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Talanta 77 (2008) 490–493

Contents lists available at ScienceDirect

Talanta

journa l homepage: www.e lsev ier .com/ locate / ta lanta

ydroxyapatite as a novel reversible in situ adsorption matrix for enzymehermistor-based FIA

alah Salmana, Srimathi Soundararajana,∗, Gulnara Safinaa,b, Ikuo Satohc, Bengt Danielssona,∗

Pure and Applied Biochemistry, Chemical Centre, Lund University, PO Box 124, Lund S-22100, SwedenFGU Federal Center of Toxicology, Radiology and Safety, 420075 Nauchnyi, Gorodok-2, Kazan, RussiaKanagawa Institute of Technology, 1030 Shimo-Ogino, Atsugi, Kanagawa 243-0292, Japan

r t i c l e i n f o

rticle history:eceived 15 November 2007eceived in revised form 19 March 2008ccepted 4 April 2008vailable online 12 April 2008

eywords:

a b s t r a c t

The application of the easily available and inexpensive chromatographic matrix hydroxyapatite forreusable and reversible immobilization of enzymes for enzyme thermistor-based flow injection analy-sis of glucose and urea was tested. The immobilization was achieved by simple affinity adsorption ofglucose oxidase and urease by a suitable pH-induced alteration of the protein charge. A linear detectionrange of 0.05–8.0 mM was observed for glucose estimation depending on the sensitivity and sample loopparameters with a detection limit of 0.05 mM. A broad detection range of 0.5–50 mM was observed for

ydroxapatitenzyme thermistorlow-injection analysislucose biosensor

mmobilization

urea using the flow injection calorimetric biosensor. Some real samples like commercial soft drink, syrups,honey and serum samples were analyzed. The novelty of the described work is the rapid set up of glucoseanalysis using hydroxyapatite as a reusable immobilization support in a flow injection thermal biosensorwithout any need for covalent immobilization or chemical cross-linking. The property of hydroxyapatiteto adsorb and desorb proteins as a function of the buffer pH and ionic strength makes in situ enzymereloading or exchange possible. The standard curves were obtained within few hours with a high degree

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. Introduction

Hydroxyapatite (HA) has been used in adsorption chromatog-aphy for many years. There are customized methods to exploitts unique characteristics. The elution of proteins adsorbed onydroxyapatite, a microcrystalline precipitate of calcium phos-hate Ca5(PO4)3OH is usually achieved by ascending gradient ofhosphate [1–4,7,9]. Hydroxyapatite chromatography has beensed for the purification and separation of acidic, neutral and basicntibodies [5,7–9]. Protein samples are normally loaded in low ionictrength buffer at higher loading concentrations of protein [2,3].asic proteins bind to hydroxyapatite strongly at lower pH dueo increased positive charge on the protein. The lower the bind-ng pH the higher will be the ionic strength of the eluting bufferequired to desorb the proteins. It has been reported that high

oncentrations of sodium chloride frequently encountered in ionxchange chromatography do not interfere with the protein absorp-ion onto hydroxyapatite [2–4,6]. The application of hydroxyapatiteor enzyme immobilization has already been reported for dextran-

∗ Corresponding authors. Tel.: +46 46 22 28 257; fax: +46 46 22 28 266.E-mail addresses: srimathi matoria@yahoo.com (S. Soundararajan),

engt.danielsson@tbiokem.lth.se (B. Danielsson).

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039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2008.04.003

me remained fully active even after 3 months.© 2008 Elsevier B.V. All rights reserved.

ucrase, glucosyl transferase, levansucrase and urease [10–13]. Inhis paper we combine hydroxyapatite with the established bioan-lytical calorimetric technique [15–18] to present a unique enzymeiosensor with the advantage and novelty of flow injection analysis.

Bioanalytical calorimetry exploits the exothermic nature of bio-ogical reactions to follow biological processes by measuring theeat released. The changes in the enthalpy associated with enzy-atic reactions is the basis of enzyme thermistor (ET) developed

nd successfully employed in a variety of practical applicationsuch as clinical diagnosis, process control, environmental and fer-entation monitoring [15–21,23]. A large number of enzymatic

eactions have been studied by this device among which glucoseetection was studied extensively using glucose oxidase mostlyovalently immobilized on controlled pore glass and packed inolumns [18–20,23]. This calorimetric sensor is a good alternativeo most of the electrochemical glucose sensors. Commonly usedlectrochemical sensors have the disadvantages such as enzymeeakage, denaturation of the enzyme on the electrode surface whichffects the life time of enzyme modified electrodes and media-

or leakage besides the interference from electroactive impurities24–26]. Drawbacks of the enzyme thermistor include relativelyigher instrumental complexity, limited commercial availability toate and use of rather expensive enzyme immobilization proce-ures (which on the other hand result in outstanding operational

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tability). Therefore, the construction of a good glucose sensortill needs improvement not only at the device level but also bymproving or innovating enzyme capturing. Most of the enzymemmobilization for biosensor applications involves covalent cou-ling to solid supports and thus requiring replacement of thentire biological recognition setup when the signal deteriorates.n the present work we have exploited the flow injection assistednalysis in ET (ET-FIA) for the first time to carry out on-site immo-ilization and sample analysis. We have chosen the well-knownhromatographic material ceramic hydroxyapatite as a reusable, initu adsorption support for enzyme immobilization and subsequentnalyte determination using the enzyme thermistor.

In this paper, we explore the use ceramic hydroxyapatite (CHT)lso known as macro-prep ceramic hydroxyapatite as a renewableatrix for repeated immobilization of enzymes for calorimetric

stimation of glucose and urea. Enzymes of clinical importanceuch as glucose oxidase/catalase couple (GOD/CAT) and ureaseere the candidate enzymes for the application. We emphasize the

uitability of this method for glucose estimation and a brief accountf how this method could be adopted for urease is also given. Themmobilization methodology, sensitivity, detection range, stabilitynd reproducibility are investigated.

. Materials and methods

.1. Materials

Glucose oxidase (1.1.3.4) 324–396 units/mg from Aspergillusiger from Biozyme, UK, catalase (E.C.1.11.1.6) 19,000 units/mg fromeef liver and jack bean urease (E.C.3.5.1.5) 63,000 �M units fromigma were used as obtained from the manufacturer. Ceramicydroxyapatite type I (80 �m in diameter, surface area 50 m2 g−1)as obtained from Bio-Rad laboratories. Glucose and urea (Sigma)ere used as standards. All other reagents were of analytical grade

rom Merck, Germany. All the solutions were prepared in ultra pureater (18.2 M�). Human blood sample was taken from a healthy

olunteer. Honey, soft drink and sodium saccharine were purchasedrom a local store. HemoCue Glucose 201+ analyzer was purchasedrom HemoCue AB, Sweden.

.2. Enzyme thermistor

Briefly, the enzyme thermistor employs enzymes as specific bio-ogical recognition elements. The biocatalyst is brought in closeroximity to a transducer, thermistor in this case, which can mea-ure the biological reaction and convert it into a physical signal. Theevice combines the selectivity of the biosensor with flow injec-ion analysis (FIA) for continuous monitoring of analytes. A detailedccount of the design, construction of the enzyme thermistor andhe principle behind the calorimetric determination can be foundlsewhere [15,16,18,20].

.3. Enzymatic reaction

The principle behind the calorimetric estimation of glucoses based on heat generated in the following enzymatic reactions18,20,23]. Glucose oxidase catalyses the oxidation of glucoseccording to the reaction:

H O + H O + O → H O + C H O (gluconicacid) + �H

6 12 6 2 2 2 2 6 12 7 1

he hydrogen peroxide is eliminated by the following reactionatalysed by catalase:

H2O2 → 2H2O + O2 + �H2

byans

7 (2008) 490–493 491

here �H1 and �H2 are the enthalpy changes during the enzy-atic reactions with glucose oxidase and catalase, respectively.

eduction of H2O2 by catalase provides additional heat thatncreases the sensitivity of the assay. Also this eliminates theresence of hydrogen peroxide which could otherwise affect thetability of the immobilized enzyme [16,18,20]. The other enzymeested namely urease catalyses the hydrolysis of urea according tohe following reaction [22]:

NH2)2CO + 3H2O → HCO3− + 2NH4

+ + OH− + �H

.4. Immobilization procedure

Ceramic hydroxyapatite (CHT) was thoroughly washed withmM phosphate buffer containing 0.3 mM CaCl2 at pH 6.5. Glucosexidase and catalase were non-covalently adsorbed/immobilizedn ceramic hydroxyapatite by ion exchange according to the follow-ng procedure: 4 mg of GOD (1500 U) and 2 �l of catalase (13,000 U)

ere dissolved in 2 ml 5 mM phosphate buffer (pH 6.5) and 1 mlf this suspension was mixed with 130 mg of CHT. The suspen-ion was gently mixed for 3 h at 4 ◦C. The protein concentrationn the solution was measured before and after immobilization byowry method to determine the immobilization yield [14]. Thenzyme thermistor column was packed with this suspension andhoroughly washed with the running buffer to remove the unboundnzyme and equilibrated in the same buffer. Immobilization of ure-se was carried out by shaking 130 mg of pre-washed CHT with thenzyme at a concentration of 1 mg/ml in 5 mM phosphate buffer atH 7.4 supplemented with 0.3 mM CaCl2. Both GOD/CAT and ureaseere also immobilized in situ by recirculating the enzyme solutions

or 2 h with the help of the flow injection set up in ET.

.5. Glucose and urea estimation

Glucose and urea standard solutions (0.005–60 mM) were pre-ared in 5 mM phosphate buffer at pH 6.5 and 7.4, respectively. Allhe measurements were carried out by injection of samples at aow rate of 100 �l/min. Sample loop sizes of 50 �l and 500 �l wereested. The thermal response to the enzyme reaction was measuredy integration of ET with a PC. The linear change in the thermalignal to varying substrate concentration was used to constructtandard curves. Average thermal response for three consecutivenjections was used in standard curve construction. For estimationf glucose in real samples, all the samples (commercial soft drink,oney and serum) were diluted in the working buffer and filteredefore analyzing. The soft drink was diluted and degassed prioro analysis. For glucose estimation in blood, the plasma requiredas prepared from whole blood by centrifugation at 15,000 rpm

or 10 min.

. Results and discussion

.1. Use of hydroxyapatite as a renewable immobilization matrixor FIA

In the present work hydroxyapatite as the cheap, quick andeusable support for the thermistor-based enzymatic determina-ion of glucose is examined. Urease was also tested to demonstratehat the method could also be used for other enzyme systems just

y changing the binding pH. Enzyme attachment to the hydrox-apatite support was facilitated by the chosen immobilization pHnd the protein charge. Depending on pH, HA has both positive andegative overall surface charge and hence provides multi-adsorbingites [1,5–7,27]. By knowing the pI of the enzymes of interest it was

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ossible to use an optimum pH without much trade off betweenhe immobilization yield and enzyme activity. The isoelectric pointf glucose oxidase is 4.2 and that of urease is 4.9. Acidic proteinsind to HA mainly by the carboxyl groups. The carboxyl groups areepelled electrostatically from the negatively charged sites and bindpecifically to the Ca++ sites of HA [6]. Therefore, co-immobilizationf glucose oxidase and catalase was achieved at pH 6.5. The immobi-ization yield was 80% for GOD/CAT couple and 70% for urease. Sincehe adsorption is due to electrostatic interaction it was possible toesorb the proteins bound on ceramic hydroxyapatite support byimply changing the pH or increasing the ionic strength of the elut-ng buffer. The enzyme columns placed in a flow injection enzymehermistor helps in easy desorption of the bound protein. The majordvantage is that when the enzyme column shows reduced activ-ty, it is possible to reload a fresh batch of GOD/CAT couple. The usef flow injection analysis makes the present work versatile sincenzyme reloading can be done essentially hands free. The immo-ilization can be carried out in the ET system by flow injectionechnique without the need to do a separate immobilization involv-ng time consuming activation, incubation and cleaning proceduress with covalent immobilization. We confirmed this by carryingut the adsorption in situ by circulating the GOD/CAT mixture at aow rate of 50 �l/min for 1 h. The thermal response obtained formM standard glucose for the flow injection assisted immobiliza-

ion (peak height = 43.5 mm) was comparable to that obtained foranually immobilized GOD/CAT (peak height = 44 mm). The appli-

ation of this method could be exploited to the maximum for therequently used enzymatic determinations of clinical and diagnos-ic importance.

.2. Protein adsorption and recovery

We have optimized the binding conditions for GOD/CAT-HT interaction. The quantity of protein bound on the surfaceas calculated by subtracting the amount of protein recovered

n the combined washings after adsorption from the proteinoncentration present before immobilization. At pH 6.5 wheremmobilization was carried out we found that 85% adsorptionccurred and the protein recovery was also >90% as measuredy Lowry assay. In the case of urease 70% of the protein loadedas adsorbed and >90% of the loaded protein was recovered. Pro-

ein recovery was achieved by washing the enzyme column with00 mM phosphate buffer at the same pH. The recovery of thenzymes by desorption is attractive for expensive enzymes andasy optimization of binding conditions without any wastage ofhe enzyme.

.3. Determination of glucose and urea

The linear range of detection for a sample volume of 50 �l and00 �l were found to be 0.5–8.0 mM and 0.05–1.0 mM, respec-ively (Fig. 1). The corresponding limits of detection (LOD) were.5 mM and 0.05 mM, respectively. The increase in sample vol-

(eruT

able 1iosensor characteristics of the CHT-ET-FIA for glucose estimation

nalyte Sample volume (�l) Linear range (mM) LOD (mM) Glucose in di

Softdrink (1:

lucose 50 0.5–8.0 0.5 0.57 ± 0.02b

500 0.05–1.0 0.05 (3.11 ± 0.06)c

a Dilution ratio used in the estimation. All dilutions were done in the running buffer.b Glucose concentration in pure, diluted samples.c Glucose concentration after spiking the diluted samples with 2.5 mM standard glucosd Value obtained using HemoCue Glucose 201+ analyzer, a commercial glucose estimat

ig. 1. Calibration curve for glucose. The sample volumes used were 50 �l and00 �l. Inset. Stability of thermal response over time for 5 mM glucose.

me by 10-fold though improved the LOD, 0.05 mM as opposed to.5 mM, resulted in reduced linear detection range. However, whensample volume of 50 �l was used the linear detection range was

xtended up to 8.0 mM. The increased sensitivity when using higherample volumes was evidenced from a higher signal response forhe same concentration of the analyte (Fig. 1). Thus, it is possi-le to improve the sensitivity of detection in dilute solutions justy increasing the sample volume to be injected. This is also use-ul when glucose needs to be estimated in dilute solutions. Thetandard curves were highly reproducible. Each data point in thetandard curve is the average of triplicate measurements. The oper-tional characteristics of the biosensor are presented in Table 1.omparison of our previous studies where GOD/CAT was covalently

mmobilized on to controlled pore glass (linear range 0.5–16 mM,OD = 0.5 mM) [19] showed that the upper limit of detection is low-red to 8.0 mM for CHT immobilization. Estimation of glucose ineal samples such as the commercially available soft drink, honey,rtificial saccharine and blood serum produced excellent reliabil-ty and reproducibility. These samples were diluted, spiked with anown concentration (2.5 mM) of standard glucose and comparedith the calculated glucose concentration in the pure samples. The

rtificial saccharine contained no glucose as specified by the man-facturer. No thermal response was observed for pure saccharines opposed to a significant and corresponding thermal responsehen spiked with various concentrations of standard glucose. The

xcellent agreement between the glucose estimated in pure sam-les and the spiked samples proves the reliability of the techniqueTable 1). The specificity and interference characteristics of the

nzyme system GOD/CAT has been shown in many of our previouseports [16–19,23]. The linear range of detection for urea estimationsing hydroxyapatite immobilized urease was 1.0–50 mM (Fig. 2).he sample volume used was 50 �l. Urease was used as another

luted real samples (mM, mean ± S.D.)

200)a Honey (1:2500)a Saccharine (0.5 mg/ml)a Blood serum (1:100)a

1.25 ± 0.03b 0b 5.44 ± 0.16b

(3.76 ± 0.09)c (2.50 ± 0.18)c (5.7)d

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ig. 2. Urea calibration curve. The data points are an average of triplicate measure-ents.

nzyme system to demonstrate the use of hydroxyapatite in theT-FIA. Immobilization of jack bean urease on HA has already beeneported to be efficient at pH 7.0–8.0 where the activity was mea-ured by a titrimetric procedure [13]. Urease immobilization on soilonstituents is of special interest because of the universal pres-nce of plant and microbial urease in the soil and their role inhe hydrolysis of urea used as a fertilizer [13]. The calorimetricIA method proposed herein could be of use in determining theydrolytic activity of soil urease for agricultural applications.

.4. Storage and operational stability

The enzyme system GOD/CAT is a robust bienzyme combina-ion and is known to be stable when immobilized [15,16]. However,he adsorption of these enzymes on hydroxyapatite is a new con-ept for flow injection analysis and hence we tested the columntability for a total period of 3 months. The detachable enzyme col-mn was stored at 4 ◦C in between measurements. It was foundhat the thermal response for the injection of 5 mM standard glu-ose was unaltered for 80 days (Fig. 1 inset). Therefore, a goodtorage stability of 3 months is guaranteed. In our previous workshere GOD/CAT was covalently immobilized to controlled pore

lass (CPG) by glutaraldehyde cross-linking we have achieved bet-er stability. The thermal response of CPG immobilized enzymesemained unaltered for 6 months [15,16]. However, the advantageith hydroxyapatite that it is easy to reload the enzyme and con-

truct standard curves in 2–3 h overcomes this lowered stability.his enzyme sensor also gave reproducible activity response forultiple sample injections (>150 samples) at room temperature

ndicating a useful operational stability.

. Conclusion

The novelty of the described work is the rapid glucose analysisn a flow injection mode without any need for covalent immobiliza-ion or chemical cross-linking. The immobilization can be carriedut in the ET system by flow injection technique without the need too a separate immobilization involving time consuming activation,

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7 (2008) 490–493 493

ncubation and cleaning procedures. The property of hydroxyap-tite to adsorb and desorb proteins as a function of the buffer pH andonic strength makes in situ enzyme reloading or exchange possible.he use of flow injection analysis makes the present work versa-ile since enzyme reloading can be done essentially hands free. Thetandard curves were obtained within 2–3 h and the system showedhigh degree of reproducibility and stability for up to 3 months.ompared to covalent immobilization the proposed method may

ead to higher loss of enzyme activity with time or higher risk forontamination from crude samples. The main anticipated applica-ion of the method is however, in cases such as studies of enzymereparation or inhibition and clinical applications, where a quicknd simple technique is desirable without the need for long-termse such as in. The use of hydroxyapatite in biosensors thoughorks well with the two enzymes studied, is limited by the protein

harge characteristics and pH dependence of activity and requiresn enzyme to enzyme variation in methodology to avoid biocat-lyst bleeding in a flow through system. But once established theethod will be suitable for regular analysis just like the proven

hromatographic procedures.

cknowledgements

SS and GF acknowledge Swedish Institute for the Guest Researchcholarship. Financial assistance from SIDA and Vinnova is alsocknowledged.

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