BIOTECHlVOLOGY TECHNIQUES Volume 10 No. 9 (September 1996) ~~-673-678 Received 16 July 1996

A CATALASE MICROBIOSENSOR FOR DETECTING HYDROGEN PEROXIDE

David Emerson”#, Serban F. Peteu’ and R. Mark Worden”“‘*

‘Center for Microbial Ecology, #Department of Microbiology, and ‘Department of Chemical Engineering, Michigan State University, East Lansing, MI 48824.

Fax: (517) 432-l 105, E-mail: worden@che.msu.edu.

A microbiosensor for hydrogen peroxide (H,O,) was constructed by immobilizing catalase in a polyacrylamide gel on the tip of a Clark-type oxygen microelectrode. The outer tip diameter was 15-40 urn. The sensors had response times of 0.7-1.2 s, and could detect as little as 2-4 uM H,O,. They could measure with a spatial resolution of about 100 pm and remained operational for up to three weeks.

Hydrogen peroxide (H,O,) is an important oxidant that is widely used in industrial

processes. It is also a common product of a number of enzymatic biochemical

oxidations. For example, it is thought to play an important role in the respiratory burst

utilized by phagocytes as an antimicrobial defense (Tizard, 1992). It has also been

implicated in neurodegenerative disorders such as Parkinson’s disease (Garguilo et al.,

1993).

The methods commonly used for H,O, detection are titrimetry,

spectrophotometry, and chemiluminenscence (Stein and Hain, 1995). While some of

these methods are exquisitely sensitive, they all require sample processing, and are not

suitable for ‘real-time’ process studies. Enzyme-based biosensors offer a highly specific

method for measuring substrates in situ without sample preparation. There have been

several designs reported for biosensors for H,O, (Aizawa et al., 1974; Mascini et al.,

1981; Garguilo et al., 1993). Microbiosensors, which have tips of less than 100 pm,

offer several advantages over macrosensors. They have extremely rapid response times

of 1s. or less, can be used with very small sample volumes, and can measure with high

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spatial resolution (Peteu, et al., 1996). We report here, a novel design for an

amperometric,internally referenced microbiosensor for H,O, detection.

The microbiosensor design was based on attaching catalase in a polyacrylamide

matrix to the tip of a Clark-type oxygen microelectrode. The catalase reacts specifically

to dismutate 2 moles of H,O, to 2 moles of H,O and 1 mole of 0,. The 0, diffuses

through the silicone membrane in the 0, microelectrode and is reduced

electrochemically at the cathode, resulting in an electrical current that can be measured

with a picoammeter. Due to the small size of the sensor tip (15-40 pm), the

measurements are very rapid and the sensor is quite sensitive. The use of an internally

referenced Clark-type oxygen microelectrode makes the biosensor insensitive to

electrical noise. Furthermore, the silicone layer used in this design is essentially

impermeable to most common interferents (e.g. uric acid, ascorbic acid) and shields the

cathode from their influence (Peteu et al., 1996).

EXPERIMENTAL

Chemicals Catalase EC 1.11.1.6 from bovine liver (2x crystallized, suspension in water) with an activity of 41,000 units/mg solid was purchased from Sigma Chemical Co. (St. Louis, MO). The polyacrylamide came as a premixed stock solution containing 30% high tensile strength Duracryl@ and 0.8% Bis (Millipore, Corp. New Bedford, MA) . Polyurethane (PU; solution grade, Thermedics, Inc., Woburn, MA) was prepared as described previously (Peteu, et al., 1996). Hydrogen peroxide was purchased as a 30% stock solution and diluted appropriately.

Construction of oxygen microelectrodes and microbiosensors The construction of the glass Clark-type oxygen microelectrodes and the application of enzyme to the tip to make the biosensor have been discussed in detail elsewhere (Peteu et al., 1996). To briefly summarize, 20 ul of the enzyme solution, containing a total of 2050 catalase activity units, was added to 30 1.11 of polyacrylamide mixture. Just prior to application to the sensor tip, the catalysts tetra-ethyl-methylenediamine and ammonium persulfate were added, and this mixture was drawn up in an applicator constructed from a Pasteur pipet. A small bulb of the polyacrylamide/catalase mixture was coated on the tip of the microelectrode where it polymerized within a few minutes. This latter step was carried out on a microscope stage using a 20X long-working-distance objective. The diameter of the bulb was between 15 and 40 urn. After the polyacrylamide had polymerized overnight, a thin (l-2 urn) PU layer was added to the bulb.

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Calibration and use of the microsensors The experimental set-up is shown in Figure 1. The tip of the microsensor was placed in a beaker containing 65 ml of O.lM PO, buffer, pH 7.4, in deionized water. In some cases, an oxygen electrode was also placed in the beaker to determine if there were any spontaneous breakdown of the H,O, into 0,. The sensor was connected to a Chemical Microsensor meter (model 1201, Diamond-General, Ann Arbor, MI) that provided a polarization voltage (-0.75 V) to the sensor and measured the current output from the sensor in pA. This signal was recorded on a strip chart. For activity determinations, aliquots of H,O, were added to the beaker from a stock solution with a pipettor. The beaker was held on a magnetic stir plate and the solution was constantly mixed with a magnet. Calibrations and tests with microsensors were done at room temperature,

Dissolved oxygen microelectrode Anode Cathode Casing

Figure 1. Diagram of the microbiosensor and an oxygen microelectrode placed as a control. See text for details. Dimensions are not to scale.

21fl C. No special precautions were required to avoid electrical noise, since this type of microsensor is electrically very stable (Peteu, et al 1996).

RESULTS AND DISCUSSION

A typical calibration curve for a H,O, sensor is illustrated in Fig. 2. This sensor had a

linear range from 0 to 7.5 n&I H,O,. The results for linear range, detection limit, and

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0 0 IO 0

0 1000 2000 3000 4000 5000 6000 7000

Peroxide concentration, uM

Figure 2. Calibration curve for hydrogen peroxide. The slope (S) from the linear portion of the

curve is included as a measure of sensor sensitivity. The dissolved oxygen value was also recorded

as a control.

response time for five different H,O, microsensors are shown in Table 1. Sensor #l was

not coated with PU; the other four sensors had l-2 urn thick PU coatings. The most

sensitive microsensors had a detection limit of 2 uM H,O,, which is among the highest

sensitivity for H,O, biosensors reported in the literature, to the best of our knowledge.

Table 1. Characteristics for five different H,O, microsensors.

Sensor

1 2 3 4

Detection Limit, pM

4 2 2 4

Range

0 - 7.5 o-9 O-8 o- 10

Slope pA/IllM

450 330 185 100

RZ 0.995 0.999 0.999 0.999

Response time, s

0.7 0.9 1.0 0.9

These microsensors had different output currents because they were based on hand-

made 0, microelectrodes. The response time of these microbiosensors was extremely

rapid, with 90% response times of 0.7-1.2 s. A copy of a chart recorder tracing from a

H,O, sensor is shown in Figure 3. The response time was rapid enough that the vortex

stirring characteristics of the calibration buffer could be followed when each aliquot of

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the H,O, stock solution was added. Over a three week period, the sensors remained

operational, although their sensitivity decreased by lo-35%. This trend was similar to

what has been observed for glucose microbiosensors, which initially lost up to 50% of

their sensitivity within four weeks, but then remained quite stable for an additional 20

weeks (Peteu, et al, 1996). The long-term stability of our microbiosensors was

previously shown to increase with the specific activity of the immobilized enzyme.

Since catalase has a high specific activity, it might be expected to produce a long-lived

biosensor. Similarly, the thickness of

J w2 160 pM - s 50 pl

15 s

\ 50 pl

s

1

lby+%-d

50 pl

cc

Figure 3. Chart recorder trace of microsensor response to hydrogen peroxide. The sensor tip was

placed in a beaker containing buffer that was stirred with a magnet. Each arrow indicates when an

aliquot of H,O, was added. Due to vortex mixing, there was a high, initial concentration of H,O, in

the vortex which caused a large transient increase in the sensor signal. Afterwards, the signal

rapidly fell to a new equilibrium level as the H,O, was uniformly mixed.

the PU layer was found to affect both the longevity and sensitivity of the sensors.

Sensors not coated with PU lost activity after several uses. Sensors with thin (l-4 urn)

PU coatings retained activity better, but maintained high sensitivity. Sensors with

thicker (4-8 urn) PU coatings lost sensitivity, but had an increased linear measuring

range (Peteu, et al., 1996).

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CONCLUSIONS

Amperometric microbiosensors for H,O, have been constructed by coupling catalase to

Clark-type oxygen microelectrodes using a polyacrylamide matrix and a polyurethane

coating. These microbiosensors had tip diameters of 15-40 pm. They had sensitivities of

< 5 pM, and 90% response times of about one second. They remained operational for at

least 3 weeks and were insensitive to electrical noise. These microsensors should find a

wide range of applications for in-situ measuring of peroxide in industrial or clinical

applications, including in-situ food toxicology and in-vivo neuro-physiology.

ACKNOWLEDGEMENTS

This work was funded by National Science Foundation grant BIR 9 120006 to the Center for Microbial Ecology, the MSU Crop and Food Bioprocessing Center, and Fulbright Grant to S.P. Partial support for D.E. was also provided through a subcontract from Koh Development, Inc., Ann Arbor MI to MSU on a NSF-SBIR grant.

REFERENCES

1. Aizawa, M., Karube, I. and Suzuki, S. (1974). Analytica Chimica Acta 69: 41-437.

2. Garguilo, M.G., Huynh, N., Proctor, A. and Michael A.C. (1993). Analytical Chemistry, 65: 523-528.

3. Mascini, M., Iannello, M. and Palleschi, G. (1981). Analytica Chimica Acta 138: 65-69.

4. Peteu, S.F., Emerson, D., and Worden, R.M. (1996). Biosensors & Bioelectronics ll(10): 1059-1071.

5. Stein, K. and Hain, J.-U. (1995). Mikrochimica Acta 118: 93-101.

6. Tizard, I.R. (1992). Immunology: An Introduction. 3rd edition, Saunders College Publishing, New York.

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