screen-printed amperometric microcell for proline iminopeptidase enzyme activity assay

Biosensors & Bioelectronics 15 (2000) 265–272

Screen-printed amperometric microcell for proline iminopeptidaseenzyme activity assay

Geza Nagy a,b, Robert E. Gyurcsanyi a,c, Alessandra Cristalli a, Michael R. Neuman a,Erno Lindner a,*

a Joint Graduate Program in Biomedical Engineering, The Uni6ersity of Memphis and Uni6ersity of Tennessee Health Science Center,Herff College of Engineering, Memphis, TN 38152-6582, USA

b Department of General and Physical Chemistry, Uni6ersity of Pecs, 7624 Pecs, lfjusag u. 6, Hungaryc Research Group of Technical Analytical Chemistry of the Hungarian Academy of Sciences, Department of Analytical Chemistry,

Technical Uni6ersity of Budapest, 1111 Budapest, Gellert ter 4, Hungary

Received 29 November 1999; accepted 29 June 2000

Abstract

A microfabricated amperometric microcell was designed and used for the determination of proline iminopeptidase (PIP) enzymeactivity in 2–10-ml samples. The measurements were made in the range of 10.3–841.5 mU/ml enzyme activities. The sensitivity ofthe determinations was between −0.0195 and −0.0203 mA ml/mU per min. The coefficient of variation of the determined valuesranged between 2.8 (at 561.2 mU/ml) and 24.1% (at 10.3 mU/ml). The microcell was manufactured on an alumina substrate usingscreen-printed graphite working and Ag/AgCl reference electrodes. Elevated PIP activity in the vaginal fluid is a biochemicalindicator of bacterial vaginosis. The method is appropriate to differentiate between normal (669145 mU/ml) and elevated,diseased (7049145 mU/ml), values. © 2000 Elsevier Science S.A. All rights reserved.

Keywords: Proline iminopeptidase activity; Screen-printed graphite electrode; Microfabricated amperometric cell; Enzyme assay

www.elsevier.com/locate/bios

1. Introduction

In recent years, we have been interested in the simpleand cost effective electrochemical detection of metabo-lites associated with Bacterial Vaginosis (BV). BacterialVaginosis (BV) is linked with increasing confidence toidiopathic premature labor (Hauth et al., 1995; Hillieret al., 1995). Association of BV with increased levels ofdiamines in the vaginal fluid has been widely confirmed(Chen et al., 1982). For the determination of diaminesin the vaginal fluid, a miniature biosensor (Xu et al.,1997) and a simple flow injection assay (Marzouk et al.,1998) were developed. Leaking amniotic fluid with in-creased levels of diamine oxidase enzyme activity (Try-ding and Willert, 1968; Tornqvist et al., 1971) mayinterfere with the diamine determination. Similarly, in-creased diamine levels in the vaginal fluid may interfere

with the determination of diamine oxidase activity inthe cervicovaginal secretions, which can be a diagnosticindicator of premature rupture of the fetal membranes(Nagy et al., 1998c). Elevated proline iminopeptidase(PIP) activity has been identified as a reliable biochem-ical indicator of the bacterial vaginosis (Thomason etal., 1988, 1989; Schoonmaker et al., 1991; Schwebke,1999). Proline iminopeptidase (EC 3.4.11.5) is a specifichydrolase enzyme. It catalyses the proline liberatinghydrolysis of Pro-NH-R type peptides or molecules.Patogeneous microorganisms, e.g. Spirochete treponemacontain it in the outer cell envelope. This suggests thatthe enzyme has a vital function in the propagation ofthe cells within their biological niche (inflamed humantissues; Makinen et al., 1996).

Anaerobically grown cells often show higher prolineiminopeptidase activity than those grown aerobically(Syed et al., 1993). Because of the extracellular locationof PIP in bacterial cultures (Smacchi et al., 1999) thedischarges of the infected tissues often have high PIPactivity.

* Corresponding author. Tel.: +1-901-6785641; fax: +1-901-6785281.

E-mail address: [email protected] (E. Lindner).

0956-5663/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.PII: S 0 9 5 6 -5663 (00 )00081 -6

G. Nagy et al. / Biosensors & Bioelectronics 15 (2000) 265–272266

The PIP activity can be determined by measuring thereactants or the products of the enzyme-catalyzed reac-tion. No electrochemical detection for the assay of PIPhas been reported.

In the spectrophotometric PIP assay L-proline p-ni-troanilide (Scheme 1) (Yoshimoto et al., 1983; Fujimuraet al., 1985), L-proline b-naphthylamide (Prol b-NA)(Scheme 2) (Ninomiya et al., 1982; Waters and Dalling,1983; Makinen et al., 1987), and L-Pro-4-(pheny-lazo)phenylamide (Senkpiel et al., 1974) are used asenzyme substrates. Using the Scheme 1 the PIP activityis measured through the appearance of p-nitroaniline at410 nm (Yoshimoto et al., 1983). Schoonmaker et al.(1991) adapted the method for vaginal wet preparations.By inoculating concentrated patient specimen into mi-crotiter plate wells, containing L-proline p-nitroanilide,the samples were incubated for 4 h and read visually.A yellow color indicated a diagnosis of BV while a clearcolor was scored as negative for BV. Thomasson et al.(Thomason et al., 1988) performed the PIP assay withProl b-NA as enzyme substrate. After incubation, thefree arylamine was determined by adding Fast GarnetGBC Salt, as an indicator, to each test well. The resultswere read after 5 min. A red or pink color indicateda diagnosis of bacterial vaginosis while yellow andorange color was scored negative for BV. However, theproducts of the enzymatic hydrolysis of the abovesubstrates (b-NA, p-nitroaniline, phenylazopheny-lamine) can also be measured electrochemically. Theanodic oxidation 1-naphthylamine has been studied inaqueous acid media and in different organic solvents(Marioli et al., 1981; Vettorazzi et al., 1981; Arevalo etal., 1990). Different oxidation mechanisms were pro-posed and evaluated. In solvents such as acetonitrile andmethylene chloride, C�C and C�N coupled solubledimers are the principal products through a monocationradical produced in the heterogeneous charge transferstep. In aqueous acid media, the electrooxidation ofAr�NH2 shows the formation of a film adhered to theelectrode surface. The anodic oxidation of para substi-tuted anilines in aqueous media has a common patternof paragroup elimination and head to tail couplinggiving the corresponding substituted 4-aminodipheny-lamine in the oxidized form (Bacon and Adams, 1968).

Recently, we reported a microfabricated amperomet-ric microcell for glucose oxidase, putrescine oxidase andcreatine kinase enzyme activity measurements (Nagy etal., 1998a,b,c). The two-electrode cell was fabricatedwith conventional thin film microfabrication technologyon a flexible polyimide substrate (Kapton®) with a Ptworking and Ag/AgCl reference electrode. In theseassays, the enzyme-catalyzed reaction takes place in athin solution film. The uniform spread of the reactantfilm is realized by a replaceable, porous hydrophilicmembrane, resting on the working and reference elec-

trode surface. The hydrogen peroxide generated in theenzyme-catalyzed reaction is detected amperometrically.The amperometric current–time curves are used forevaluation.

In this work, the amperometric microcell was evalu-ated for PIP enzyme activity measurements. The PIPactivity was assessed through the amperometric detec-tion of b-naphthylamine (b-NA), the reaction productof the enzyme catalyzed hydrolysis of Prol b-NA. Sincethe flexibility of the polyamide substrate is not anadvantage in the case of planar, single use devices, a newversion of the amperometric microcell was prepared onceramic support. Thick-film screen-printing was used toshape the reaction well and the two-electrode cell. Ascreen-printed graphite and silver/silver chloride filmswere used as working and reference electrodes, respec-tively. Screen-printing is a very cost-effective way ofmanufacturing uniform electrochemical sensors. A largenumber of reports is published about well functioning,and reproducible biosensors based on this relativelysimple, thick film technology (Alvarez-Icaza andBilitewski, 1993; Schmidt et al., 1994; Hart and Wring,1997).

2. Experimental

2.1. Materials and methods

2.1.1. Chemicals and reagentsThe proline iminopeptidase enzyme (EC 3.4.11.5,

from Bacillus coagulans in 50% glycerol solution), theenzyme substrates (Prol b-NA hydrochloride, L-prolinep-nitroanilide trifluoroacetate), the electroactive species(b-NA, p-nitroaniline), and bovine serum albumin(BSA) were all products of Sigma Chemical Co. (St.Louis, MO, USA). Potassium ferrocyanide (II) trihy-drate was purchased from Aldrich Chemical Co. (Mil-waukee, WI, USA). All other chemicals (Tris-(hydroxymethyl)aminomethane, Potassium chloride,Hydrochloric acid) were from Fluka (Buchs, Switzer-land). Buffer solutions were prepared with Milli-Q Gra-dient A10 system water (Millipore Corp., Bedford, MA,USA).

To construct a calibration curve and determine thedetection limit of the new method, the activity of prolineiminopeptidase enzyme (EC 3.4.11.5) was determinedspectrophotometrically at 30°C. L-Proline-p-nitroanilidewas hydrolyzed in the presence of the enzyme and theappearance of p-nitroaniline was measured at 410 nm(Yoshimoto et al., 1983).

2.1.2. The fabrication procedure of the screen-printedelectrodes

The sequentially printed layers and the final design ofthe amperometric microcell are shown in Fig. 1 with a

G. Nagy et al. / Biosensors & Bioelectronics 15 (2000) 265–272 267

cross sectional profile of the printed layers across thex–x cut. The cells were manufactured on a laserscribed, alumina substrate (Coors Ceramic, GrandJunction, CO, USA). The 114.3×114.3 mm-size, 0.635mm-thick wafer can accommodate 60 individual cells(9×22 mm). The single cells were easily separatedalong the laser-scribed lines. A model MC810-C (C.W.Price Co., Bloomsbury, NJ, USA) screen printer wasused to deposit the thick film layers through stainless-steel wire mesh screens.

First, the alumina substrates were carefully cleaned.The wafers were sonicated in an ultrasonic bath for 10min in detergent, 10 min in acetone and finally for 10min in methanol. After each cleaning step, the waferswere rinsed with DI water, acetone and methanol,consecutively. Next, a silver layer was deposited ontothe alumina surface to form the electrical contacts and

the internal reference electrode (Fig. 1a). A Metech3571 silver ink (Metech, Inc., Elverson, PA, USA) wasused in combination with a 325 mesh-count per in.screen (Microcircuit Engineering Corp., Mount Holly,NJ, USA) and Five Star (Autotype Americas, Schaum-burg, IL, USA) photosensitive emulsion film to definethe pattern. The silver layer was fired for 9 min in aprogrammable muffle furnace at a peak temperature of850°C. After the wafer returned to room temperature,the graphite layer was deposited to form the workingelectrode surface (Fig. 1b). A C10903D14 graphite ink(Gwent Electronic Materials Ltd., Pontypool, UK) wasscreen-printed using a 200 mesh-count per in. screen.After the ink leveled for 10 min at room temperature,the layer was dried in an oven at 80°C for 30 min.Finally up to five layers of a thick film UV curabledielectric (type 5018, Dupont, Research Triangle Park,

Scheme 1.

Scheme 2.

Fig. 1. Left, the printed patterns in succesion of the graphite working electrode based amperometric microcell, (a), silver electrode and connectionpads; (b), pattern after the deposition of the graphite over the silver pattern in (a); and (c), final structure with the insulation layer over the patternin (b). Right, cross sectional profile over the x–x cut in (c).


Fig. 2. Cyclic voltammograms of 0.5 mM b-NA (1) and p-nitroaniline(2) in 0.1 M, pH 7.3 Tris buffer solution (0.1% BSA) at 20 mV/s scanrate. The cyclic voltammogram of the background electrolyte solution(0.1 M, pH 7.3 Tris buffer) is marked (3). The screen-printed graphiteand the electrochemically chloridized Ag/AgCl electrodes were usedas working and reference electrodes, respectively.

respectively. The surface roughnesses of the silver andgraphite layers were found to be 1.190.2 and 2.8490.6 mm. The insulating layer thickness (the well depth)was 118.198.4 mm (N=9) (Fig. 1b).

Cyclic voltammetry was used to evaluate the electro-chemical surface area of the screen-printed graphiteworking electrodes. The measurements were made at 20mV/s scan rates in 4 mM ferrocyanide solution using1-M KCl as background electrolyte. The surface areacalculated by Randles-Sevcik equation (2.97 mm2) wasfound to be significantly smaller than the geometricalarea (4.34 mm2). It is assumed that the binder in thegraphite ink covers a part of the active surface. Thecoefficient of variation of the peak current measuredwith a single cell in 16 consecutive scans was 1.0%.

2.1.3. Procedure for enzyme acti6ity measurementsThe procedure to measure the enzyme activity was

the same as we have described in our earlier paper(Nagy et al., 1998a). The depth of the reaction well wasenlarged using a PVC insulating tape of about 200 mmthickness with a 6 mm diameter circular openingmatching the diameter of the reaction well. A smallcircular disk (f=5.5 mm) of absorbent paper(Kimwipes Ex-L delicate task wiper) was introduced inthe well. Buffered solution (5–10 ml) of the enzymesubstrate was injected onto the absorbent disk using aHamilton microsyringe, and +0.6 V was applied be-tween the working and the reference electrode withsimultaneous recording of the current (Model 283 Po-tentiostat/Galvanostat, EG&G Instruments, PrincetonApplied Research, Oak Ridge, TN, USA). The cell wascovered with a cell cap to avoid solution evaporation.After the transient current decayed to about 50 nA,2–10 ml of enzyme solution was added, and the cur-rent–time curves were recorded. The initial slopes ofthe curves were determined and plotted as a function ofthe enzyme activity.

3. Results and discussions

3.1. Preliminary electrochemical experiments

In Fig. 2, the cyclic voltammograms of p-nitroanilineand b-NA are shown. The curves were recorded withthe screen-printed graphite working electrode by im-mersing the planar amperometric cell into 5-ml 5×10−4 M solution (0.1 M; pH 7.3 Tris buffer, 0.1%BSA). The peak current is larger and the oxidation canbe made at significantly lower potential (0.6 V) whenb-NA is oxidized on the graphite electrode comparedwith p-nitroaniline (1.0 V). During their electrochemi-cal oxidation, both substrates form resistive surfacefilms that result in decreasing peak current in consecu-tive scans (Arevalo et al., 1990). Fortunately, the elec-

Fig. 3. Consecutive scans with the screen-printed electrode in 10−4 Mb-NA in 0.1 M, pH 7.3 Tris buffer (0.1% BSA) solution, (1),background electrolyte; (2,3,4), consecutive scans; (5), repeated scanafter thoroughly rinsing the electrode surface with methanol. Scanrate, 20 mV/s.

NC, USA) were printed through a 200 mesh screen toform the reaction well and to insulate the conductivepaths of the electrodes. Each layer was cured separatelyunder a UV lamp (Model, XX-15A, Spectronics Corpo-ration, Westbury, NY, USA) for 60 min after the inkwas allowed to level for 10 min at room temperature.The layer thicknesses were determined with a profi-lometer (model Alpha-Step 500, KLA-Tencor, SanJose, CA, USA). The thickness of the silver and thegraphite layers were about 1091.0 and 16.891.0 mm,


trode surface can efficiently be restored and the screen-printed graphite electrode can be reused if a simplewashing with methanol follows the b-NA oxidation asshown in Fig. 3. The 10−4 M b-NA concentration isthe upper limit of the expected b-NA concentrationwhen the amperometric microcell is used to detectphysiologically relevant PIP activities. The enzyme sub-strates (Prol b-NA and L-proline p-nitroanilide) do notshow electroactivity on the graphite electrode surface inthis potential range. Since the oxidation of b-NA canbe made at lower potentials and higher sensitivitiesthan that of the p-nitroaniline and because the elec-trode surface could easily be renewed after its oxida-tion, Prol b-NA was used as the enzyme substrate inthe electrochemical PIP assays.

Calibration curves of b-NA were linear only in themicromolar range with a detection limit of 5×10−7 M(Fig. 4). At higher concentrations the sensitivity de-creased and no stable, steady state conditions could beestablished due to the electrode fouling.

Preliminary experiments indicated the presence ofelectrochemically active impurities in the Prol b-NAand proline iminopeptidase enzyme solutions as shownin Fig. 5. Prol b-NA (100 ml; 3 mg/ml) was added to a0.5 ml pH 7.2 TRIS buffer solution (Fig. 5A) or a 1 mlPIP enzyme solution (12.5 mU total activity) was in-jected into the planar microcell filled with 15 ml ofbuffer solution. The recordings show the presence ofsome kind of electroactive compounds in both solu-tions. The comparison of the cyclic voltammograms ofthe Prol b-NA and b-NA suggests that the recordedelectroactivity of the Prol b-NA is due to b-NA impuri-ties. In the commercial enzyme solution, some sort ofenzyme stabilizer may be responsible for the small Fig. 5. Current–time profiles in pH 7.3 Tris buffer solutions contain-

ing initially only the Prol b-NA (A) or the proline iminopeptidaseenzyme (B) After the steady state current is reached, the solutionswere completed with the enzyme (A), or the enzyme substrate (B).Working electrode potential was 0.6 V vs. Ag/AgCl.

Fig. 4. Calibration curve of b-NA in the full concentration range andat low concentrations (insert). Background electrolyte, 0.1 M Trisbuffer, pH 7.3 (0.1% BSA), working electrode potential 0.6 V vs.Ag/AgCl.

oxidative current. Adding PIP enzyme to the substratesolution (Fig. 5A) or Prol b-NA substrate to the en-zyme solution (Fig. 5B) initiates the enzyme-catalyzedreaction and a rapidly increasing current is recorded.

3.2. Enzyme acti6ity measurements

The screen-printed amperometric microcells weretested for PIP enzyme activity determinations in a wideenzyme activity range. Generally, 10 ml of enzymesolution was added to 5 ml of substrate solution having1 mg/ml concentration. Typical current–time record-ings and the calibration curves for three individualmicrocells are shown in Fig. 6. The initial slope of thetransients is proportional to the enzyme activity andwas used for the evaluation. Each curve in Fig. 6 oreach point on a selected calibration curve (insert in Fig.


6) was determined with a new paper disk and a newaliquot of substrate solution, i.e. the porous membranewas replaced and the cell was washed with methanoland distilled water between measurements. The threeparallel calibration curves in the insert of Fig. 6 weredetermined with three separate cells. The slopes of thecalibration curves (sensitivity) ranged between −0.0195 and −0.0203 mA ml/mU per min (coefficient of

variation, 2.2%). The electrode fouling does not signifi-cantly affect the reproducibility of the determinationsbecause the generated b-NA concentration is small atthe start of the enzyme-catalyzed reaction (initial slopesection of the current–time transients). The reproduci-bility of the single measurements depends on the en-zyme activity level and volume of the enzyme solutioninjected into the microcell. As expected, the scatteringof data is larger at low enzyme activities. The coeffi-cient of variation (CV) of parallel measurements inindividual cells (N=3) is almost an order of magni-tude larger at low enzyme activities (24.1 and 17.1% atenzyme activities of 10.3 and 30.9 mU/ml) as comparedwith higher enzyme activities (3.3 and 2.8% at enzymeactivities of 421.5 and 561.2 mU/ml). The experimentsmade with 2 ml of enzyme solutions have shown thehighest uncertainty. The use of larger volumes of en-zyme solutions decreases the coefficient of variation.However, there is no difference between the CV whensuccessive measurements are made in one amperome-tirc microcell (N=5) or when each measurement wasmade in a new, separate cell (N=5). For this experi-ment, five individual cells with very similar electro-chemical surface areas were selected (1.6% CV in theirpeak current in 4 mM ferrocyanide solution). Whenthe same microcell was used for all five successivemeasurements, the CV was found to be 1.7% at 365mU/ml enzyme activity. This value is hardly largerthan the CV determined in 4 mM ferrocyanide solu-tion. The small CV indicates also that the electrodesurface, in fact, can effectively be renewed throughwashing with methanol. Under optimal conditions, theCV of the enzyme activity measurement only slightlysurpasses the CV of peak currents in the electrochemi-cal surface determination.

3.3. Substrate dependency of enzyme acti6itymeasurement

In Fig. 7, the initial rates of the PIP response curvesare plotted as a function of the Prol b-NA concentra-tion. The measurements were made at 421.5 mU/mlPIP activity level by adding 10 ml of enzyme solutionto 5 ml of substrate solution in the cell. The substrateconcentration was varied between 1.25 mM and 14.4mM in 0.1 M tris buffer (pH 7.3, 0.1% BSA). Above4.0 mM Prol b-NA concentration, the initial slope ofthe current–time transient is independent from thesubstrate concentration. The Michaelis–Menten con-stant KM=0.13 mM, calculated from the Lineweaver–Burk plot, is in good agreement with the valuesreported in the literature (Waters and Dalling, 1983;Yoshimoto et al., 1983; Makinen et al., 1987). Thesaturation substrate concentration (1 mg/ml=3.6mM\10×KM=1.3 mM) was used for the enzymeactivity measurements.

Fig. 6. PIP responses of amperometric microcells with the screen-printed graphite electrode at different enzyme activity levels, (a) 10.3;(b) 30.9; (c) 93.4; (d) 280.2; (e) 421.5; (f) 561.2; (g) 841.5 mU/ml. Theresulted calibration curves of three individual amperometric micro-cells are shown as an insert. The reaction volume consisted of 5 ml, 1mg/ml, Prol b-NA and 10 ml PIP solution (a–g).

Fig. 7. PIP enzyme activity measurements at different concentrationlevels of Prol b-NA substrate and the dependence of the initial rate ofthe enzymatic reaction on substrate concentration (insert). The reac-tion volume contained 5 ml Prol b-NA (concentration range, 1.25–14.4 mM) in pH 7.3, 0.1-M Tris buffer (0.1% BSA) and 10 ml 546mU/ml enzyme solution. Working electrode potential was 0.6 V vs.Ag/AgCl.


3.4. Electrochemical PIP enzyme acti6ity measurementsfor BV

The PIP activity in vaginal wash solutions rangesbetween 25 and 850 mU/ml (Schoonmaker et al.,1991). The PIP calibrations were done at even broaderPIP activity range between 10.3 and 841 mU/ml. Thesmall coefficient of variation of the data suggests areliable discrimination between PIP activities associ-ated with negative (66941 mU/ml) and positive(7049145 mU/ml) BV samples (Schoonmaker et al.,1991). The amperometric microcell is envisaged as asingle use device with a reaction well filled with screen-printed absorbent material (e.g. silicagel, cellulose ace-tate or alumina powder using carboxymethyl celluloseas binding material). This porous supporting materialcould be impregnated with the enzyme substrate (Prolb-NA in this example).

4. Conclusions

Screen-printed amperometric microcells withgraphite working and Ag/AgCl reference electrodewere used for the determination of proline iminopepti-dase enzyme activity, a biochemical indicator for bacte-rial vaginosis. The sensitivity of the determinations isabout −0.020 mA ml/mU per min (CV=2.2%) whilethe coefficient of variation of the measurements rangesbetween 1.7 and 24.1% depending on the enzyme activ-ity level, sample volume and the reproducibility of theworking electrode surface area. Compared with themicrotiter based spectrophotometric PIP assays, usedin medical practice, this novel amperometric methodoffers distinct advantages, i.e. shorter analysis time andquantitative information on the enzyme activity levels.However, until this method is tested on a large numberof human specimens, it is not clear how much of theseadvantages can be realized in simple, single use devices.

Acknowledgements

This work was supported by NSF/Whitaker Foun-dation Cost-Reducing Technologies Grant BES-950526and by the OTKA T 030968 grant from Hungary.

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screen-printed amperometric microcell for proline iminopeptidase enzyme activity assay

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