Enzyme-based fiber-optic device for the determination of total and free cholesterol

Analytzca Chmcu Acta, 256 (1992) 263-268 Elsevler Science Pubhshers B V , Amsterdam

Enzyme-based

263

fiber-optic device for the determination of total and free cholesterol A Krug ‘, A A Sulelman and G G Gmlbault *

Depariment of Chemrstry, Unrvemty of New Orleans, New Orleans, LA 70148 (USA) (Received

10th June 1991, revised manuscnpt

received

12th September

1991)

Abstract A fiber-optic devxc for the determmatlon of total and free cholesterol was developed Cholesterol oxldase and cholesterol esterase are co-unmobdued on a preactlvated nylon membrane The membrane 1s fiied around the common end of a bifurcated fiber-optic bundle After munerslon of the fiber with the membrane m the sample cell, which contams dmmmomum 2,2’-azmobls(3-ethylbenzothlazohne-6-sulfonate), peroxldase and the cholesterol sample, the enzymatic reactlons take place The cholesterol concentration can be related to the mtenslty of the color development with kmetlc evaluation The result of one measurement IS obtamed wlthm 2 nun The present method was compared with an enzymatic procedure based on the Trmder reactlon by assaymg 28 human serum samples Keywords

Blosensor,

Blood, Cholesterol,

Enzyme reactor,

Wlthm the last two decades, an mcreasmg number of pubhcatlons have dealt with the determmatlon of cholesterol m blood [l] The Interest m cholesterol assays developed with the dlscovery of the relatlonshlp between the cholesterol level m the plasma and the frequency of coronary heart disease [21 The earher non-enzymatic assays were replaced with enzymatic methods mvolvmg the following reaction scheme cholesterol ester + H,O

cholesterol esterase

A

cholesterol + fatty acid cholesterol + 0,

cholesterol oxldase

A

cholest-4-en-3-one + H,O,

’ Present address Institute of AnalytIcal Chenustry, s~ty of Technology, Getreldemarkt 9, 1060 Menna, 0003-2670/92/$05

UmverAustna

00 Q 1992 - Elsevler Science Pubhshers

Flbre-optx

sensor,

Serum

The oxygen consumption or the productlon of hydrogen peroxide 1s measured either by electrochemical methods, e g , amperometry [3-51 or potentlometry [6], or by optlcal methods, e g , spectrophotometry [7], fluorescence [8] or chemllummescence [91 Recently, cholesterol enzymatic measurements have been coupled w&h fiber-optic technology Trettnak and Wolfbels [lOI developed a blosensor with an oxygen optrode as transducer, which explolts the fluorescence quenching of molecular oxygen of a fluorescent dye Blum et al [ll] demonstrated the feaslblllty of combmmg the lummol chermlummescence reactlon with the enzymatlc oxldatlon of cholesterol In this study, the approach of an enzymatic fiber-optic device for glucose determmatlon [12] was adapted to the detection of total and free cholesterol Cholesterol oxldase 1s either nnmoblhzed on a preactlvated membrane or co-lmmoblhzed with cholesterol esterase for the free and total cholesterol, respectively The concentration

B V All ngbts

reserved

264

of the analyte IS correlated with the color mtenslty of the oxldlzed dye duunmonnun 2,2’-azmobls(3-ethylbenzothlazolme-6-sulfonate) (ABTS) formed m the reaction with hydrogen peroxide The apphcablhty of ABTS as a chromogen for the quantlficatlon of enzymatically generated hydrogen peroxide has been investigated by Gawehn et al [13], Werner et al [14] and Kahle et al [15] The use of ABTS m a kmetlc enzymatic procedure for the determination of cholesterol was introduced by MaJkic and Berkes [161

EXPERIMENTAL

Materials Cholesterol oxldase (ChOD) (E C 1 13 6) from Streptomyces species with a specific actlvlty of 23 5 U mg-’ solid and cholesterol esterase (ChE) (EC 3 1 1 13) from Pseudomonas species with a speclflc activity of 112 U mg- ’ sohd were obtamed from Toyobo (Osaka) Cholesterol, cholesteryl undec-lo-enoate, Trlton X-100, ABTS, blhrubm and horseradish peroxldase (HRP) (E C 1 11 1 7) with a specific activity of 120 U mg-’ solid were purchased from Sigma (St Louis, MO) Immunodyne unmunoaffuuty membrane, which has a chemically preactlvated surface, 1s a product of Pall (Glen Cove, NY) All other chemicals were of analytical-reagent grade Apparatus The experimental set-up consists of a light source (quartz lamp), fiber-optic bundles, monochromator and detection system, all from OneI (Stratford, CT> The light-tight sample cell housmg was machined from grey PVC Disposable glass vials (15 X 45 mm> from &mble (Toledo, OH) were used as sample cells The design of the cell compartment and set-up were described prev1ously 1121 Preparation of cholesterol solutions Cholesterol solutions m the concentration range 0 5-5 mmol l- ’ were prepared as described by Trettnak and Wolfbeis [lo] The cholesterol was dissolved m 12 8 ml of propan-2-

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ET Al.

01, 3 85 ml of Trlton X-100 were added and, after homogemzatlon, the volume was made up to 100 ml with 0 1 M phosphate buffer (pH 7 0) The solutions that were needed for the assay of total cholesterol contained cholesterol and cholesteryl undec-lo-enoate m such amounts that the ratlo of free to esterlfled cholesterol was 30 70 Owing to the low solub&y of cholesteryl undec-lo-enoate m propan-2-ol-buffer mvrtures, the solvent used was propan-2-01 contammg 4% of trlton X-100 for all concentrations from 1 to 8 mmol I-’ A 10 mM AJ3TS solution was prepared by dlssolvmg 549 mg m 100 ml of water An HRP solution with an activity of 10 U ml-’ was achieved by dlssolvmg 8 3 mg of solid enzyme m 100 ml of 0 1 M phosphate buffer (pH 7 0) A buffer mixture similar to the cholesterol solvent was prepared by dlssolvmg 38 5 ml of Trlton X-100 m 128 ml of propan-2-01 and finally dllutmg to 1000 ml with 0 1 M phosphate buffer (pH 7 0) The resulting pH of this murture was about 73

Immobduaaon of enzymes ChOD was lmmoblhzed on an lmmunodyne lmmunoaffuuty membrane, which does not require any pretreatment of the membrane owmg to Its chemically activated carboxyhc groups, as described by Assolant-Vmet and Coulet [17] A 1 mg amount of enzyme was dissolved m 50 ~1 of 0 1 M phosphate buffer (pH 7 0) This solutlon was applied to the surface of the membrane (2 7 X 2 2 cm), which was then stored m a Parafllm-covered beaker to maintain constant humidity for 24 h at 4’ C The co-unmoblhzatlon of ChE and ChOD followed the same procedure Amounts of 1 mg each of ChE and ChOD were combined and dissolved m 80 ~1 of 0 1 M phosphate buffer (pH 7 0) before lmmoblhzatlon Followmg washing with buffer, the membrane was fixed on the common end of the bifurcated fiber-optic bundle with a strip of Parafdm To allow sufficient mass transfer m the sample cell the membrane was mounted asymmetncally, m fact It did not span the whole cmxunference of the fiber

DETERMINATION

265

OF CHOLESTEROL

Assay procedure The sample cell was filled wrth 4 ml of the buffer containing propan-2-01 and surfactant, then 0 5 ml of the cholesterol sample, 0 4 ml of ABTS solution and 0 2 ml of HRP solution were pipetted mto the cell By closmg the sample cell housmg the enzyme membrane was Immersed m the sample reagent solution and the enzymatic reactions took place immediately on stirring All measurements were made at 23 o C The absorption m the backscattered light contingent upon the oxldatlon of ABTS by hydrogen peroxide m the presence of HRP was followed at a wavelength of 425 nm The fiber-optic lllummator was operated at 9 2 V and a potential of -600 V was applied to the photomultlpher tube

RESULTS AND DISCUSSION

Enzyme membranes The lmmunodyne lmmunoaffmlty membrane has chemically activated carboxyhc groups, which allows the unmoblhzatlon of the enzymes to proceed slowly under mild condltlons [17] The density of the activated sites allows linkage of sufficient amounts of enzymes to utilize kmetlc evaluation The unmoblhzatlon procedure consists of one step only, IS performed easily and yields a highly active enzyme preparation Unfortunately, the oxldlzed ABTS 1s irreversibly adsorbed on the surface of the membrane wrth contmuous use Thus may be a factor m the decrease m the enzyme actMy with time Therefore, the lifetime of the mnnoblhzed ChOD was investigated The enzyme activity decreases below 80% of its mltlal value after 10 days or ca 500 measurements However, the simple preparation of the enzyme-loaded membranes makes this a tolerable problem Calrbratron for free cholesterol Table 1 gives the data for a typical cahbratlon graph for measurements of free cholesterol usmg a membrane with unrnoblhzed ChOD only The values are given m microamps, the readout of the detector As it 1s a kinetic readout, the difference 111the slgnal at two certain times 1s taken Any

TABLE 1 Cahbratlon data for free and total cholesterol Cholesterol (mm01 1-l)

Free cholesterol (/.LA) =

Total cholesterol (pAI a

0

-022 030 0 88 193 300 3 98 5 18

-015

0.5

10 20 30 40 50 60 70 80

030 0 9.5 164 192 254 288 3 30 3 66

a The detector readout was m pA

time interval longer than 10 s and shorter than 120 s can be evaluated, from the time the reaction 1s started by unmersmg the membrane until about 3 mm have elapsed Evidently the readout 1s not available m the first few seconds Two reasons are responsible for this First, the sample cell has to be closed entirely before the shutter of the photomultlpher tube IS opened, and second, the mxnerslon of the membrane 1s done manually and hence IS not reproducible enough for the fast start of the reaction To take account of these problems, the period was chosen to be 60 s, starting 60 s after the membrane was immersed The calibration graph IS linear m the concentration range 0 5-5 mmol l-‘, wth a correlation coefficient of 0 998 and a detectlon lmut of ca 0 05 mm01 1-l This range corresponds to the interesting clmlcal range accordmg to Tletz [18] The reference ranges of total cholesterol m serum or plasma are 181-4 53 mm01 1-l for infants, 3 11-5 44 mmol I-’ for adolescents and 3 63-8 03 mm01 I-’ for adults Takmg mto conslderatlon that ca 30% of the cholesterol IS unesterifled, the free cholesterol ranges from 0 54 to 2 41 mmol 1-l over all the mentioned categories The signal difference IS negative for concentration values below 0 2 mm01 I-‘, owing to adsorption of the dye on the membrane Table 2 shows the linear regression data for the calibration of a membrane which was used for 10 consecutive days, performmg 10 measurements with each test concentration dally The decrease

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266 TABLE

ET AL

2

Statlskal data for the free cholesterol sensor (r = correlation coefficient, R S D = relative standard deviation with a 3 mmol cholesterol solution, y = PA mm-‘, x = mmol cholesterol I-‘) Day

Equation of hnear regresslon

r

RSD (%I

1 4 7 10

y=107x-028 y=O97x-021 y=O92x-019 y=ossx-018

> 0 99 >099 >099 > 0 99

24 32 35 51

m the slope m the cahbratlon graphs correspondmg to the dally sets of assays comcldes with the decrease found m the lmmoblhzed enzyme actlvlty

05

1

15

2

wetght mtlo

Cahbrat8on for total cholesterol The calibration graph for total cholesterol 1s achieved by usmg membranes with co-lmmoblhzed ChE and ChOD The solutions used imitated the ratio of esterlfled to free cholesterol m real serum or plasma samples Before calibration was established, the membrane was tested with a cholesteryl undec-lo-enoate solution to make sure that the free cholesterol alone would not contribute to the color reaction Further, the proportlon of ChOD and ChE m the lmmoblhzatlon step was investigated m order to optimize the sequence of the enzymatic reactions It is shown m Fig 1 that the enzymatic degradation proceeds fastest when a ratio of ChOD to ChE from 1 1 to 1 2 1s applied m the lmmoblhzatlon process The theoretically expected weight ratio of ChOD to ChE is 1 2 5, taking mto account the tenfold molecular weight of ChE over ChOD and the approximately fourfold activity of free ChE over free ChOD NatuTABLE

Fig ratlo two at 2

25

1 Actwlty of total cholesterol sensor depending on the of unmolnllzed ChE to ChOD The total weight of the enzymes m the unmobdlzat~on solution was held constant mg and then ratlo was varied

rally, the lmmoblhzatlon of the enzymes mfluences their actlvltles to different extents However, it was not the aim of this work to investigate the effects of decreasing activity on lmmoblhzatlon The reproducibility of membrane fabrication was checked by evaluating the response of dlfferent batches and was determined to be 88% or better The co-mnnoblhzatlon of peroxldase resulted m a lower response and a less stable enzyme preparation, hence it was decided to use soluble peroxldase owmg to its avallablhty and low cost The calibration graph obtained (Table 1) with the cholesterol-cholesteryl undec-lo-enoate solutions is lmear m the desired range from 1 to 8 mm01 1-l total cholesterol usmg the same data

of bdirubm

Parameter

Concentration 1 at 2 mm01 1-l concentration at 5 mm01 1-l (%)

of cholesterol

3

ChOD

3

Interference

Difference Dtfference

ChE

(%) -

of Lxhrubm (mg dl-‘) 10

20

30

-05

-17 -1

-25 -24

DETERMINATION

267

OF CHOLESTEROL

collectlon procedure as described for free cholesterol The detection hmlt 1s 1 mmol 1-l at a slgnalto-noise ratio of 3 The relatwe standard devlatlon for ten rephcate measurements on an 8 mM solution 1s 5 8%, and 1s 4 2% at 2 mm01 1-l The correlation coefficient 1s 0 995 Cholesteryl undec-lo-enoate was chosen for calibration owing to its good conformity of actlvlty with serum samples A steady state 1s reached wlthm 3-5 mm and at least 10 mm when sensing free and total cholesterol, respectively The longer time 1s obvlously due to the mtroductlon of an additional enzymatic reaction m the case of total cholesterol Accuracy

The accuracy of the fiber-optic cholesterol procedure was demonstrated by comparing it with the Paramax cholesterol reagent This cholesterol test lut utlhzes the Trmder reactlon, m which hydrogen peromde couples with 4-ammo-antlpyrme and 3,5-dlchloro-2-hydroxybenzenesulfomc acid to yield a chromogen, which 1s monitored photometrically Twenty-eight serum samples were analyzed for cholesterol usmg the present method and compared with the provided values of hospital analyses The present values were converted to the more common mg dl-’ units used m chmcal laboratories The resulting correlation graph gave a slope of 0 958, an intercept of 8 75 mg dl-’ and a correlation coefficient of 0 981 Interference studm

The recommended maximum allowable error of a method for the determmatlon of serum cholesterol 1s expected to be 3% or less Therefore, various compounds that could lead to erroneous results have to be considered as mterferences According to Zak and Artlss [l], either endogenous or exogenous sources of interference could occur Here the endogenous class of mterference was considered In this case the serum matrix could contam severely elevated levels of tnglycendes, hemoglobm resultmg from hemolySISor high blhrubm concentrations The effect of

several blhrubm levels at constant concentrations of cholesterol was mvestlgated (Table 3) The complex interference mechamsm of blhrubrn consists manly of two opposing effects [19] On the one hand bllirubm has a broad absorption band around 450 nm, which could contribute as a posltlve interference m the present procedure, and on the other bilirubm consumes hydrogen peroxide, which should result m an apparent depression of the cholesterol value However, the results showed that normal blhrubm levels lower than 1 mg dl-’ [20] do not disturb the method Elevated concentrations cause negative interference under these condltlons The interference of high trlglycende levels was not determined, although two serum specunens with hypertnglycendemla could not be analyzed properly owing to turbidity Concluson

This work has demonstrated the apphcablhty of a fiber-optic device based on a spectrophotometric prmclple for the determmatlon of free and total cholesterol For the first time total cholesterol was measured with a fiber-optic sensor based on co-munoblhzatlon of ChE and ChOD The procedure 1s accurate and the kmetlc evaluation permits results to be obtained rapldly Automation of the procedure by mJectlon of the cholesterol sample through a syrmge would permit data evaluation from the first second of the reaction It should be mentloned that the sample volume 1svery large compared with estabhshed cluucal tests, but this could be overcome easily by muuaturlzatlon of the sample cell geometry Although several disadvantages anse, this work should encourage the contmuatlon on research on fiber-optic cholesterol devices with nnmoblhzed enzymes This should lead to a reduction m cost per cholesterol determmatlon when one set of lmmoblhzed enzymes can be employed contmuously for the assay of 500 or more cholesterol samples This work was supported by a grant from the Louisiana Education Quahty Support Fund (LEQSF-RD-B-17) AK thanks the Austrian

A KRUG

268

Bundesmuustermm fur Wlssenschaft und Forschung for a research stipend and the Fond zur Forderung der Wlssenschafthchen Forschung (Project 7181~CHE) for addltlonal support Dr W T Wu, Dlrector of the Clmlcal Laboratory of the Charity Hospital of New Orleans, IS gratefully acknowledged for provldmg human blood serum samples mcludmg their total cholesterol values measured with the Paramax cholesterol reagent system

REFERENCES B Zak and .I D Artlss, Mlcrochem J ,41 (1990) 251 Improvmg Cholesterol Measurement, US Department of Health and Human Servxes, Natlonal Institute of Health, Bethesda, MD, 1990, Ch 1, p 1 H K. Kuang, S S Kuan and G G Gullbault, Chn Chem , 23 (1977) 671 I Karube, K. Hara, H Matsuoka and S Suzuki, Anal Chum Acta, 139 (19821, 129 M Mascuu, M Ianello and G Palleschl, Anal Chum Acta, 146 (1983) 135 D S Papastathopoulos and GA Rechmtz, Anal Chem , 47 (1975) 1792

ET AL

7 CC Allam, L C Poon, C S G Chan, W Richmond and P C Fu, Chn Chem, 20 (1974) 470 8 H Huang, J W Kuan and G G Gullbault, Chn Chem ,21 (1975) 1605 9 A Tamguchl, Y Hayash] and H Y&I, Anal Chum Acta, 188 (1986) 95 10 W Trettnak and 0 S Wolfhels, Anal Blochem, 184 (1990) 124 11 LJ Blum, J M Plaza and P R Coulet, Anal I.&t, 20 (1987) 317 12 M S Abdel-L&f, AA Sulelman and G G Gudbault, Anal Lett , 21 (1988) 943 13 K Gawehn, H Wlelmger and W 2 Werner, Fresenms’ J Anal Chem , 252 (1970) 222 14 W Werner, H G Rey and H Z Wlehnger, Fresenms’ J Anal Chem , 252 (1970) 224 15 K Kahle, L Weiss, M Klarwem and 0 Z Wleland, Fresenms’ J Anal Chem , 252 (1970) 228 16 N MaJklc and I Berkes, Chn Chum Acta, 80 (1977) 121 Assolant-Vmet and P R Coulet, Anal Lett, 19 17 CH (1986) 875 18 NW Tletz, Fundamentals of Chmcal Chemistry, Saunders, Phdadelphla, 3rd edn , 1987, p 948 19 M T Perlstem, R J Thlbert and B Zak, Microchem J , 22 (1977) 403 20 G Schettler, Innere Medlzm, Vol 1, Thleme, Stuttgart, 7th edn , 1987, Ch 19, p 700