Thin-film polyamine biosensor: substrate specificity and application to fish freshness determination

Anafytzca Chmca Acta, 263 (1992) 93-100

93

Elsevler Scteoce Pubhshers B V , Amsterdam

Thin-film polyamine biosensor: substrate specificity and application to fish freshness determination Gabrlele C Chemmtms, Masayasu Suzulo and Knmyasu Isobe ’ GBF-Gesellschaft fir Bwtechnologzsche Forschung, Department of Enzyme Techmbgy, Mascheroak Weg 1, W-3300 Braunschwerg (Ge-y)

Jun I(lmura NEC Corporatwn, l-l, MtyazakzCchome, Myanwe-ku, kkwasakr 213 (Japan)

Isao Karube Research Centre for Advanced Scmce and Technology, Unrversay of Tokyo, 4-6-l

Komaba, Meguro-ku, Tokyo 153 (Japan)

Rolf D Schnud * GBF-Gesekhafi

fiK Bwtechnologmche Forschung, Department of Enzyme Technology Mascheroder Weg I, W-3300 Braunwhwetg (Germany)

Ah&net

A polyamme bloseosor was developed by unmoblllvng putrescloe oxldase on nucro planar thm-fko hydrogen peromde electrodes The sensor showed a rapld response to putrescme (90% response 40 s) and the relatlonshlp between putrescme concentration and output current was linear between 0 03 and 3 PM under the optlmum reactloo coodlttoos (pH 8 0, 30°C) The substrate specificity of the polyaoune sensnr was determtoed usmg a flow-mJection analysis system 10 addlttoo to the mam substrate putrescme, the sensor also converted cadaverme (46%), spemodloe (45%), agmatme (13%), 1,6-dlammohexane (3%), 1,7-dlammoheptaoe (2%) and tyraoooe (13%, non-eozymatlc response) These cooversloo rates were high compared wth those of soluble putrescme oxldase This ISprobably due to broadeomg of the enzyme’s actlve ceotre durlog the monobdlzat~oo procedure The sensor was apphed to fish freshness deterounafioo durlog a fish storage expenmeot The sensor response obtaloed m fish extracts increased with mcreaslog storage tooe and agreed wth hqmd chromatographtc aouoe deteromtatlons Keywords Amperometry, Flow system, Bloseosors, Eozyme electrodes, Rsh, Polyaounes, Putrescme

Simple and rapld techmques for the estrmatlon of food freshness are m great demand by the food Industry Dunng storage of foodstuffs blogemc ammes may be formed owing to the degradation of proteins and ammo acids by nucroblal proteolytlc and decarboxylatmg actlvlty [ll In fish and

1 On leave from Amano Pharmaceutical Co, Ltd , Nagoya, Japan

processed meat products mcreasmg levels of blogemc ammes, of which putrescme and cadaverme are the major malodorous compounds, are regarded as chemical mdlcators for decreasmg freshness and quality [21 Many methods for the determmatlon of polyamme concentrations m foodstuffs and blological flulds are based on chromatographlc separations of the different ammes, either by gaschromatography (GC) [3] or hqmd chromatography

0003-2670/92/$05 00 8 1992 - Elsevler Scleoce Pubhshers B V All nghts reserved

94

G C Chemmtuu et al /Am!

(LC) [4-61, combmed with their pre- or postcolumn derlvatlzatlon As these methods are time consummg and require complicated and expensive mstrumentatlon, several methods for the measurements of ammes using amme oxldases have been proposed The enzymes catalyse the oxldatlon of ammes to the corresponding aldehydes RCH,NH,

+ 0, + H,O m

RCHO + NH, t H,O, To reduce the costs of enzymatic amme assays, unmobdlzed enzymes should be applied which can be recovered from the assay solution Therefore, blosensors using various kmds of transducers and unmoblhzatlon techmques have been developed For their construction dlamme oxldase (from pea seedlings) has been incorporated mto carbon paste [7] or mnnoblhzed on controlledpore glass [81, platinum [91 or oxygen electrodes [lo] Putrescme oxldase (from M~rococcus dens) has been used m its soluble form m combination with an oxygen electrode [ill and has been nnmobdlzed on thick-film platinum electrodes [ 121 Mmaturlzatlon of the blosensors offers several advantages First, only a small amount of enzyme 1s required for sensor construction Second, mass production of such mlmaturrzed blosensors is possible and consequently disposable-type blosensors may be reahzed Third, mtegratlon of several different enzyme electrodes on one sensor will be possible Hence, mmlaturlzed electrodes produced by thick- [13] and thm-film [14] technology will become mcreasmgly unportant for the construction of mmlaturlzed blosensors In this study, putrescme oxldase from Mlcrococcu.s rubens was used to construct a micro putrescme sensor based on a nucro planar thin-film hydrogen peroxide electrode The selectivity of the electrode to hydrogen peroxide was enhanced by deposition of a cellulose acetate membrane The sensor was tested wth respect to the substrate speclfiaty of the unmoblhzed putrescme oxldase m a flow-mJectlon system and applied to fish freshness determmatlon

Chun Acta 263 (1992) 93-100

EXPERIMENTAL

Reagents

Putrescme oxldase (E C 1 4 3 10, from Mzcrococcus rubens) was kmdly donated by Amano Pharmaceutical (Nagoya) Ammes and amme hydrochlorldes were purchased from Sigma (St Louis, MO) Amine standard solutions (0 1 M) were prepared by dlssolvmg the ammes or their hydrochlorldes m 0 1 M HCl (Merck, Darmstadt) Cellulose acetate and glutaraldehyde (25% aqueous solution) were obtained from Fluka (Buchs) and Serva (Heidelberg), respectively Bovme serum albumin (fraction V (BSA), 2,2’-azmobls (3-ethylbenzthlazolmesulphomc acid) (ABTS) and horseradish peroxldase, grade II (POD), were purchased from Boehrmger (Mannhelm) All other reagents were of analytical-reagent grade Micro hydrogen peroxuie electrode The procedure for the fabrication of the micro

planar hydrogen peroxide electrodes has been described previously [15] As shown m Fig 1, the electrode has two gold workmg electrodes and a

1 6 mm

b-b’

2

4

2ihY a-a’

Rg

1 Structure of the nucro hydrogen peroxide electrode 1 = Counter electrode (gold), 2 = workmg electrode (gold), 3 = photoreslt membrane, 4 = sapphire substrate

G C Chemmtauset al /Anal Chm Acta 263 (1992) 93-100

surroundmg gold counter electrode A gold layer (1 pm thlck) was deposited on the sapphire substrate by the sputter method, through the presputtered tltanmm layer To Improve the selectlvlty to hydrogen peroxIde, a cellulose acetate layer was formed on the electrode surface Cellulose acetate (0 2 g) was dissolved m a mtiure of acetone (3 ml) and cyclohexanone (2 ml) The electrodes were dipped mto the polymer solution and were removed and dried overnight

Testfor specrfic@~detennrnatwnof solubleputrescmeoxuiase For the determmatlon of the speclflclty of soluble putrescme oxldase, the hydrogen peroxide generated by the enzymatic conversion was measured spectrophotometrlcally at 420 nm using ABTS as the chromogenic reagent The reaction mixture was thermostated at 35°C m the spectra-photometer and contamed 25 ~1 of 0 1 M amme solution, 525 ~1 of 02 M Na,B,O,KH,PO, buffer (pH 8 5) and 400 ~1 of 5 mM ABTS-20 U ml-’ POD dissolved m this buffer The measurement was started by the addition of 50 ~1 of diluted putrescme oxldase solution (1 1000) The increase m absorbance was recorded over a period of 1 mm One umt of putrescme oxldase catalysed the generation of 1 Fmol of H,O, per rmnute using the substrate putrescme

95

Enzyme rmmobduatwn The enzyme was mnnoblllzed by a method based on glutaraldehyde-albumm crosslmkmg Before nnmobtiatlon, the enzyme solution was dlalysed against 0 1 M phosphate buffer (pH 8 0) overnight A lo-p1 volume of dlalysate and 10 ~1 of 10% BSA solution were well mixed, then 10 ~1 of 5% glutaraldehyde were added After rapid mung, 5 ~1 of the mixture were dnpped onto the electrode and allowed to dry for 1 h Finally, the electrode was washed wth phosphate buffer and stored at 4°C Measurementeqwpmentand procedure A two-electrode system was employed The amperometrlc measurement system consisted of a rmcro H,O, electrode, a polarizer (Type E585, Metrohm, Herrsau, Switzerland), an x-t recorder (Type 2045, Lmseq Selb, Germany) and a water-bath (Type 000-8700, Haake, Karlsruhe) with a thermostated cell Prehmmary experunents using cychc voltammetry showed that the optlmum polarlzatlon potential was 10 V for this system (gold workmg and gold counter electrodes) Thus the workmg electrode of the nucro H,O, electrode was polarized at + 10 V against the counter electrode The enzyme-nnmobdlzed rmcro H,O, electrode was dipped mto 30 ml of 0 1 M Clark and Lubs solution (0 1 M H,BO,-KC1 + NaOH 1161) (pH 8 0) at 30°C After the output current had

waata waste amin solut

Fig 2 FL4 set-up for the detenmnatlon of the selectlvlty of the thin-fdm polyamme sensor The sensor was Incorporated mto a flow-through cell Every 7 mm amme samples were injected mto the tamer stream [flow rate 1 ml mm-‘, 0 1 M Clark and Lubs solution (pH 811 Amine samples were automatically supphed by the sur-wayvahre

G C Chemmtruset al /Anal Cham Acta 263 (1992) 93-100

96

become stable, standard amme solutions of 0 1, 1 or 10 mM concentration were added stepwlse and the output current was recorded For real sample measurements, 5 ~1 of 0 1 mM putrescme solution were added to 10 ml of buffer followed by 50 ~1 of fish extract (see below) and a second addltlon of 5 ~1 of 0 1 mM putrescme standard

30.

-$

25.

Flow-zn]ectwn andyszs system

A modular flow-mJectlon analysis (FIA) system developed at the GBF [17] according to Fig 2 was used for the determmatlon of the speclflclty of the lmmoblhzed putrescme oxldase The enzyme electrode was placed m the flow-through cell The flow-rate of the carrier stream (0 1 M Clark and Lubs solution, pH 8) was set at 1 ml mm-’ Every 7 mm amine solution was inJected mto the carrier stream Ahquots of each amme sample were inJected ten times Automatic sample exchange was accomplished by means of a six-way valve (Latek, Heidelberg) The switching of the inJectIon valve and the six-way valve was controlled by an industrial timer (Klockner and Moller, Bonn) Fzsh storage expenments

Pollack was purchased from a local fish shop and stored at 4°C for 10 days Extracts of fish sample were prepared m 10% trlchloroacetlc acid for amme determination For LC measurements the extracts were cleaned up by ion-exchange chromatography [6] Llquzd chromatography

Polyamme concentrations m cleaned up fish extracts were determined using ion-panmg reversed-phase LC [6]

RESULTS

Batch system

Hydrogen peroxide generated by the enzymatlc reaction was detected amperometrlcally by the micro gold thm-film electrode Using the batch system, the electrode response was optlmlzed with respect to pH and temperature using

05

00’

1

, 70

I

,

75

80

I

85

PH

Fig 3 Dependence of the polyamme sensor response on pH Putrescme concentration, 17 PM, 0 1 M Clark and Lubs solutlon. 37°C

the mam substrate putrescme of the putrescme oxldase from Mzcrococcus rubens The sensor responded to putrescme very rapldly, the 90% response time was ca 40 s Effects of pH and temperature The influence of pH on the output of the sensor was exammed at various pH values (Fig 3) The optimum pH was found to be 8 0, and was adopted m subsequent expernnents The Influence of temperature on the output of the sensor was also mvestlgated (data not shown) The sensor response increased with mcreasmg temperature, but above 35°C the rate of Increase dlmuushed In subsequent experiments, a temperature of 30°C was employed for maintenance of sensor stab&y Putresczne cahbratzon The cahbratlon graph for the micro putrescme sensor IS shown m Fig 4 The 2a detection lnmt was 0 03 PM A linear detectlon range up to putrescme concentrations of 3 PM was demonstrated The sensltlvlty of the sensor was 19 nA PM-’ FL4 system Calzbratzon The sensor response was evalu-

ated by peak-height measurements Figure 5 depicts cahbratlon graphs of the amme sensor for putrescme, cadaverme and spermldme obtamed with the FIA system The sensor response was

G C Chemnrturset al /Anal Chm Acta 263 (1992) 93-100

xfj

60-

:: e P ; L

40-

20 -

0-l 0

50

100

150

200

0

250

linearly dependent on the amme concentration over the range of 0 l-l mM for putrescme kensitlwty 25 3 nA mM_‘), 0 1-O 8 mM for spemudme (sensltlvlty 8 4 nA mM_‘) and 0 1-O 9 mM for cadaverme (sensltWy 8 5 nA mM_‘) The detection lnmt for putrescme was 0 01 mM Selectivity of the micro putrescine sensor The response of the polyamme sensor towards various ammes was Investigated at an amme concentration of 0 2 mM, which 1swlthm the linear cahbratlon range The mean values and the standard

I

4 tmw

putresclneconcentmtlon [ pk.4 I Fig 4 Cahbratlon graph for putrescme obtamed m the batch system 0 1 M Clark and Lubs solution (pH 8),3O“C

I

2

-i 6

6

[days]

Rg 6 Stab&y of the nucroputrescme sensor Putrescme cuncentratlon, 17 PM, other condltlons as m Fig 4 Imtral response (lOa%), 3 3 nA

devlatlons of the selectlvltres of five mdlvldually prepared sensors are gwen m Table 1 For each sensor and each amme a sequence of ten mjectlons of putrescme (100% value), ten injections of the amme to be tested and a further ten mJections of putrescme were used for selectlvlty determmatlon In addition to putrescme the sensor responded to spendme, cadaverme, tyramme, 1,6-dtammohexane, l,%dlammoheptane and ag-

zooLlo. %

50 -

150 E E z3

40-

z

; x)-

i! 3 Q

zo-

zt x 8

-2

e

5

10 -

100 -

50-

01 0

0 0

12 amme

I

I

I

3

4

5

concentratwx7

I

,

6 [ mM

7 ]

Fig 5 Cahbratlon graphs for (0) putrescme, (4 1 cadaverme and ( n) spemudme obtamed wth the FIA system

2

4 storage tlme

6

6

10

[ daya ]

Fig 7 Fsh storage expenment comparison of amme concentratlons m fish flesh obtamed by (0) the thm-film polyamme sensor and (0) LC Values are given as apparent putrescme content m pg g-’ fish flesh

98

G C Chemmttus et al /Anal

Chtm Acta 263 (1992) 93-100

TABLE 1 Selectlvlty of soluble and unmoblhzed Amme

putrescme

Compound

type

oxldase from ~crucoccus

rubens a

Selectlvlty (o/o) Response of enzyme electrode in FIA b 0 2 mM amme

0 2 mM putrescine

Soluble enzyme 25mMamme

+02mMamme 1,2-Diammoethane (ethylenedlamme)

Dlammes

1,3-Dlammopropane 1,4-Dlammobutane (putrescme)

0 0 100

loo*2 100 200

0 0 100

1,5-Dlammopentane (cadaverme)

46 f 8

130 f 15

6kl

1,6-Duunmohexane 1,7-Dlammoheptane 1,8-Dlamumoctane

3*1 2 0

99* 1 102 100*2

05 0 0

Monoammes

Methylamme Ethylanune n-Propylamme n-Butylamme n-Pentylamme n-Hexylamme Dlmethylamme Tnmethylamme

Aromatlc

Benzylamme Phenylethylamme mamme Hlstamme

1*1 0 13 f 3 1*1

101 f 4 1cQfl 107 f 7 99 f 7

1 0 0 0

Spernudme Spermme

45 f 6 lltl

124f15 80 f 20

11*3 0

Agmatine

13*4

112 f 6

5*1

Polyammes

ammes

102*2 102f4 98 f 3 104*4 102 f 6 101 f 4 102 99*5

B The selectwlty of the munobdlzed putrescme oxldase was determined using the thin-film polyamme sensor m the FIA system The response towards various ammes (0 2 mM) was tested m the absence and presence of putrescme (0 2 mhf) The selectwlty of the soluble enzyme was determmed by the spectrophotometrlc method m cuvettes b Mean f S D (n = 50, 5 sensors Hrlth 10 uqectlons each)

matme, but not to ahphatlc monoammes The selectlvlty of the sensor was also tested m the presence of 0 2 mM putrescme Almost addltlve responses were obtamed to murtures of putrescme with tyramme, cadaverme, agmatme or spernudme In the absence of putrescme oxldase most of the ammes, mcludmg putrescme, cadaverme, agmatme and spendme, showed no sensor response Benzylamme, spermme and histamme, however, showed a slight response and tyramme was oxldlzed at the electrode to an extent of 13% compared wrth an equnnolar hydrogen peroxide sample

Stabzhty of the putrescme sensor Figure 6 shows the micro putrescme sensor response over 1 week m the batch system The sensor response was still above 80% of the uutlal value after 8 days In the FIA system the enzyme electrode was used contmuously over a period of 2 weeks with a sampling frequency of seven mjectlons per hour and dally cahbratlon Applrcatzon of the method to jkh extracts As 1s evident from selectlvlty measurements, the amme sensor provided a combmed response for several ammes The amme sensor wdl re-

G C Chemmtrus et al /Anal

Chum Acta 263 (1992) 93-100

spond enzymatrcally to putrescme, cadaverme, spernudme and agmatme and non-enzymatrcally to tyramme Therefore, these amrnes were separated and quantrtattvely detected by the LC reference method Their concentrattons m fish samples were summed and used as LC data Figure 7 shows a comparison of apparent putrescme contents, given m pg g-’ fish flesh, obtained by the amme sensor and by LC during fish storage experlments The amme content of the pollack sample increased from ca 25 pg g-’ at the beginning of the storage period to ca 190 pg g- ’ by the tenth day LC and brosensor data curves showed very similar trends Trrchloroacettc acid used for fish extraction dtd not interfere with amme determination Repetrtrve brosensor measurements mdrcated high reproduabrhty (e g , seventh day 130 + 1 /.I,gg-l, n = 3) DISCUSSION

Polyammes containing a 4-ammobutyl group are known to be preferred substrates of soluble putrescme oxrdase from Muzrococcus rubens From mhtbrtor bmdmg and substrate specificity, Swam and Desa [181 and Okada et al I191 deduced that the active site of the putrescme oxrdase contams a hydrophobic bmdmg region, an anionic pomt and a separated catalytic oxtdatton site which are arranged so as to match the drmensrons of the 4-ammobutyl group As monoalkylammes are not converted by the native enzyme and the enzyme can rapidly be mactrvated by modrfrers of carboxyl groups [20], a carboxyl group seems to be mdtspensable as the amomc point for substrate bmdmg Hence, Okada et al [19] concluded that the essential structure for substrates of putrescme oxldase is an ammoalkylammo group, [H,N(CH,),_,NH]R The present fmdmg that agmatme, which contams a 4ammobutyhmmo group, was oxrdrzed by putrescme oxldase m solutron as well as by the polyamme sensor supports the above model of the enzyme’s active site, m contrast, Yamada [21] reported that agmatme was not converted by putrescme oxtdase The FIA system has proved to be advantageous for the determmatron of the selecttvrty of

99

rmmobrhzed putrescme oxtdase towards a large number of dtfferent ammes A compartson of the substrate specrftcttres of soluble and lmmobrltzed putrescme oxtdase (Table 1) reveals that putrescme was the mam substrate of both enzyme forms and that the other substrates of the soluble enzyme are oxrdrzed to a higher extent by the unmobrhzed enzyme Compared with the conversion of putrescme, which was set at lOO%,the conversion of cadaverme (lJ-dtammopentane) was shifted from 6% for the enzyme m solutron to 46% for the unmobrlrzed enzyme The conversron of 1,6-dmmmohexane was also higher for the munobrlrzed enzyme 1,7-Drammoheptane was not oxrdrzed at all by soluble putrescme oxrdase but was oxldrzed by the rmmobrlrzed enzyme Substrates such as spermrdme and agmatme, whtch have the structure [H,N(CH,),NH]R and a large substrtuent R, are also converted to a higher extent by the tmmobtlrzed enzyme than by the enzyme m solutron This might be due to an overall broadening of the enzyme’s active site during the enzyme munobrlrzatlon procedure Especially elongation of the regron between carboxyhc bmdmg site and oxtdatrve reaction site 1s likely because substrates with carbon chains longer than C, were converted more easily by the rmmobrhzed enzyme The polyamme sensor presented has a lower detection lrmrt for putrescme than other reported systems [7,9,11,121 Therefore, putrefaction can be estimated at a very early stage Although the sensor response was not specific for one amine, it has been used successfully as an mdrcator of fish freshness, because the other ammes detected are closely lurked with the putrefaction process For each amme measurement the brosensor needed only a small amount (50 ~1) of tnchloroacetrc acid extract Btosensor amme measurement was completed within 15 mm, mcludmg cahbratron, whereas LC analysrs took 70 mm Another advantage m using the bmsensor was that no further time-consuming clean-up of the fish extracts was necessary Nevertheless, LC or GC will remam the methods of choice rf analyses of amme composition are required The micro hydrogen peroxtde electrode used

100

was mass produced using semiconductor fabrxatlon technology and it could be supphed at low cost Hence the sensor represents a promlsmg step towards the reahzatlon of disposable-type freshness sensors which may fmd apphcatlon m screenmg methods for fish freshness determmatlon at the place of sale

REFERENCES S L Taylor and S S Summer, m DE Kramer and J LIston (Eds ), Seafood Quality Determmahon (Developments m Food Saence, Vol 15), Elsevler, Amsterdam, 1987, pp 235-253 A Askar and H Treptow, Biogene Amme m Lebensnutteln Vorkommen, Bedeutung und Bestlmmung, Ulmer, Stuttgart, 1986 W F Staruszklewax, Jr, and JF Bond, J Assoc Off Anal Chem ,64 (1981) 584 J Y HUI and S L Taylor, J Assoc Off Anal Chem, 66 (1983) 853 N Seder and B Knodgen, J Chromatogr , 339 (1985) 45 A lehne, Abschlussarbmt m the Department of Food Chenustry, Technical Umversrty of Braunschwelg, Braunschweig, 1991 D WlJesuriya and GA Rechmtz, Anal Ctnm Acta, 243 (1991) 1

G C Chemmbus et al /AnaL Chun Acta 263 (1992) 93-100 8 R Stevanato, B Mondow, S Sabatml and A Rlgo, Anal Chum Acta, 237 (1990) 391 9 R Gaspann], M Scarpa, M L Dl Paolo, R Stevanato and A Rlgo, Bmelectrochem Bmenerg ,25 (1991) 307 10 L Macholan and D hlkova, Collect, Czech Chem Commun , 48(1983) 672 11 S Todonlu, M TaJnna and M Senda, Anal Scl ,4 (1988) 583 12 U Bdltewslu, G C Chemmtms, P Ruger and R D Schmrd, Sensors Actuators B, 7 (1992) 351 13 P Ruger, U B&ewskt and RD Schnnd, Sensors Actuators B, 4 (1991) 267 14 M Suzuki, H Suzulu, I Karube and R D Schmld, m R D Schrmd and F Scheller (Eds 1, Blosensors Apphcatlons m MedIcme, Envnonmental Protection and Process Control (GBF Monographs, Vol 131, VCH, Wemhelm, 1989, pp 107-111 15 T Murakanu, S Nakamoto, J hmura, T Kunyama and I Karube, Anal L&t, 19 (1986) 1973 16 R M C Dawson, D C Elhott, W H Elhott and K M Jones (Eds ), Data for Bmchenucal Research, Oxford Umversity Press, Oxford, 3rd edn , 1986, p 438 17 J Flossdorf, D Hamsch and F Papanuchael, Ger Pat, P 3737604-7(1987) 18 W F Swam and R J Desa, Bmchlm Acta, 429 (1976) 331 19 M Okada, S Kawashuna and K Imahoq J Blochem, 86 (1979) 97 20 M Okada, S Kawashuna and K Imahon, J Bmchem, 88 (1980) 481 21 H Yamada, Methods Enzymol , 17B (1971) 726