Amperometric flow injection determination of putrescine and putrescine oxidase

Analytica Chimica Acta 363 (1998) 57±65

Amperometric ¯ow injection determination of putrescine and putrescine oxidase Sayed A.M. Marzouka, Clarke X. Xua, Bogdan R. Cosofreta, Richard P. Bucka,*, Saad S.M. Hassanb, Michael R. Neumanc, Robert H. Sprinkled a

Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA b Department of Chemistry, Ain Shams University, Cairo, Egypt c Department of OB/GYN, Metro Health Medical Center, Case Western Reserve University, Cleveland, OH44109-1998, USA d School of Public Affairs, University of Maryland, College Park, MD 20742, USA Received 8 July 1997; received in revised form 1 December 1997; accepted 8 December 1997

Abstract Flow injection methods for sensitive, accurate and rapid determination of diamines, mainly putrescine (PUT) and cadaverine (CAD), and diamine oxidase (DAO) are described. Putrescine determination is based on an immobilized enzyme reactor (IER) loaded with putrescine oxidase and amperometric detection of the produced hydrogen peroxide at a platinum electrode. Putrescine oxidase (PUO) was assayed by simple injection of putrescine oxidase solution into a carrier buffer containing a ®xed concentration of putrescine followed by similar detection of hydrogen peroxide. In PUT measurements peak currents were linearly related to putrescine concentration in the range from 10 mM to 1 mM with sample frequency of 70 samples/ hour. No change in sensitivity and detection limit were observed after more than 1000 injections over a two week period. PUO was determined in the range of 50±2500 mU/ml, with sample frequency of 40 samples/hour. The lower detection limit of the present method (1.5 mU/ml) is suitable for determining the expected low levels of PUO in the amniotic ¯uid as a possible marker for premature rupture of membranes (PROM). The effects of ¯ow rates, sample sizes, and substrate concentrations in the detection limits and assay ef®ciencies are given in the present work. # 1998 Elsevier Science B.V.

1. Introduction Measurements of diamine levels, mainly putrescine (PUT) and cadaverine (CAD), and diamine oxidase (DAO) activity are of clinical importance for different diagnostic purposes. Bacterial vaginosis (BV) is the most common type of vaginal infection. Almost 35% of women visiting sexually transmitted diseases

*Corresponding uthor. Fax: 0019199622388. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00043-9

clinics, 15±20% of pregnant women, and 5±15% of women visiting gynecology clinics are infectecd with BV [1±3]. The presence of diamines, mainly putrescine and cadaverine, in the vaginal ¯uids is con®rmed to be associated with BV, which in turn, has been linked with increasing con®dence to idiopathic premature labor [4±8], the predominant cause of premature delivery in USA [9,10]. The literature conclusion that the presence of these diamines could be an effective clinical marker in BV diagnosis is very reasonable.

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The currently available clinical screening diagnostic methods are considered to be inadequate in terms of sensitivity and speci®city. Those methods include examination of the vaginal odor, discharge character, density of clue cells, and pH measurements with nitrazine-paper. The current research methods for measuring diamines are based on relatively complicated techniques suitable only as research tools. These include electrophoretic [11], chromatographic [12±15] and enzymatic spectrophotometric methods [16±22]. In an attempt to introduce a more practical approach for possible in vitro and in vivo diamine detection, we reported recently a miniaturized diamine biosensor based on an immobilized diamine oxidase and realized on a ¯exible Kapton substrate [23]. The sensor provided the desired sensitivity and selectivity required for reliable detection of diamines in the vaginal ¯uids. The preparation for clinical in vivo assessment of this sensor as a screening tool for reliable BV detection is in progress. On the other hand, measurement of diamine oxidase, speci®cally putrescine oxidase activity is of clinical importance as well, for diagnosis of premature rupture of membranes (PROM). PROM, de®ned as rupture of fetal membrane before onset of labor, occurs in 3±14% of all pregnancies [24,25] and is followed by spontaneous labor within 24 h in 80% of patients. The prenatal mortality doubles after a latent period of 24 h and again after 48 h. PROM is also associated with increased incidence of amnionitis, fetal immaturity and maternal and fetal morbidity and mortality [26±28]. The management of PROM cases is a controversial issue and the clinical diagnosis is often dif®cult [29]. The classical diagnostic procedures for PROM include, determination of the vaginal pH with nitrazine paper [30], staining for fetal fat globules [31], identi®cation of fetal squamous cells [32], and examination of amniotic ¯uid crystallization (ferning) [33]. None of these procedures proved entirely satisfactory on its own, and a combination of them is required for good accuracy. In 1974, another method was introduced [34] for resolving the doubt about whether or not the membranes have ruptured. The method is based on the radioenzymatic assay of diamine oxidase (DAO) with 14 C-putrescine as substrate. The principle of the method relies on the fact that amniotic ¯uid

contains DAO in high concentration, whereas normal vaginal secretions or urine do not contain any DAO. Therefore, the detection of any DAO activity in vaginal secretions gives a direct indication of ruptured membranes. The speci®city of the assay is high in the absence of vaginal bleeding. The reliability of this method was investigated 5 years later by others [35] and a high accuracy of 96% was reported. DAO activity in vaginal ¯uid is present for not more than 2 h after the amniotic ¯uid was discharged into the vagina. Therefore, the long assay time, 4±5 h, has limited the usefulness of the method. Two more recent approaches were introduced for diagnosis of PROM based on detecting other amniotic components, i.e., fetal ®bronectin (fFN) [36] and alphafetoprotein (AFP) [37]. Several papers were published to evaluate these methods as a reliable alternative for diagnosis of PROM [38±40]. The reported accuracy of these methods was varied. The advantage over DAO methods in terms of the test accuracy is not pronounced [37]. These kit methods are more advantageous in terms of simplicity, but they seem to be less speci®c [41]. The objective of this work was to develop a rapid, sensitive and selective amperometric ¯ow injection method based on an immobilized enzyme reactor (IER), as a complementary technique, for in vitro determination of putrescine and putrescine oxidase to be used for diagnosis of either BV or PROM, respectively. The enzyme reactor is based on an immobilized PUO on controlled pore glass beads. This system will be important for preliminary in vitro screening of diamines in vaginal secretion samples before attempting in vivo measurements using the previously reported diamine sensor. 2. Experimental section 2.1. Materials and reagents Putrescine oxidase (EC 1.4.3.10), 36 U/mg from micrococcus roseus was received from Toyobo (Japan). Glutaraldehyde (25% aqueous solution), putrescine dihydrochloride, cadaverine dihydrochloride, L-ascorbic acid, acetaminophen, uric acid (sodium salt), and L-cysteine were purchased from Sigma

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(St. Louis, MO). All other chemicals were of analytical reagent grade. Stock putrescine solutions (0.1 M) were freshly prepared every week, in a 0.1 M phosphate buffer (PB) of pH 7.2 and stored at 48C when not in use. Putrescine oxidase stock solutions (15 U/ml) were prepared, in the same buffer, immediately before use and the activity was measured using a standard assay method [42]. All solutions were prepared with water from a Barnstead Nanopure II system. 2.2. Apparatus A computer-controlled electrochemical measurement system was used throughout the experiments. The system contains an EG & G PARC M273A Potentiostat/Galvanostat, IBM PC-AT compatible computer, and M270 (v4.0) software for remote experimental control and data acquisition. All potentials were applied against a miniature Ag/AgCl reference electrode with ¯exible barrel (Cypress System, Lawrence, KS). The sensor was allowed to stabilize, and steady-state current values were obtained in all experiments. Data were collected at a frequency of 1 Hz while a constant polarization potential of ‡0.6 V was applied. All experiments were performed at 250.18C. Some data were recorded on Yokogawa 3025 X-Y-T recorder.

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2.3. Preparation of the immobilized enzyme reactor (IER) PUO was immobilized by using the common controlled pore glass (CPG) method [43,44]. Controlled pore glass (aminopropyl-CPG pore size 700 A, particle size 80±120 mesh), obtained from Sigma (St. Louis, MO), was packed into a glass column (40 mm2.5 mm i.d.). glutaraldehyde solution (5% v/v) in 0.1 M sodium carbonate was circulated to activate the glass. A portion of 0.1 M phosphate buffer (pH 7.2) was used to wash out the excess glutaraldehyde for 1 h. A 15 mg portion of PUO was dissolved in 2 ml of phosphate buffer and circulated through the column for two hours. The excess enzyme was removed by washing with phosphate buffer for 1 h. The enzyme reactors were stored at 48C in phosphate carrier when not in use. 2.4. FIA of PUT and PUO based on the IER The ¯ow system used for either PUT or PUO measurements is shown in Fig. 1. A carrier solution was propelled by means of the peristaltic pump through PTFE tubing (0.8 mm i.d.) at the desired ¯ow rate. A manual injection valve (Rheodyne, Model 7125) ®tted with a 20 ml injection loop was used for sample injection. Three electrode con®guration was used for on line amperometric detection of the

Fig. 1. FIA set up based on IER for PUT and PUO determination.

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enzymatically produced hydrogen peroxide. Two platinum wires (1 mm diameter) were sealed into a 4 cm glass capillary tube (1.0 mm i.d. and 6.5 mm o.d.) and used as working and counter electrodes, respectively. The working electrode was polarized at 0.6 V throughout the experiments. The miniature Ag/AgCl electrode was used as a reference. The reference electrode was inserted through a hole drilled in the tube wall between the working and the counter. The three-electrode detection system was placed within 2 cm inside capillary. Before assembling the system, the working electrode was coated with an electropolymeric layer of poly(1,3±DAB) as described in another paper [23], with the exception that the monomer solution was circulated through the glass tube during electropolymerization. For PUT determination, the IER was inserted immediately after the sample injection valve and the tubing length from the valve to the reactor was 2 cm, and PB of pH 7.2 was used as a carrier solution. In the assay of PUO activity, the carrier solution was replaced by 1 mM putrescine solution in PB, and the IER was removed and replaced by a 10 cm delay coil (0.8 mm i.d.) as shown in Fig. 1.

achieved by using immobilized peroxidase and a glassy carbon electrode. The reported logarithmic scales for both current and concentration were used to show an apparent high linear dynamic range, which is not common for biosensors measurements. Such scales, produce large errors in sample quanti®cation. Moreover, utilization of a second enzyme (i.e. peroxidase) in the detector fabrication eliminated the advantage of the simplicity of ¯ow measurements with the IER. In this case, the stability and reproducibility will be dependent on two enzyme systems, i.e., the reactor and the detector. To promote a more convenient detection method, we employed an electropolymeric layer on the platinum working electrode. The only advantage of H2O2 detection based on peroxidase is the lower applied potential which leads to more selective H2O2 detection. In the present work, a permeselective layer based on electropolymerized of 1,3-DAB on the working electrode surface was used to allow selective H2O2 detection in the presence of easily oxidizable interferents, present in the biological ¯uids, such as ascorbic acid. Our approach is more advantageous in terms of simplicity, reproducibility, cost, and ease of automation for mass production.

3. Results and discussion

3.2. Flow injection determination of Putrescine (PUT) based on IER

3.1. Immobilized enzyme reactor (IER) Putrescine oxidase, immobilized on the reactor, catalyzes the oxidation of putrescine according to Eq. (1). H2 N…CH2 †4 NH2 ‡ O2 ‡ H2 O ! H2 N…CH2 †3 CHO ‡ NH3 ‡ H2 O2

(1)

The generated H2O2 is detected amperometrically at the Pt working electrode. The ef®ciency of putrescine conversion inside the IER was estimated by comparing the signals corresponding to injection of equal concentration of putrescine and hydrogen peroxide, respectively. The reactor showed ef®ciencies of 97 and 94% for 100 and 500 mM putrescine, respectively. A literature search showed that only one paper for ¯ow injection determination of putrescine with an IER was published. The amperometric H2O2 detection was

Flow injection of putrescine, based on immobilized PUO, was easily accomplished by using a carrier phosphate buffer solution as shown in Fig. 1. In this case the putrescine carrier was stopped and the ¯ow was switched to the IER only. The peak heights were independent of ¯ow rate up to 3 ml/min. This trend is common with IER [44], because of the rapid conversion of the analyte, inside the reactor, into products producing analytical signals at the down stream detector. This is not the case when an immobilized enzyme sensor is used as detector where the signal height is very dependent on the ¯ow rate. In this case, lower ¯ow rate allows longer residence time for the sample in the vicinity of the sensor and gives higher response. Typical ¯ow injection peaks obtained by injecting 20 ml of putrescine solution into the carrier solution (2 ml/min) are shown in Fig. 2. The selectivity of hydrogen peroxide detection in the presence of common interferents is presented in Fig. 3. Only ascorbic

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Fig. 2. Typical flow injection peaks for putrescine determination obtained with the IER.

Fig. 3. Flow injection peaks obtained by injecting 20 ml of 50 mM PUT solution and 500 mM of uric acid (UA), L-cysteine (Cys), ascorbic acid (AA) and acetaminophen (AP) solutions, respectively.

acid gave rise to a noticeable interfering effect at tenfold 500 mM level. This level is much higher than the biological level [45,46]. The system has a detection limit of 10 mM PUT and the peak current was

linearly related to the putrescine concentration up to 500 mM with sample frequency of 70 sample/h. No change in sensitivity was observed after more than 1000 injections over a period of two weeks.

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Fig. 4. Effect of putrescine concentration on the flow injection peaks sensitivity for PUO measurement. Flow rateˆ1.0 ml/min. CPUOˆ6.5 U/ml.

3.3. Flow injection determination of putrescine oxidase (PUO) activity PUO can be determined by direct sample injection into a carrier solution containing putrescine. In this

case, the putrescine solution (Fig. 1) will be pumped through the delay coil. A 10 cm coil was found to be optimum in terms of high sensitivity and fast response time. The effect of substrate putrescine concentration, in the carrier solution, was tested in the range of 0.1±

Fig. 5. Effect of flow rate on the sensitivity of flow injection determination of PUO activity. CPUTˆ1 mM, CPUOˆ6.5 U/ml.

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50 mM at a ¯ow rate of 1.0 ml/min and 6.5 U PUO/ ml. The results presented in Fig. 4 show that the peak heights are almost constant (plateau) in the range of 0.5±50 mM putrescine. This is the reason that 1 mM concentration level was selected as a good compromise between low background current, highest possible sensitivity.

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The effect of ¯ow rate of substrate on the ¯ow injection peak current is shown in Fig. 5. The increase in the ¯ow rate reduces the incubation time of the enzyme sample with the carrier substrate. The extent of enzyme reaction will be smaller which explains the decrease in the response. Therefore, a high ¯ow rate is recommended for relatively high PUO activity to

Fig. 6. Typical flow injection peaks obtained for PUO assay. Flow rateˆ1 ml/min and Tˆ2518C.

Fig. 7. Calibration plot for the assay of PUO at 0.3 ml/min and 3718C.

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minimize the assay time, and a low ¯ow rate is recommended for low PUO activity. The ¯ow injection peaks corresponding to different PUO activities were obtained under two different conditions. Fig. 6 shows the typical ¯ow injection peaks obtained at ¯ow rate of 1.0 ml/min at 2518C. The linear range was up to 2.5 U/ml and the lower limit of detection was 50 mU/ml (S/Nˆ2). A more sensitive detection was obtained by using a ¯ow rate of 0.3 ml/min and a higher temperature (370.18C) as shown in Fig. 7. Under these conditions, the lower limit of detection improved to 1.5 mU/ml. This value is very close to the reported sensitivity for the radiochemical methods, i.e., 1 mU/ml. This difference is not signi®cant because the expected activity is well above these limits [34,35,47].

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4. Conclusions

[14]

A simple ¯ow injection system based on immobilized putrescine oxidase has been developed and its simplicity, sensitivity, and ef®ciency for diamine detection has been demonstrated. This system will be important for preliminary in vitro screening of diamines in vaginal secretion samples for bacterial vaginosis diagnosis before attempting in vivo measurements using the previously reported diamine sensor. The system can also be used for quick putrescine oxidase determination with replacement of the enzyme reactor with a delay coil. The sensitivity of 1.5 mU/ml for the present method is suitable for detecting the expected low levels of PUO in the amniotic ¯uid as a possible marker for premature rupture of membranes. Since patients with either BV or PROM are being treated in OB/GYN clinics, the technique described in this work may provide the health care providers quick, simple, sensitive and low cost alternatives to the current clinical diagnosis of BV and PROM.

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Acknowledgements This work was supported by NSF/Whitaker Foundation grant BES-9520526.

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