Bienzymatic Creatine Biosensor Based on Reflectance Measurement For

Sensors and Actuators B 269 (2018) 36–45

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Sensors and Actuators B: Chemical

jo urnal homepage: www.elsevier.com/locate/snb

Bienzymatic creatine biosensor based on reﬂectance measurement for

real-time monitoring of ﬁsh freshness

a b,∗ a

Farah Faiqah Fazial , Ling Ling Tan , Saiful Irwan Zubairi

School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul

Ehsan, Malaysia

Southest Asia Disaster Prevention Research Initiative (SEADPRI-UKM), Institute for Environment and Development (LESTARI), Universiti Kebangsaan

Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

a r a

t i c l e i n f o b s t r a c t

Article history: In view of the growing need for rapid and non-destructive method for monitoring of ﬁsh freshness to

Received 22 December 2017

secure the safety standard of food, a reﬂectometric biosensor kit has been developed for in situ and real-

Received in revised form 18 April 2018

time analysis of creatine as freshness marker. The optical bienzymatic creatine biosensor was fabricated

Accepted 24 April 2018

by covalent grafting of both creatinase (CI) and urease (Ur) on the pH chromoionophore ETH 5295-doped

Available online 30 April 2018

poly(styrene-co-acrylic acid) (PSA) latex microspheres via EDC/NHS coupling chemistry. An optical ﬁber

reﬂectance spectrophotometer was employed for reﬂectance measurement of the biorecognition phase

Keywords:

for quantiﬁcation of creatine levels, which can be used as the indicator of the extent of ﬁsh spoilage

Creatinase

Creatine for direct evaluation of ﬁsh freshness. The dynamic linear response range of the reﬂectance creatine

biosensor was obtained from 16 to 48 mM, which corresponds to the physiological range of creatine in

Optical biosensor

Reﬂectometric ﬁsh body surface metabolite with a reproducibility of less than 2% relative standard deviation (RSD). The

Urease biosensor response time was about 7 min and did not show interference by some important biogenic

amines (BAs), amino acids and nitrogen compounds normally co-exist with creatine in the bioﬂuid of

ﬁsh surface such as histamine, phenylalanine, tyrosine and taurine except sarcosine and uric acid.

1. Introduction mining the quality and the remaining life span of ﬁsh products

[1–3].

Monitoring of ﬁsh freshness is crucially importance to ensure Generally, ﬁsh spoilage involving a complex process in which

safety of ﬁsh for human consumption as the handling procedures physical, chemical and microbiological mechanisms could result

and storage conditions of fish and seafood products may influ- in the changes of the protein and lipid fractions of the flesh. The

ence their spoilage patterns. Conventional approaches by physical chemical and biological changes take place in the dead ﬁsh leading

examination through sensory evaluation of ﬁsh freshness attributes to the formation of nitrogen compounds (e.g. amino acid, uric acid,

based on the appearance of eyes, gills, skin, smell and colour as purines, methylamine, hypoxanthine, taurine, imidazoles, creatine

well as texture of meat can sometimes cause confusion as preser- and creatinine) and biogenic amines (BAs, e.g. histamine, tyramine,

vation of ﬁsh by treatment with certain chemical preservatives cadaverine, putrescine and phenylethylamine) as a result of pro-

such as formaldehyde, ammonia, sodium chloride, sodium nitrite tein degradation by endogenous enzymes into amino acids (e.g.

and sodium hydroxide can alter the physical appearance of ﬁsh. phenylalanine, tyrosine, sarcosine, etc.) as well as increase in the

Microbiological analysis method involves inoculation and incuba- total volatile basic nitrogen (TVBN, e.g. trimethylamine, ammonia

tion of samples in petri plates at appropriate temperature followed and dimethylamine). Determination of TVBN in ﬁsh ﬂesh consists

by colony counting to estimate the speciﬁc spoilage bacteria (SSB) of extraction of basic spoilage volatile amines by perchloric acid

count, however, requires a lengthy time to produce results in deter- followed by steam distillation of the extract collected in boric

acid with subsequent titration against a strong acid, such as sul-

phuric acid (H2SO4) or hycrocloric acid (HCl) [4,5]. Chun et al.

[6] had reported a pH sensor based on bromocresol green dye

immobilized in a polytetraﬂuroethylene (PTFE) membrane to qual-

∗

Corresponding author. itatively examine the TVBN level in the packaging headspace. A

E-mail addresses: [email protected] (F.F. Fazial), [email protected]

deﬁnite colour change of yellow to blue was adopted to monitor

(L.L. Tan), [email protected] (S.I. Zubairi).

https://doi.org/10.1016/j.snb.2018.04.141

F.F. Fazial et al. / Sensors and Actuators B 269 (2018) 36–45 37

the spoilage of ﬁsh product as the pH level changed in correlation the deprotonation of the immobilized pH chromoionophore ETH

with bacterial growth. Recent studies on the determination of ﬁsh 5295, and thus triggered a visible colour change of the immobilized

freshness have been focused on the investigation of BAs by high- pH indicator from blue (protonated form) to violet (deproto-

pressure lipid chromatography (HPLC), which is time-consuming nated form) at the latex micro-substrate surface, which could

and required specific instrument. Electrochemical BAs biosensor be determined by optical fiber reflectance spectrophotometer for

based on enzymatic electrodes, on the other hand, are limited quantiﬁcation of creatine concentration. So far, there has been

by their low sensitivity and high working potential leading to a no study reported on the creatine determination based on opti-

high level of interference and false results in the quantification cal biosensor method. Optical reflectance measurement of biofluid

of creatine concentration. In view of the different mechanisms metabolite concentrations i.e. creatine levels from ﬁsh surfaces

have been found responsible for ﬁsh deterioration during chilled could be a useful and non-destructive alternative marker of ﬁsh

storage, lipid hydrolysis and oxidation analysis has been reported freshness grading [24]. The proposed optical enzymatic biosensor

according to different lipid quality indexes i.e. lipid hydrolysis; can be utilized as portable colorimetric sensor for real-time, rapid

primary, secondary and tertiary lipid oxidation. However, the prin- and in situ monitoring of creatine in the bioﬂuid from ﬁsh body

ciple of above-mentioned analyses are destructive in application as surface as freshness marker simply based on colour change of the

the forces and chemicals exerted on the fish flesh are destructive sensor that is visible to the naked eyes. The schematic reflectomet-

prior to the measurement of the products resulting from ﬁsh post ric creatine sensor probe design is illustrated in Fig. 1.

mortem spoilage [7–9].

Creatine is a nitrogenous organic acid that occurs naturally in

2. Experimental

vertebrates. It is abundant in metabolically active tissue such as

muscle, heart, brain, blood and urine [10]. Creatine is classiﬁed as

2.1. Reagents and solutions

uremic toxin upon hydrolysis of creatine phosphate and adeno-

sine triphosphate (ATP) by phosphatase, thus creatine level in blood

Chromoionophore reagent (ETH 5294) from Fluka was pre-

serum and urine is an important factor in the assessment of muscle

pared by dissolving 0.1 mg chromoionophore in 1 mL of 99.5%

damage [11,12]. Huss [13] and Haard & Simpson [10] reported that ◦

ethanol, and vortexed at room temperature (25 C) for 10 min

the creatine content in ﬁsh muscle varies from 160 mg/100 g mus-

to obtain a homogenous blue-coloured chromoionophore solu-

cle to 720 mg/100 g muscle (i.e. 10–50 mM). FDA [14] advocated

◦ tion. Potassium phosphate buffer solution (PBS) was prepared by

the refrigerated ﬁsh (4 C) is safe for consumption for 2 days maxi-

mixing monopotassium dihydrogen phosphate (KH2PO4, Fluka)

mum. Nevertheless, there are ﬁshmongers and hypermarkets store

with dipotassium hydrogen phosphate (K2HPO4, Fluka). Ur (type

and sell ﬁshes up to 10 days as reported by Prince [15]. Various ana-

III from Jack Beans, SIGMA) and CI (from Pseudomonas, SIGMA)

lytical methods have been proposed for the creatine determination

enzymes’ solutions were prepared by dissolving 2 mg of the respec-

in human blood sample [11,16–18]. The most common approach

tive enzymes in 1000 ␮L of 20 mM PBS (pH 7.0), and kept in

is through spectrophotometric Jaffé reaction based on chemical ◦

the refrigerator at 4 C until further use. Styrene, acrylic acid, N-

reaction of alkaline sodium picrate with creatine [14]. The great-

hydroxysuccinimide (NHS), N-(3-Dimethylaminopropyl)-N’-ethyl-

est disadvantage of this method is that the non-speciﬁc biological

carbodiimide hydrochloride (EDC) and ammonium persulfate (APS)

substances present in the bioﬂuids may act as false positive in Jaffé

were obtained from SIGMA for preparing PSA microspheres.

reaction [18]. In addition, instrumental methods such as HPLC [19],

HPLC–MS [20], capillary electrophoresis [21] and IR spectropho-

tometry [22] for creatine determination require highly equipped

2.2. Instrumentation

instrument, extensive analysis time due to the special pre- and

post-ﬂushing procedures, high reagent consumption, specialized

A miniature ﬁber optic spectrophotometer (Ocean Optic SD

training and sample pre-treatment. Alternative methods by using

2000) was employed in this study for all reﬂectance measurements.

enzyme electrodes (i.e. creatinase and sarcosine oxidase) for poten-

This instrument consists of a tungsten halogen lamp as the light

tiometric and amperometric quantitation of creatine, whereby the

source, a CCD array detector, optical ﬁber as a light guide and com-

product of enzymatic reaction due to the oxidation of H2O2 dif-

puter software for data processing. Measurements of pH were made

fuses to the platinum electrode transducer and causes the electrical

by pH meter (Metrohm). Fourier transform infrared (FTIR) spec-

response [16]. However, one of the most challenging disadvantages

trum was recorded on ATR-FTIR spectrophotometer (Perkin Elmer,

of amperometric biosensor detection method is signal reduction −1 −1

UK) ranging from 4000 cm to 500 cm with 4 scans. Surface

from fouling agents and interference from chemicals present in the

morphology and size distribution of the dried PSA microspheres

sample matrix.

were examined with Zeiss Merlin/Merlin Compact/Supra 55VP ﬁeld

In this study, we report a new bienzymatic optical crea-

emission scanning electron microscope (FESEM) working at 3 kV

tine biosensor by using poly (styrene-co-acrylic acid) (PSA) latex

acceleration voltage and 30 kx magniﬁcation.

microspheres as the enzyme supporting matrix containing chro-

moionophore ETH 5294 pH indicator. Both creatinase (CI) and

urease (Ur) were covalently immobilized on the PSA copolymer 2.3. Synthesis of PSA copolymer microspheres

microspheres via carbodiimide linkage between amine (-NH2)

functional groups of enzymes and carboxyl (-COOH) functional PSA latex microspheres were prepared by emulsion copoly-

groups of latex particles [23]. The immobilized CI ﬁrst catalyzed the merization reaction as described by Yan et al. [25] with some

hydrolysis of creatine to sarcosine and urea [Eq. (1)]. The immobi- modiﬁcations. In brief, deionized water (190 g) was purged with

lized Ur then rapidly decomposed urea into ammonium ion (NH4 ), nitrogen gas in a three-necked ﬂask submerged in a water bath for

− − ∼

bicarbonate ion (HCO3 ) and hydroxide ion (OH ) [Eq. (2)]. 30 min at ambient temperature under stirring at 350 rpm. Some

0.5 g of acrylic acid was then added followed by adding 20 g of

+ creatinase ◦

Creatine H O → sarcosine + urea (1)

2 styrene after adjusting the temperature to 70 C. After that, about

urease + − − 0.2 g of APS was added into 10 mL of deionized water, and poured

+ → + +

Urea 2H2O 2NH4 HCO3 OH (2)

into the latex formulation in the three-necked ﬂask with vigorous

As the bienzymatic reactions involved a pH change to a more stirring for 7 h under nitrogen atmosphere to initiate the radical

−

basic condition ascribed to the production of OH ion, it led to polymerization reaction. The resultant PSA latex colloidal parti-

38 F.F. Fazial et al. / Sensors and Actuators B 269 (2018) 36–45

Fig. 1. Reﬂectance creatine biosensor probe developed from chemical immobilization of CI and Ur enzymes and physical adsorption of chromoionophore ETH 5294 on the

PSA latex microparticles surface.

cles were washed with deionized water twice by centrifugation at phenylalanine, sarcosine, tyrosine, uric acid and taurine towards

◦

13000 rpm for 20 min and oven dried at 40 C. the biosensing of 24 mM creatine in different molar concentration

ratios (i.e. 1:0, 1:3, 1:5 and 1:7) between creatine and interferent.

Signiﬁcant deviation in the reﬂectance response resulted by the

2.4. Fabrication of creatine biosensor

presence of interfering agent was estimated based on ± 5% devi-

ation from the relative reﬂectance intensity at 755 nm when no

The optical creatine biosensor was constructed by depositing

interfering compound was present in the determination of 24 mM

10 mg of PSA microspheres into the round cap of a 200 ␮L Eppen-

creatine by the enzymatic optical biosensor.

dorf microcentrifuge tube (with 8 mm diameter and 5 mm height)

and compressed manually to obtain a ﬂat surface. About 40 ␮L of

0.1 mg/mL chromoionophore ETH 5294 was then dispensed onto

3. Results and discussion

◦

the PSA microspheres, and dried overnight at 25 C. Some 10 ␮L of

200 mM EDC and NHS was later carefully smeared onto the chro-

3.1. Characterization of biochemically functionalized PSA

moionophore ETH 5294-coated latex microspheres before loading microspheres

the mixture of CI and Ur enzyme solution at a volume ratio of 1:1

(40 ␮L) onto the microspheres matrix. The enzyme-based biosen-

In general, the PSA microsphere has a core-shell structure

◦

sor was left overnight in a refrigerator (4 C) under dry condition to

mainly consists of hydrophobic polystyrene at the core segment,

permit covalent cross linking of the enzyme molecules onto the car-

and the shell composed of hydrophilic polyacrylic acid due to

boxylated PSA matrix surface to take place. Those physically bound

the presence of the carboxylic acid moieties and the hydrophilic

probes were then rinsed off with an ample amount of 20 mM PBS

residues of APS [25,26]. The particle size distribution of the immo-

at pH 7.0.

bilization supporting material largely depends on the APS initiator

loading and the ratio of styrene to acrylic acid monomers used [25].

2.5. Optimization of reﬂectometric creatine biosensor response When higher amount of hydrophobic styrene monomer is loaded,

the resulting latex microspheres tend to aggregate easily due to

The optical creatine biosensor was optimized with respect to pH gravity, and become sticky colloidal particles. Thus, surfactant is

effect, buffer capacity, dynamic linear range, response time, long sometimes needed to avoid hydrophobic surface bonding in order

term stability, reversibility and interference effect. The reﬂectance to obtain a well-dispersed colloidal suspension of microspheres.

reading was taken after a maximum reﬂectance response attained The FESEM image of PSA colloidal particles (Fig. 2a) shows

by the optical ﬁber reﬂectance spectrophotometer before and after uniform spherical shape and smooth surface morphology with

the enzymatic biosensor reacted with creatine. All the reﬂectace diameter range from 167 nm to 216 nm. The spherical morphology

measurements were carried out in triplicates. The effect of pH on of the latex microsphere resembles the desired three dimensional

the biosensor response was carried out by using 20 mM PBS in the (3D) shape that could serve to maximize the enzyme binding capac-

pH range of pH 4.0–10.0 towards the detection of 24 mM creatine. ity with large speciﬁc immobilization surface area. Additionally,

The PBS concentration effect on the creatine biosensor response the homogenous size distribution of the carboxylated latex micro-

was conducted by varying the PBS concentration between 10 mM spheres allow consistent amount of enzyme immobilized at the

and 100 mM at pH 7.0 in the presence of 24 mM creatine. The microspheres matrix surface, and promotes effective diffusion of

response time of the creatine biosensor was determined by measur- reactants and products to enhance the biosensor sensitivity per-

ing the reﬂectance response at 755 nm every minute for one hour formance [27].

in the determination of 24 mM creatine at pH 7.0. Shelf life study The FTIR spectra for styrene, acrylic acid, PSA latex microspheres

of the creatine biosensor was conducted by batch fabricating some and enzymes-immobilized latex microspheres are shown in Fig. 2b.

50 individual creatine biosensors, and three units of them were FTIR spectra of styrene and acrylic acid monomers show speciﬁc

examined with reﬂectance spectrophotometer using 24 mM crea- bands which correspond to their molecular characteristics absorp-

tine until a signiﬁcant decrement in the relative reﬂectance signal tion band, and similar to those reported by Boyer et al. [28] for

was obtained. Dynamic linear range of the creatine biosensor was the respective monomers. The PSA copolymer microspheres’ FTIR

determined by changing the creatine concentration in the range of spectrum displays several characteristic bands of both styrene and

−1

0.8 mM to 72 mM in 20 mM PBS at pH 7.0. The regeneration test acrylic acid. The absorption peak at 1709.24 cm is attributed

was performed by alternate incubating the creatine biosensor into to the non-bonded and hydrogen bonded carbonyl groups in the

24 mM creatine solution at pH 7.0 for 7 min and 20 mM PBS regen- acrylic acids segment of the copolymer [29,30]. The absorption

−1 −1 −1

eration solution at pH 7.0 for 10 min at room temperature, and this peaks at 1601.55 cm , 1583.77 cm , and 1492.84 cm are char-

procedure was repeated for ﬁve times. Interference study was con- acterized as the benzene ring stretching modes of the aromatic

−1

ducted by using a range of foreign compounds such as histamine, styrene. The absorption peak at 753.97 cm is due to the out-of-

F.F. Fazial et al. / Sensors and Actuators B 269 (2018) 36–45 39

Fig. 2. (a) FESEM image at 30 kx magniﬁcation of PSA latex microspheres synthesized via emulsion polymerization and (b) FTIR spectra of styrene, acrylic acid, PSA latex

−1

microspheres and enzymes-immobilized latex microspheres in the wavenumber range of 500–4000 cm .

plane C H bending mode of the monosubstituted benzene. The the PSA microsphere is an opaque substance, which is optically

methyl group of styrene is indicated by the C H bending vibra- non-transparent, and that it transmits no light, therefore it reﬂects,

−1

tion at 1451.86 cm [30]. The formation of C C covalent bond scatters or absorbs all of it. As such, measurement of its reﬂectance

from the polymerization reaction between vinyl functional group light intensity using reﬂectance spectrometry transducer is the best

(-CH CH2) of styrene and vinyl group of acrylic acid can be per- approach for solid-state biorecognition of creatine. The reﬂectom-

−1

ceived at the wavenumber of 1492.84 cm . Similar C C covalent etry transducer quantiﬁes the colour intensity whereby it reﬂects

bond was also acquired by Kosinska´ et al. [31] in the emulsion greater reﬂectance response with bright colour object compared to

polymerization to produce styrene-butadiene rubber copolymers. dark background colour substance.

When enzyme molecules (i.e. CI and Ur) were immobilized on Chromoionophore ETH 5294 is a lipophilic pH-sensitive indi-

the carboxylated latex particles surface, C O stretching band at cator thus having high compatibility with styrene, a non-polar

−1

1647.60 cm (Amide I) and C N stretching and N H bending at monomer of PSA microspheres. The cascade bienzymatic hydrol-

−1

1563.53 cm (Amide II) were observed. A broad peak obtained ysis of creatine and urea by the respective immobilized CI and

−1 −

at 3394.94 cm corresponds to the free O H stretch and N H Ur can result in the net release of OH ions causing deprotona-

stretch associated with the hydrogen bonded secondary amino tion of immobilized chromoionophore ETH 5294 pH indicator dye,

group [32,33]. and accompanied by a visible change in colour of the biosensor

from blue to violet. By loading a total of 40 ␮L of the mixture of CI

and Ur enzymes’ solution at a volume ratio of 1:1 onto the latex

3.2. Characterization of reﬂectance response between enzymatic

micro-substrate surface, it was sufﬁcient to evenly diffuse through

biosensor and creatine

the pH chromoionophore ETH 5295-doped PSA microparticles, and

gave uniform colour change of the biosensor from blue to violet

The PSA copolymer microspheres were used as enzyme support-

upon reaction with 24 mM creatine (pH 7.0). However, depositing

ing matrix, whereby the CI and Ur were chemically immobilized

the enzyme solution mixture at a volume lower than 40 ␮L was

on the carboxylated latex particles surface by using EDC/NHS cou-

noticed to be insufﬁcient to evenly cover the latex microspheres

pling strategy to form neutral amide bonds between carboxyls and

surface, whilst loading of the enzyme solution mixture above 40 ␮L

amines [11] to prepare the enzymatic biosensor for creatine. As

40 F.F. Fazial et al. / Sensors and Actuators B 269 (2018) 36–45

Fig. 3. Reﬂectance spectra of the creatine biosensor in the wavelength range of

350–850 nm (a) before and (b) after reaction with 24 mM creatine at pH 7.0 and

maximum reﬂectance wavelength of 755 nm.

onto the microspheres surface resulted in blocking of the enzyme

active sites, thus making some of the enzyme not participating in

the enzymatic reactions. Furthermore, excessive loading of enzyme

will also create a diffusion barrier to the movement of substrates

and reaction products. This was afﬁrmed as non-uniform violet hue

was observed with bare eyes after the addition of 24 mM creatine

at pH 7.0 onto the pH chromoionophore ETH 5295-modiﬁed latex

microspheres when the volume of enzymes’ solution mixture at

above or below 40 ␮L was used. Fig. 3 depicts the reﬂectance spectra

of the enzymatic biosensor based on PSA copolymer microspheres

in the presence and absence of creatine. The optical creatine biosen-

sor exhibited maximum reﬂectance intensity at the wavelength of

755 nm after reaction with creatine as bright violet colour back-

bround reﬂects better than dark blue. The maximum reﬂectance

wavelength at 755 nm was later used as the working wavelength in

the next optimization studies of the reﬂectance creatine biosensor.

3.3. pH effect, buffer capacity, response time and reversibility of

the creatine biosensor

The effect of buffer pH on the biosensor reﬂectance response

has been performed towards the reﬂectometric determination of

24 mM creatine in the pH range of pH 4.0-10.0 as both CI and Ur

enzymes have an optimum pH range between pH 6.0 and pH 8.0

[34,35]. The results obtained in Fig. 4a shows that there was an

increase in the reﬂectance response of the biosensor from pH 4.0-

7.0, after which the biosensor response decreased up to pH 10.0.

The decrement in the biosensor reﬂectance responses at pH values

below and above pH 7.0 were attributed to the change in enzyme

conformation with change in pH, whereby the enzyme active site

become inactivated, thus decreasing the enzyme activities [16].

Since maximum creatine biosensing response was obtained at pH

7.0, it was then chosen as the optimum pH in all subsequent

analytical biosensor measurements. This optimum pH value is con-

sistent with previously reported studies based upon potentiometric

and voltammetric creatine biosensors’ works reported by Karakus

et al. [11] and Tiwari & Shukla [32], respectively. However, differ-

Fig. 4. (a) pH trending of the creatine biosensor towards the reaction with 24 mM

ent optimum pH values were employed by Madˇ arasˇ ¸ et al. [17],

creatine in 20 mM PBS within pH 4.0-10.0. (b) Buffer concentration effect on the

Ramanavicius [18] and Stefan et al. [36] for creatine quantiﬁca- optical biosensor response at 755 nm using PBS concentration range of 10–100 mM

tion via amperometric electrochemical method due to different containing 24 mM creatine at pH 7.0. (c) Response time of the biosensor for the

determination of 24 mM creatine in 20 mM PBS at pH 7.0 and 755 nm and (d) regen-

polymeric substrates and immobilization methods were used.

eration proﬁle of the creatine biosensor for the detection of 24 mM creatine using

The effect of PBS concentration (pH 7.0) on the reﬂectance inten-

20 mM PBS (pH 7.0) as the biosensor regeneration solution (n = 3).

sity of the bienzymatic creatine biosensor is presented in Fig. 4b.

The optimum buffer capacity for optical detection of creatine was

acquired at 20 mM PBS. Due to the zwitterionic characteristic of the

enzyme molecules, the bienzymatic catalysis of hydrolysis of both

creatine and urea were higyly dependent on the local ionic strength

F.F. Fazial et al. / Sensors and Actuators B 269 (2018) 36–45 41

of the reaction medium. The use of PBS at concentrations lower or centration. The biosensor reﬂectance response reached a plateau

higher than 20 mM were found to be unfavourable for optimum between 48 mM and 72 mM creatine as the degree of violet colour

enzyme catalytic reaction. This was due to the fact that in both developed on the creatine sensor remained unchanged, which

low and high ionic strengths’ PBS media, the charge distribution at implies saturation of immobilized enzyme active sites with sub-

the enzyme active sites were disrupted, thus reducing the overall strates. The reﬂectance biosensor demonstrated a reproducible

enzymatic reaction rate [37], and that lower optical responses were linear response range to creation concentration from 16 mM to

obtained. 48 mM with a promising relative standard deviation (RSD) attained

The response time of the biosensor determines the length of at 1.8%. As the linear working range of the reﬂectometric biosensor

real-time analysis duration [38]. The response time curve in Fig. 4c is fell within the physiological range of creatine in ﬁsh body sur-

shows that up to 7 min, the creatine biosensor response increased face metabolite, therefore it holds enormous potential for real-time

abruptly with time at 755 nm, and the biosensor reached a steady and in situ colorimetric assay for creatine detection as freshness

state response thereafter until one hour of experimental time marker for ﬁsh using the naked eye. The biosensor limit of detection

period. The steady state condition could be assumed as the rate of (LOD) is deﬁned by the lowest concentration of creatine in which no

enzyme-substrate formation that is equal to its rate of breakdown changes in biosensor reﬂectance response was calculated accord-

resulting in plateau graph [27,39]. The biosensor response time at ing to the equation LOD = 3s/m, where s is the average of standard

7 min was later applied as the optimum exposure time that yields deviation of seven blanks and m is the slope of the calibration curve

the best biosensor response for the detection of creatine concentra- [47], which was obtained at 1.43 mM creatine.

tion. Reversibility study showed that the developed reﬂectometric Kinetic parameters of the enzymatic reaction can be esti-

creatine biosensor was capable to undergo two consecutive crea- mated by the direct linear method of the Lineweaver-Burk plot

tine analyses after regenerated with 20 mM PBS (pH 7.0) for 10 min, from the experimental data. The apparent Michaelis-Menten con-

and gave a promising reversibility RSD calculated at 2.9% (Fig. 4d). stant (Km) estimated from the Lineweaver-Burk plot was 16.2 mM

The creatine biosensor performance was noticed to drop to about creatine, and the maximum rate achieved by the system, at sat-

90% of its initial response after the second successive assay of cre- urating substrate concentration i.e. Vmax = 666.67 a.u. (Fig. 5b). In

atine, which may be attributed to the low enzyme stability and terms of biosensor stability, the optical creatine biosensor showed

activity after two consecutive regeneration cycles [40,41]. rather constant maximum biosensor response over a period of

19 days, whereby the enzyme activity was still maintainable to

3.4. Determination of dynamic linear range and biosensor shelf a greater degree in the immobilized state on the PSA copolymer

life microspheres solid surface, and it maintained its activity at approx-

◦

imately above 90% of its initial response after being kept at 4 C for a

We have previously reported on the LC–MS/MS analysis of tuna month (Fig. 5c). Nevertheless, the biosensor response declined sub-

ﬁsh (Euthynnus afﬁnis) surface metabolite composition, and cre- stantially after 19 days of storage period, which could be attributed

atine appeared to be one of the dominant nitrogen compounds to the degradation of the enzyme component of the biosensor

◦

throughout 7-day of strorage period under 4 C with ice ﬂakes to device during storage. A variety of spontaneous reactions and enzy-

mimic the storage condition applied by the ﬁshmongers and super- matic processes can occur in the solid-state biosensor at different

market ﬁsh counter. Generally, the physiological changes in ﬁsh rates, and resulted in chemical and enzymatic degradation of the

after it is caught encompass pre-rigor, rigor-mortis and post-rigor reagent or bioreceptor phase at the substrate surface. As such, the

stages. In pre-rigor stage, most of the creatine is phosphorylated in reﬂectance creatine biosensor has a satisfactory shelf life of close

the form of creatine phosphate, which would react with adenosine to 3-week of operational duration.

diphosphate (ADP) to form ATP i.e. the energy carrier molecule in

cells to render the ﬁsh body remains soft. Pre-rigor stage can vary 3.5. Interference study

from 1 day up to 3 days depending on the ﬁsh species, size, catching

method, feeding habit, temperature, handling of ﬁsh, storage con- Interference study was conducted to evaluate the effect of the

dition and the physical condition of ﬁsh [42–44]. Then, rigor-mortis presence of some potential interfering species commonly found

starts immediately or shortly after death, whereby the muscles of in the ﬁsh bioluids on the reﬂectance response of the biosensor

the ﬁsh begin to stiffen and harden due to the degradation of ATP against the detection of creatine concentration. In general, ﬁsh

and creatine phosphate by phosphatase as the proteolytic enzyme bioﬂuid is a mixture of mucus, serum, blood, urinogenital and

started to degrade the ﬁsh protein, and producing the creatine intestinal excretions [48,49], which contains a dominant substance

[13,45], which can be used as the indicator of the extent of ﬁsh of amines-synthesized nitrogen compounds especially amino acids

spoilage for evaluation of freshness of ﬁsh. Quantiﬁcation of the (i.e. phenylalanine, sarcosine and tyrosine), BAs like histamine and

creatine concentration in the residual liquid from tuna ﬁsh by HPLC some other nitrogen-containing organic compounds (i.e. uric acid

with UV detector showed that the creatine concentration increased and taurine). However, the amount of these potential interfering

from 30.7 mM on day-0 to 43.1 mM on day-4 during the rigor- substances may vary according to the type of ﬁsh [50]. Based on the

mortis process. After some hours, the muscles gradually begin to results tabulated in Table 1, only uric acid and sarcosine interfered

soften and become limp again, and the muscle was in the post-rigor with the detection of 24 mM creatine with the proposed bienzy-

condition due to the decrement of ATP content in cells, thereby matic optical biosensor at molar concentration ratio of 1:7 between

reduced the phosphatase enzymatic activity, and a decline in crea- creatine and interferent. Uric acid is the end product of purine

tine level between 25.2 mM and 39.5 mM from day-5 and onwards metabolism commonly present in ﬁsh serum in large amount

was observed [46]. (i.e. > 23.00 mM) in tuna-type ﬁsh species such as Euthynnus afﬁnis.

As Fig. 5a indicates, the reﬂectance intensity of the creatine Sarcosine, on the other hand, is the by-product of creatinase enzy-

biosensor increased with the increasing of the creatine concentra- matic reaction, whereby the typical physiological range of sarcosine

tion from 0.8 mM to 48 mM. The blue-coloured chromoionophore in ﬁsh is between 0.005 mM and 1.00 mM [51], which is about 100

ETH 5294 immobilized on the PSA latex microspheres surface times lower than physiological creatine levels in ﬁsh. Therefore, the

slowly become deprotonated, and changed to violet due to the presence of both uric acid and sarcosine in ﬁsh at high levels to give

−

increasing OH ion concentration that promoted a more alkaline signiﬁcant deviation in the optical creatine biosensor is not possi-

environment as a result of the bienzymatic catalytic reactions by ble. It was noticed that the presence of other potential interferents

immobilized CI and Ur with the increasing of the creatine con- at high levels (i.e. from 24 to 168 mM) gave no signiﬁcant devia-

42 F.F. Fazial et al. / Sensors and Actuators B 269 (2018) 36–45

Fig. 5. (a) Reﬂectance response of the creatine biosensor with (i) creatine concentration increased from 0.8 mM to 72 mM and (ii) the dynamic linear concentration range

of the reﬂectometric biosensor from 16 to 48 mM creatine.2 (b) Lineweaver-Burk plot of biosensor based on CI and Ur enzymes immobilized on the pH chromoionophore

ETH 5294-modiﬁed PSA latex microparticles. (c)3 Long term stability proﬁle of the creatine biosensor response over a 30-day experimental period using 24 mM creatine in

20 mM PBS at pH 7.0 (n = 3).

tion in relative reﬂectance signal during creatine detection with the ical molar ratio of taurine over creatine in ﬁsh is slightly higher

developed optical biosensor, and the typical levels of these interfer- (i.e. 24.00-64.00 mM) compared to the other interferents. However,

ing species in ﬁsh are far usually low e.g. histamine (0.18-9.00 mM), the presence of high levels of taurine did not signiﬁcantly interfere

phenylalanine (0.17–0.30 mM) and tyrosine (∼6.00 mM). The typ- with the quantitative determination of creatine. This indicates the

F.F. Fazial et al. / Sensors and Actuators B 269 (2018) 36–45 43

Table 1

The relative reﬂectance intensities obtained from bienzymatic biosensor developed using 10 mg PSA microspheres for the determination 24 mM creatine in the absence and

presence of various potential interfering species commonly found in ﬁsh bioﬂuids at pH 7.0 and wavelength of 755 nm (n = 3).

Interferent Relative reﬂectance intensity at different molar concertation ratios between creatine and potential interferent (a.u.)

1:0 1:1 1:3 1:5 1:7

Histamine 336.20 ± 3.08 325.18 ± 1.31 332.81 ± 3.81 338.15 ± 3.35 337.80 ± 2.93

Phenylalanine 336.20 ± 3.08 328.01 ± 2.68 348.03 ± 3.36 348.78 ± 4.71 332.55 ± 4.73

± ±

Tyrosine 336.20 3.08 333.33 4.74 333.99 ± 3.67 335.26 ± 4.45 337.55 ± 3.03

Sarcosine 336.20 ± 3.08 329.98 ± 4.18 333.80 ± 2.63 332.92 ± 3.97 304.69 ± 2.53*

Uric acid 336.20 ± 3.08 328.57 ± 2.68 330.93 ± 2.80 332.36 ± 2.96 379.13 ± 1.16*

Taurine 336.20 ± 3.08 345.24 ± 2.59 326.79 ± 3.14 335.99 ± 3.73 340.72 ± 3.95

a b c d

PSA 336.20 ± 3.08 337.76 ± 3.32 341.58 ± 4.28 345.53 ± 3.58 357.68 ± 1.52 *

*Biosensor response deviated at ± 5% from the relative reﬂectance intensity at 755 nm of the creatine biosensor without the presence of interfering substance.

relative reﬂectance intensity obtained using bienzymatic biosensor prepared from 14 mg PSA latex microspheres.

relative reﬂectance intensity obtained using bienzymatic biosensor prepared from 16 mg PSA latex microspheres.

relative reﬂectance intensity obtained using bienzymatic biosensor prepared from 18 mg PSA latex microspheres.

relative reﬂectance intensity obtained using bienzymatic biosensor prepared from 20 mg PSA latex microspheres.

Table 2

Comparison of creatine with other spoilage indicators for ﬁsh freshness grading and speciﬁc limitations associated with the respective methods.

Fish spoilage indicator Detection method Sample matrix type Limitations Reference

Creatine Bienzymatic reﬂectance Fish surface bioﬂuid Loss of functional conformation of enzyme at This study

biosensor extreme temperature and pH.

TVBN LED-based optical sensor Flesh TVBN amine compounds easily volatile at Pacquit et al. [5]

elevated pH and involves strong acid

extraction step using perchloric acid, which

may give rise to ﬁre and explosion risks.

Histamine Amperometric biosensor Flesh Requires reference electrode and high Pérez et al. [50]

signal-to-noise ratio.

Gill and eye colour changes Colorimeter and machine Gill and eye Colour intensity of gills and eyes may vary Dowlati et al. [54]

vision system between ﬁshes for the same species.

BAs HPLC Flesh Requires sample pretreatment, extraction and Park et al. [55]

derivatization steps (2–24 h).

Free fatty acid GC-FID Flesh Requires sample pretreatment, extraction and Aubourg et al. [56]

derivatization steps (2–24 h).

Bacterial count Total plate count Flesh Long incubation time for agar plate cultures Hernández et al. [57]

(18 h–5 days).

high selectivity of the reﬂectometric enzymatic biosensor towards value of ∼12.0 [53] may also change the pKa of the polymeric

creatine determination. system. Therefore, the carboxyl functional groups of carboxylated

The effect of PSA latex microspheres loading on the interference PSA microspheres have undergone deprotonation process during

of bienzymatic optical biosensor response towards the detection of polymerization reaction as well as during the dye loading process.

creatine was also examined due to the carboxylated PSA copolymer

microspheres surface which may undergo deprotonation process

3.6. Comparison of creatine marker with other indicators for ﬁsh

during deprotonation of the immobilized pH chromoionophore

spoilage evaluation

ETH 5295 whilst the bienzymatic redox reactions are taking place.

Based on the results tabulated in Table 1, there is no signiﬁcant dif-

Quality assurance in the ﬁsh sector involves monitoring and

ference between the relative reﬂectance responses obtained from

documenting deﬁned quality criteria as required by regulations,

the bienzymatic biosensors developed using 10 mg to 18 mg PSA

product speciﬁcations and consumer demands. Fish freshness is

micro supporting matrix. The biosensor response started to devi-

fundamental to ﬁsh quality which can be assessed by various indi-

ate at ± 5% from the relative reﬂectance intensity of the creatine

cators [54]. Table 2 outlines several freshness indicators, which

biosensor without the presence of interfering substance at 755 nm

have been adopted for evaluation of ﬁsh spoilage based on ﬂesh,

when the PSA microspheres loading increased to 20 mg, where

gill and eye sample matrices using sensory, physical, and instru-

the PSA latex microspheres become dominant compared to the

mental techniques. The levels of those indicators are depending

immobilized pH chromoionophore ETH 5295 and that the latex

on different biological and processing factors that inﬂuence the

microspheres surface was not fully occupied with the pH dye, and

degree of various physical, chemical, biochemical and microbiolog-

a high reﬂectance intensity was attained. It is important to note

ical changes occurring post mortem in ﬁsh [13]. Although physical

that when acrylic acid reacts with styrene to form poly(styrene-

technique based on gill and eye colour observation is often used for

co-acrylic acid), the pKa value of acrylic acid would increase from

rapid fresh ﬁsh quality assessment, however the colour changes

pKa = 4.20 to a higher pKa value as a result of reaction of vinyl func-

of gills and eyes may vary between ﬁshes for the same species

tional groups of acrylic acid with styrene. Lienkamp [52] has shown

[54]. Fish freshness assessment based on BAs and free fatty acid

the increase in pKa of acrylic acid from pKa = 4.25 to pKa = 6.80

indicators employing HPLC [55] and gas chromatography–ﬂame

after polymerization process forming polyacrylic acid. Besides, the

ionization detector (GC-FID) [56], respectively are labour-intensive

reaction between COOH of acrylic acid and the bulk water (H2O,

and time-cnsuming due to the inevitable sample pretreatment,

pKa = ∼15.74) during polymerization process also contributed to

+ extraction and derivatization steps. Optical measurement of TVBN

the increment of the pKa value, whereby the H ion from car-

[5] is almost instantaneous, however, TVBN amine compounds are

boxylic acid group is donated to H2O and forms carboxylate ion.

easily volatile and require strong acid extraction step that is suscep-

Chromoionophore ETH 5294, on the other hand, having a pKa

tible to explosion risks. Microbiological quality assessment of ﬁsh

44 F.F. Fazial et al. / Sensors and Actuators B 269 (2018) 36–45

Table 3

The comparison of analytical performance between the proposed reﬂectometric creatine biosensor and other previously reported creatine biosensors.

Immobilization matrix Detection method Linear range (mM) Detection limit (mM) Response time (min) Reference

PSA Reﬂectometric 16.0–48.0 1.43 7.0 Present study

Carbon rod electrode Amperometric 0.2–3.5 – < 1.0 Ramanavicius [18]

−9 −6 −9

Carbon paste electrode Amperometric 1.0 × 10 –1.0 × 10 2.00 × 10 0.5–2.0 Stefan et al. [36]

PVC Potentiometric 0.001−1.000 1.00 1.0–2.0 Karakus et al. [11]

PVC Potentiometric 0.1–30.0 0.01 1.5–4.0 Koncki et al. [58]

Chitosan-g-PANI Potentiometric – – – Tiwari & Shukla [32]

Polyurethane hydrogel Amperometric Up to 0.25 0.02 6.2 Madˇ arasˇ ¸ et al. [17]

−3 −1

poly(carbamoyl)sulfonate (PCS)-hydrogel Amperometric 1 × 10 –1.5 × 10 0.03 0.3 Schneider et al. [59]

by total plate count requires long incubation time for agar plate higher reﬂectace response with the increasing of the creatine

cultures [57] and electrochemical method prone to electrical inter- concentration as the biosensor changed to a bright-coloured

ference [50]. In general, most of the methods used for ﬁsh spoilage background. The reﬂectance optical detection through coloured-

indicator determination require the flesh of fish to be cut or sliced derivative method is beneficial for rapid and on-site detection of

away from the bone followed by chemical pretreatment or strong ﬁsh freshness. The proposed creatine biosensor could serve as a

acid digestion, and extraction procedure via ultrasonication and miniature biosensor kit for ﬁsh freshness application based on

centrifugation, which are destructive. The developed reflectomet- assessment of biological fluid from the fish body surface.

ric biosensor based on PSA copolymer microspheres would provide

an easy-to-operate means and non-destructive alternative method

Acknowledgments

for rapid and real-time estimation of fresh ﬁsh quality based upon

the creatine levels in ﬁsh surface bioﬂuid.

This work was supported by the National University of Malaysia

(UKM) via ‘Dana Impak Perdana’ research grant (DIP-2016-028) and

3.7. Comparison of reﬂectometric creatine biosensor performance

Malaysian Ministry of Science, Technology and Innovation through

with other reported creatine biosensors

E-Science Fund (06-01-02-SF1271).

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Biographies

and in samples collected from the roads using high-performance gel

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Farah Faiqah Fazial is a PhD student pursuing research on the physicochemical

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Ling Tan and Dr Saiful Irwan Zubairi in Universiti Kebangsaan Malaysia.

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Poly(acrylic acid-sodium styrene sulfonate) organogels: preparation,

Dr Ling Ling Tan received her ﬁrst-class honours BSc Degree in Chemical Technol-

characterization, and alcohol superabsorbency, J. Appl. Polym. Sci. 119 (2011)

ogy in 2006 and PhD Degree in Chemistry in 2009 in the areas of optical chemical

2759–2769.

sensor and electrochemical biosensor from Universiti Kebangsaan Malaysia. During

[34] M. Fidaleo, R. Lavecchia, Kinetic study of enzymatic urea hydrolysis in the pH

her PhD study, she attached to National University of Singapore for a collaborative

range 4–9, Chem. Biochem. Eng. Q. 17 (2003) 311–319.

research work in the areas of electrochemistry and analytical chemistry. She used

[35] D. Tsuru, I. Oka, T. Yoshimoto, Creatinine decomposing enzymes in

to be a technical assistant in the Department of Reliability and Integrity Engineer-

pseudomonas putida, Agric. Biol. Chem. 40 (1976) 1011–1018.

ing, Material, Corrosion and Inspection Section, Production and Corrosion Chemistry

[36] R.I. Stefan, R.G. Bokretsion, J.F. Van Staden, H.Y. Aboul-Enein, Simultaneous

Unit, PETRONAS Carigali Sdn Bhd-Sarawak Operations for 2 years. She was a Postdoc-

determination of creatine and creatinine using amperometric biosensors,

toral Researcher in UKM Chemical Sensor and Biosensor Research Group in 2011.

Talanta 60 (2003) 1223–1228.

Besides, she has been serving as a Senior Lecturer in Universiti Malaysia Pahang

[37] F. Kuralay, H. Ozyoruk, A. Yıldız, Potentiometric enzyme electrode for urea

from 2011 to 2013. Presently she is a Senior Lecturer/Research Fellow, and serving

determination using immobilized urease in poly(vinylferrocenium) ﬁlm,

as the Coordinator of Technological Hazards Programme in Southest Asia Disas-

Sensor. Actuat. B Chem. 109 (2005) 194–199.

ter Prevention Research Initiative (SEADPRI-UKM), Institute for Environment and

[38] B. Kovacs, G. Nagy, R. Dombi, K. Toth, Optical biosensor for urea with

Development (LESTARI), UKM. She has 12 years of research experience in the optical

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and electrochemical sensor/biosensor developments. Her research focuses on devel-

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