PVC-based membrane electrodes. Furthermore,

improvement of chitosan biocompatibility could be achieved by adding heparin

(anticoagulant) to chitosan CuO nano particles.

2- Experimental

2.1. Reagents and Materials

Sodium ionophore-X, o-nitrophenyl octy ether (o-NPOE), potassium

tetrakis(chlorophenyl) borate KTClPB , bis (2-ethylhexyl) sebacate (DOS), Tridodecyl-

methylammonium chloride (TDMAC) and porcine intestinal mucosal heparin were obtained

from Fluka (Ronkonkoma, NY), and poly(vinyl chloride) was from Polysciences

(Warrington, PA). Tetrahydrofuran (THF) was purchased from Fisher (Fair Lawn, NJ) and

tris-(hydroxymethyl) amino methane (Tris) was obtained from sigma (St. Louis, MO).

Anticoagulant solution CPDA-1 was obtained as a gift from the central laboratory of the

4

armed forces (Cairo, Egypt). Each 100 ml of CPDA-1 contains: 3.1900 g dextrose, 2.6300 g

sodium citrate dihydrate, 0.2990 g of anhydrous citric acid, 0.2220 g of monobasic sodium

phosphate monohydrate, and 0.0275 g of adenine. Blood samples were collected from jugular

vein of healthy sheep (Ministry of agricultural, Cairo, Egypt). All standard solutions and

buffers were prepared with de-ionized distilled water.

2.2. Preparation Heparin-selective electrode:

Polymer membrane were prepared [Meyerhoff, Yang, (1992); Meyerhoff, Yang,

(1992)] by dissolving 4mg (crossponding 2%) of TDMAC, 32.5% (65mg) DOS, 65.5 % (131

mg) PVC in 2.00 ml of tetrahydrofuran (THF). This cocktail was poured into 25-mm internal

diameter glass ring placed onto a glass plate, and the membrane was formed after evaporation

of THF overnight. Working electrode was designed by cutting out disks from the membrane

using a cork borer (7mm diameter) and then the small disks are glued to a PVC tubing using

THF. Working electrodes were soaked in 15 mM NaCl overnight prior to use. 15 mM NaCl

also use as the internal filling solution. A double-junction Ag/AgCl electrode (Orion Model

90-02-00) with an Orion (90-02-02) internal filling solution was used as the reference

electrode. The outer compartment of the reference electrode was filled with saturated

potassium nitrate. All measurements were carried out in 0.12 M NaCl. Membrane potentials

were monitored at room temperature using data acquisition system, eight-channel electrode-

computer interface (Nico2000 Ltd., London, UK) controlled by Nico-2000 software. All

potentiometric calibrations shown are the average of three trials.

2.3. Study of released Heparin using Heparin-selective electrode:

Heparin-selective electrode was used to detect amount of heparin released,

Approximately 0.1 g from every of chitosan-CuO- heparin membrane of different heparin

loadings (0.28 wt %, 1.41 wt % and 2.78 wt % heparin conc) was soaked in 20 ml of 0.12 M

NaCl (saline buffer). Membranes were left in this solution for specific time. Heparin sensor

was utilized to detect the amount of released heparin at time intervals 0, 3, 6, 12, 36, 48, 72

and 96 hours. Three electrodes were used to detect the amount of released heparin for each

measurement.

2.4 Preparation of conventional sodium selective poly (vinyl chloride) (PVC) membranes:

Sodium selective membrane was prepared by dissolving 2.0 mg of sodium ionophore-X

(corresponding to 0.70 wt%), 0.50 mg of the lipophilic salt potassium tetrakis (chlorophenyl)

borate KTClPB (corresponding to 0.20 wt%), 100.0 mg of poly vinyl chloride (corresponding

to 33.0 wt%) and 200.0 mg of plasticizer nitrophenyl octy ether (NPOE) (corresponding to

66.1 wt%) in 2.00 ml of tetrhydrofuran (THF). The cocktail was poured into 25-mm internal

diameter glass ring placed onto a glass plate, and the membrane was formed after evaporation

of THF overnight.

2.5 Preparation of sodium selective membrane electrodes based on PVC, chitosan-CuO and

modified with (CH-CuO-PVC), and Chitosan CuO-heparin (CH-CuO-H)

A schematic representation of the three sets of membrane electrodes used in this study is

shown in (Fig.1). Small disks of about 7.00 mm diameter were cut from PVC, chitosan-CuO

(CH-CuO) and chitosan CuO-heparin (CH-CuO-H) master membranes. PVC membrane

electrode was prepared by gluing the PVC membrane to a PVC tubing in using THF to give

the control PVC membrane electrodes. CH-CuO-PVC and CH-CuO-H-PVC were prepared

by physically attaching CH-CuO, CH-CuO-H film, respectively to the tip of the PVC

membrane using Teflon tape, so that the CH-CuO or the CH-CuO-H would face the sample

solution. The internal filling solution for all electrodes was 1.0 mM NaCl prepared in the

measuring buffer solution. A double-junction Ag/AgCl electrode (Orion Model 90-02-00)

with an Orion (90-02-02) internal filling solution was used as the reference electrode. All

potentiometric measurements were performed in 0.05 M Tris-HCl, pH 7.4. Membrane

potentials were monitored at room temperature using data acquisition system, eight-channel

5

electrode-computer interface (Nico2000 Ltd., London, UK) controlled by Nico-2000

software. Combination glass electrode [Orion model (8-02] was used for all pH

measurements. The detection limit, linear range, and selectivity coefficients were determined

according to the reported methods [Bakker, et al., (2000); Guilbault, et al., (1976)], and the

data presented in this paper are average of three ion–selective electrodes measurement.

Figure 1: Schematic diagram for the three sets of sodium selective membrane electrodes used in this

study: (a) conventional PVC, (b) CH-CuO-PVC, and (c) CH-CuO-H-PVC.

2.6 Biocompatibility studies

The collected blood was obtained from the jugular vein of a healthy sheep. Each pint of

blood was drawn into a blood collection bag (JMS, Singapore, PTE Ltd.) containing 70.00 ml

of CPDA-1 anti-coagulant solution. Within 30 min after collection, blood was centrifuged at

240 × g for 15 min at 4.0ᵒC. Platelet was determined by Exigo vet hematology analyzer with

a concentration of approximately 9.5×104 cells /µL. The PRP was preincubated at 37◦C for

20 min. PVC, CH-CuO, CH-CuO-H membranes were conditioned in phosphate-buffered

saline (PBS was formulated with 10 mM phosphate, 138 mM sodium chloride, and 2.7 mM

potassium chloride, pH 7.4) for three days and were immersed in PRP for 2 h at 37◦C. The

membranes were then rinsed in PBS and fixed in a PBS solution containing 2% (w/v)

glutaraldehyde for 2 h at room temperature.The membranes were dehydrated by immersion in

serial dilutions of ethanol (30, 50, 70, 90, and 95%, v/v), two immersions in absolute ethanol,

and two immersions in hexamethyldisilazane. After solvent evaporation within desiccator, the

membranes were coated with gold and examined with QUANTA FEG 250 scanning electron

microscope (SEM).

3- Results and discussion

In this study we introduce CH-CuO-H and CH-CuO to PVC electrode to enhance the

biocompatibility and reduce the thrombogenicity of PVC by sustained release of anticoagulant

heparin fom CH-CuO-H and reduce of biofilm formation and biofouling by CuO

nanoparticles (see below).

Many efforts have been focused on the development of materials that will be sufficiently

biocompatible with blood for the several days of use in critically ill hospital patients.

Utilization of more biocompatible polymers in the fabrication of sensing film [Cha and

Meyerhoff, (1989);Cosofret, et al., (1994); Cha, et al., (1995); Liu, et al., (1993); Linder,

et al. (1995); Cosofret, et al. (1996); Yun, et al. (1997); Berrocal, et al. (2001); Bratov, et

al., (1995); Ambrose and Meyerhoff (1996); Heng and Hall, (1996); Heng and Hall,

(2000), Shin, et al. (1996); Högg, et al. (1996); Tsujimura, et al., (1996); Poplawski, et al.

(1997), Badr, et.al (2014), Badr, et.al (2015)]. Coating the PVC membrane surface with a

biocompatible polymer [Zhang, et al., (1996); Berrocal, et al., (2000), Badr, et al., (2014),

Badr, et al., (2015)], or immobilization of anticoagulants to the membrane surface (to prevent

thrombosis/clot formation) [Badr, et al., (2005)]. Implantable devices suffer from biofilm

formation and biofouling. In vivo, nonspecific protein adsorption facilitates bacterial

attachment to surfaces and leads to colonization, subsequent biofilm formation, and infectious

disease. Protein fouling followed by microbial attachment with biofilm development is a

dormant factor of the failure of biomedical devices and implants. Also, microbial attachment

reduces the sensitivities and efficacies of devices [Zhang and Chiao (2015)]. Different

methodologies have been used to can enhance device biocompatibility and functionality and

6

material properties and surfaces can be modified to reduce microbial contamination and

prevent biofilm infections by different used include : antifouling coatings [Banerjee, et al.(

(2011)], antiadhesive surface modifications [Neoh and Kang (2011)], addition of

antimicrobials to the surfaces of medical devices [Shintani (2004), Kwok, et al.( (1999);

Sodhi, et al.( (2001)], Coating devices with polymer products [Molin, Yang, and Ndoni

(2013)] coating, lamination, adsorption, or immobilization of biomolecules [Khoo et al.(

(2009); Lee, et al.( (2013); Arciola, et al. (2012)] surface engineering with chemical moieties

[Antoci, et al.(2008); Gottenbos et al.( (2002); Hume, et al (2004); Pavlukhina,et

al.(2012)]. Many approaches of Nanotechnology provided a promising advancement to

prevent drug-resistant biofilm infections of medical devices and biomaterials. Copper, silver,

gold, titanium, and zinc are known to have antibacterial and antibiofilm properties that offer

alternatives to antibiotics without increasing the risk of resistance development. It has been

established that metal-based nanoparticles have much better antimicrobial activities than their

micro-sized counterparts [Colon, Ward, and Webster (2006); Jones, Ray, Ranjit, and

Manna (2008)]. Exhibit quite different bacterial adhesions, protein adsorptions, and tissue

integration characteristics [Puckett, Taylor & Raimondo (2010); Salwiczek, Qu, Gardiner

et al. (2014), Singh, Vyas, Patil et al., (2011)].

CuO NPs exhibit effective antimicrobial activity against various bacteria, CuO

nanoparticles have effective antimicrobial action against a wide range of pathogens and also

drug resistant bacteria [ Grumezescu et. al, (2016); Kelly, Coronado, Zhao, Schatz (2003),

Raffi, Mehrwan, et al. (2010)]. Therefore, introduction of CuO into Chitosan film could

reduce formation of biofilm and befouling on the surface on medical devices.

In this study the biocompatibility of membrane electrodes based on PVC was improved by

using CH-CuO and CH-CuO-H films. Heparin and CuO are expected to reduce biofilm

formation and biofouling. Thin film of CH-CuO or CH-CuO-H was sandwiched to PVC.

Furthermore, we investigated the effect of introducing CH-CuO or CH-CuO-H layer on the

potentiometric response characteristics of PVC-based sodium-selective membrane electrodes

formulated with sodium ionophore-X.

Heparin-selective electrode was used to detect amount of released heparin. A measurement of

heparin clotting time is not suitable for direct and rapid measurements. Heparin-selective

electrode enables direct, rapid and selective detection of free heparin with lower detection

limits of 0.1-1 unit/ml. (Fig 2) shows the time trace for three successive calibration of heparin

selective electrode as measured in 0.12 M NaCl. After each calibration the heparin sensor was

washed in 15 mM NaCl for 45 min. As can be seen the same heparin sensors showed good

reproducibility of each calibration. Approximately 0.1 g from each of chitosan-CuO- heparin

membrane of different heparin loadings (0.28%, 1.41% and 2.78% heparin conc) soaked in 20

ml of 0.12 M NaCl for specific time. Heparin sensor was then utilized to detect the amount of

released heparin at time intervals 0, 36, 12, 36, 48, 72 and 96 hours.

Figure 2: Dynamic response time of heparin selective membrane electrodes toward increasing heparin concentration,

as measured in 0.12 M NaCl.

7

Figure 3 Potentiometric responses of heparin selective membrane electrodes as function time for CH-CuO-H A) 0.28

wt % B) 1.41 wt % C) 2.78 wt % heparin.

Figure 3 shows, change in mV as function of time due to the heparin released from

CH-CuO-H in 20 mL of buffer 0.12 M NaCl for three different heparin loading. As shown in

this figure the potentiometric response of released heparin from the three different heparin

loading of CH-CuO-H membrane (0.28 wt %, 1.41 wt % and 2.78 wt % heparin) showed a

decrease in signal was observed with time and all membranes continue to release heparin up

to 72 hours and then reached a plateau.The amounts of released heparin were calculated after

72 hours for each membrane film as summarized in Table 1. This table Summarized

calculations of cumulative amount or heparin release form different CH-CuO-H membranes

(0.28%, 1.41% and 2.78% heparin conc) up to 72 hours. The releases after 72 hours were

respectively 64.68, 71.11 and 79.21 µg/mL. As shown in (Fig 3) the release heparin from the

three membranes was more rapid at initial stage of experiments. The highest rate of release

was at the first 10 hours after that the rate of released were decreased till reach to plateau. 72

hours. Metal oxides NPs have the ability to improve drug loading and control the drug release

rate [Xiaoli , Yanan Luo et al. (2016), AbdElhady , (2012)]. Respectively, presence of CuO

stabilizes heparin in membrane phase. This is because CuO NPs have great affinity to

carboxyl and amine groups [Ruparelia et al. (2008)]. Each disaccharide repeating unit of

heparin contains a carboxyl group and therefore, it can be attached to heparin carboxyl

function group, which in turns enhances the attachment of heparin to chitosan.

Table 1. Summary of calculations of released heparin from different CH-CuO-H film

Film weight,

g

wt % Heparin in Film

Released heparin after 72 hr

Unit/mL

Released heparin after 72 µg/mL

%Released heparin after

72

0.11 0.28 % 0.58 64.68 21.10 %

0.10 1.41 % 0.63 70.11 4.81 %

0.10 2.78 % 0.71 79.21 2.73 %

We have studied the potentiometric response characteristics of the three sodium selective

membrane electrodes prepared in this study in order to probe the effect of adding CH-CuO

and CH-CuO-H layer on the potentiometric response behaviour of PVC based sodium

selective membrane electrodes. Sensing PVC membranes in three types of electrodes (PVC,

CH-CuO-PVC and CH-CuO-H -PVC) were prepared using 0.7 wt % sodium ionophore X,

33.0 wt% polyvinyl chloride, 0.2 wt% KTClPB and 66.1 wt% NPOE. The potentiometric

responses of the three membrane electrodes (PVC, CH-CuO-PVC and CH-CuO-H-PVC)

towards different cations are shown in (Figure 4-6). The response characteristics (e.g.,

selectivity, response time, linear range...etc.) of the these sets of sodium selective membrane

electrodes (PVC, CH-CuO-PVC and CH-CuO-H-PVC) are summarized in Table 2. Sodium

selective membrane electrode based on PVC (control membrane) showed sodium detection

limit of 8.0×10-6

M and had a linear response range from 1.5×10-5

to 0.135 M as shown in

(Figure 4). Also as shown (Figure 5) and (Figure 6) the detection limits of CH-CuO-PVC

and CH-CuO-H–PVC membranes are slightly higher than PVC based sodium selective

membrane electrodes. Table 2 showed that the detection limits (CH-CuO-H–PVC >CH-CuO-

PVC > PVC) shifted to higher sodium ion concentrations. Also, the lower limit of the linear

8

range (CH-CuO-H–PVC >CH-CuO-PVC > PVC) was shifted to higher concentrations.

Although there is a shift to higher concentration in case of CH-CuO-H–PVC and CH-CuO–

PVC, it is still much lower than sodium concentrations in physiological fluid which is 135-

145 mM. In case of CH-CuO and CH-CuO-H the diffusion layer thickness is larger than in

the classical PVC membrane electrode, due to the smaller stirring effect on the thin film of

aqueous buffer solution located between CH-CuO or CH-CuO-H and PVC. Due to increasing

in thickness in the diffusion layer, it is expected that sodium concentration at PVC sample

solution interface will increase. Therefore, the lower detection limit is shifted to high

concentrations of sodium. Similarly, shifting in the detection limits to high sodium

concentrations was notices in in case of heparin-modified CTA compared to unmodified CTA

based membrane electrodes formulated with sodium ionophore-X [Badr, et al., (2005)], as

well as in heparin-modified (HCH-PVC) and unmodified (CH-PVC) membrane electrode

[Badr, et al., (2014)]. The response slopes of CH-CuO membrane electrodes towards sodium

was 55.2.mV/decade and 56.4 for CH-CuO-H which are near-Nernstian. These observed

slopes of CH-CuO and CH-CuO -H are close to the control PVC membrane, (59mV/decade)

as shown in Table 2.

Table (2): Summary of response characteristics of sodium membrane electrode

NA: not applicable, amaximal selectivity limit(<) was used for ions that exhibited minimal response

towards interfernet ions [Bakker, et al., (2000)], bRequired selectivity coefficient for blood [Oesch, et

al., (1986)], ctherabutic level of lithium ion.

The response time is an important factor for any ion selective electrode. The effect of

introducing CH-CuO-H and CH-CuO on the response time of conventional PVC membrane

was studied to control PVC. As shown in Figure 7 PVC shows equilibrium response in a

short time with t90 value of 0.5 min for 21 mM sodium concentration. While it took more time

to reach the equilibrium for CH-CuO and CH-CuO-H with t90 value of 2.5 min for both

membrane electrodes. The slight increase in the response time of PVC- CH-CuO and PVC-

CH-CuO-H compared to control PVC is consistent with the higher detection limit observed

for those membrane electrodes. This is because more time is required for sodium ions to

penetrate CH-CuO and CH-CuO-H and reach PVC sensing film. It is worth mentioning that

modified membrane electrodes exhibited good reversibility (see data in Figure 8).

Figure 4. Potentiometric responses of sodium selective PVC membrane electrode toward different ions

measured in 50 mM Tris-HCl, pH 7.4. The cations tested are: (♦) sodium, (■) potassiom, (+)lithium,

(×)ammonium, (*)magnesium, and (-) calcium.

log K ± SD a Slope,

mV/de

cade

Detectio

n limit,

μM

Linear

range, M Na+ K

+ Li

+ NH4

+ Ca

2+ Mg

2+

PVC 0 -2.0±0.1 <-

2.8±0.2

<-3.6±

0.1

-2.4±

0.2

<-3.8±

0.1 59 8.5

1.5×10-5

to

0.135

CH-CuO

PVC 0

-

1.87±0.2

<-

2.8±0.2

<-

3.4±0.4

-2.2±

0.2

<-3.3±

0.3 55.2 22

5×10-5

to

0.135

CH-CuO–H

PVC 0

-

1.99±0.2

<-

2.6±0.3

<-

3.2±0.1

-2.2±

0.2

<-3.1±

0.4 56.4 39.8

7.9×10-5

to

0.135

Required

selectivity

coefficient b

0 < -0.6 < 0.1

<2.1c

<1.6 <-1.3 <-1.2 NA NA NA

9

Figure 5 Potentiometric responses of sodium selective CH-CuO-PVC membrane electrode toward

different ions measured in 50 mM Tris-HCl, pH 7.4.

Figure 6 Potentiometric responses of sodium selective CH-CuO-H-PVC membrane electrode toward

different ions measured in 50 mM Tris-HCl, pH 7.4.

Figure 7. Dynamic response time of sodium selective membrane electrodes toward increasing sodium ion

concentrations: PVC (solid black line), CH-CuO-PVC (dashed blak-line ) and CH-CuO-H-PVC (dashed

gray line).

Figure 8 Dynamic reversibility of sodium selective electrode PVC (solid line), CH-CuO-PVC (dashed

black-line ). The concentration of sodium ions switched for m 10-4

and 10-2

several times.

The most important character of membrane electrodes is the selectivity of the

membrane so it is important to probe the effect of CH-CuO or CH-CuO-H layer on the

selectivity of PVC-based sodium selective membrane electrodes. All of membrane electrodes

selectivity coefficients (PVC, CH-CuO -PVC and CH-CuO-H -PVC) were calculated at 1.0 M

concentrations and maximal selectivity coefficients are reported for interferent ions that give

significantly lower than Nernstian slopes [Bakker, et al., (2000)]. All data of selectivity are

shown in (Table 2). and all membrane electrodes potentiometric responses towards different

ions are represented in (Figures 4-6) as recommended [Bakker, et al., (2000)] the reported

data in Table 2 showed that the selectivity coefficients of CH-CuO or CH-CuO-H are

comparable to the selectivity coefficients of controlled PVC-based sodium selective

membrane electrodes. This proves that physical attachment as a layer of CH-CuO or CH-

CuO-H to PVC sensing film did not interfere with the ion-ionophore binding chemistry.

Presence of CH-CuO or CH-CuO-H did not change the binding constants of ion-ionophore or

single ion partition coefficient. It is worth mentioning that the selectivity coefficient of the

three sets of membrane electrodes (PVC, CH-CuO-PVC or CH-CuO-H-PVC) easily meet the

selectivity requirements for sodium analysis in physiological fluids (see data in the Table 2).

Platelet adhesion to the polymeric membranes pictured using scanning electron

microscopy (SEM) can be used to compare the thrombogenicity of different surfaces. Platelets

10

play a key role in the coagulation cascade and clot formation. Therefore, the adhesion and

activation of platelets on a surface is an indicator of its thrombogenicity. Platelet adhesion is

routinely used to investigate the interactions between materials and blood in vitro [Badr et

al., 2014; Berrocal, et al., (2001); Berrocal, et al., (2000); Espadas-Torre ,et al (1995);

Espadas-Torre, et al., (1997); Frost, et al., (2003); Schenfisch, et al., (2000)]. The relative

thrombogenicity of different membranes (PVC, CH-CuO-PVC or CH-CuO-H-PVC) were

investigated using the platelet adhesion protocol. Blood was obtained from the jugular vein

of a healthy sheep at the ministry of agriculture, (Cairo, Egypt). Each pint of blood was drawn

into a blood collection bag (JMS, Singapore, PTE Ltd.) containing 70.00 mL of CPDA-1 anti-

coagulant solution. Within 30 min after collection, blood was centrifuged at 240 g for 15 min

at 4 ºC. The supernatant was platelet-rich plasma (PRP) with a concentration of

approximately 9.5 ×104 cells /µL as determined with Exigo vet hematology analyzer. The

PRP was preincubated at 37 ºC for 20 min. All membranes (PVC, chitosan, heparin-modified

chitosan) were conditioned in phosphate-buffered saline (PBS, containing 10 mM phosphate,

138 mM sodium chloride, and 2.7 mM potassium chloride, pH 7.4) for 3 days, and were

immersed in PRP for 2 h at 37 ºC. They were then rinsed in PBS and fixed in a PBS solution

containing 2 % (w/v) glutaraldehyde for 2 h at room temperature. Membranes were

dehydrated by immersion in serial dilutions of ethanol (30, 50, 70, 90, and 95% v/v), two

immersions in absolute ethanol, and two immersions in hexamethyldisilazane. After solvent

evaporation within desiccators, the membranes were coated with gold and examined using

QUANTA FEG 250 scanning electron microscope (SEM). Micrographs and results showed

below representative of various membranes materials subjected to this procedure.

In vitro blood compatibility results are shown in SEM micrographs in Figure 9 A-C.

As can be seen in this figure a great difference of thrombogenicity or platelet adhesiveness for

different polymer materials is present under the same conditions. Large number of platelets

deposition and clusters presented in conventional PVC-based sodium-selective membranes

(Figure 9A). That refers to high thrombogenicity and less compatibility of blood to the PVC

membrane. Whereas, CH-CuO (Figure 9B) and CH-CuO-H (Figure 9C) showed smaller

number of platelets adhesion to the surface. A few platelets were observed on chitosan CH-

CuO or CH-CuO-H. As chitosan has rich free amino group with pKa value of about 6.3

[Rinaudo, et al (1999)]. When the pH is above the pKa, chitosan is uncharged due to the

protonation of the amino groups (e.g., measurement conditions, pH 7.4). As reported, the

neutralization of this charge decreased the platelet adhesion [Sagnella & Mai-Ngam (2005)].

Also, it is well-documented in the literature that blending polymers with surfactants exhibited

improved blood compatibility [Amiji (1995); Espadas-Torre& Meyerhoff (1995)].

Therefore, blending chitosan-CuO with softener that has surfactant properties, also the

measurement of the platelet adhesion was done at pH 7.4 is expected to improve chitosan-

CuO biocompatibility. Moreover, CuO nanoparticles has a good antifouling performance

[Chapman, et al (2013)] that expected to improve the biocompatibility by reducing or

limiting protein and cell adsorption as show in Figure 9B. Platelets are central in the blood

clotting cascade; preventing platelets from reaching PVC would effectively prevent

coagulation on the surface of PVC and improve the blood compatibility of ISEs based on

PVC. Slow release of heparin from CH-CuO-H and presence of CuO nanoparticles will

improve biocompatibility by minimize platelet adhesion and inhibit biofilm formation or

biofouling. This was further confirmed by platelet counts of PVC, CH-CuO and CH-CuO–H

membranes. A cell count form SEM micrograghs of five membrane segments of the same

membrane yielded an average 13.5 (±5.3) × 103 cells/mm

2, 0.21 (±0.14) × 10

3 cells/mm

2 (n =

5), and 0.15 (±0.05) × 103 cells/mm

2 (n = 5) for PVC, CH-CuO and CH-CuO–H membranes,

respectively (see data in Figure 10). This indicates that the number of platelets adhered to

PVC is about 64-fold higher than that found in CH-CuO and about 90-fold higher than that

11

observed for CH-CuO–H. As expected, SEM micrographs of PVC film showed large platelet

aggregates indicating a high thrombogenicity of this membrane [Badr et al. (2014)].

Figure 9. Scanning electron micrographs of various membranes after 2-h contact with PRP: (A)

PVC, (B) CH-CuO, and (C) CH-CuO-H

Figure. 10 Scanning electron platelet count for different membranes (PVC, CH-CuO, and CH-

CuO-H) after contact 2 hours PRP.

4- Conclusion

CH-CuO, CH-CuO-H were used to improve the blood compatibility of PVC-based

membrane electrodes. CH-CuO, CH-CuO-H were physically attached to the sensing PVC

layer. Three sets of electrodes were prepared: (1) conventional PVC membrane electrode, (2)

conventional PVC membrane electrode sandwiched with CH-CuO membrane (CH-CuO-

PVC), and (3) conventional PVC membrane electrode sandwiched with Chitosan-CuO-

Heparin membrane (CH-CuO-H-PVC). All potentiometric response characterization (e.g.,

detection limit, linear range, response slope, and selectivity coefficients) were studied to all

membranes and were found to be comparable to control PVC. This indicates that physical

attachment of CH-CuO and CH-CuO-H to PVC sensing film did not affect the potentiometric

behavior of sodium electrode.

Acknowledgement

The authors are grateful to Ain Shams University Scientific Research Sector (Grant 08-010)

for providing financial support to undertake this work.

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بالجسيمات النانوية لأكسيذ النحاس اغشية الكيتوزان المحمل تحسين التوافق الحيوى لغشاء قطب الصوديوم باستخذام

والهيبارينالنانوية لأكسيذ النحاس المحمل بالجسيمات والكيتوزان

3 حساو سس2يحذ جد عبذ انحهى 1 سانى يحذعبذ انب 1 أيش سانى حسا 1إبشاى حس عه بذس

س جامعة عين شم- كمية العموم-1 انشكز انقي نهبحث -2

يعذ صحت انحا- 3

:انهخص

وغشاء الكيتوزانبانجساث انات لأكسذ انحاس في هذا البحث نقوم بدراسة تاثير ادخال غشاء الكيتوزان المحمل

نهصدو رنك كهساذ ر انغشاء الإتقائ فم انبن غشاءوالهيبارين إلىانات لأكسذ انحاس المحمل بالجسيمات

كهساذ عه تخثش انذو فم نتحس انتافك انح تقهم انقذسة انعانت نهبن

( 2)كهساذ ، فم انغشاء الانكتشد انتقهذ نهبن( 1): تمت دراسة استجابة القوة القياسية لثلاثة مجموعات من المجسات

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جساث انات لأكسذ انحاس الكيتوزاني مركب كهساذ انغط بغشاء فم انغشاء الانكتشد انتقهذ نهبن( 3)

. انباس

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