Ž .Sensors and Actuators B 66 2000 269–271www.elsevier.nlrlocatersensorb

A lactate needle-type biosensor for in vivo detection in muscular tissues

Meng Wu a,), Zhihong Lin a, Yiyong Li a, Shu Ren b

a Department of Chemistry, College of Basic Medical Sciences, Tongji Medical UniÕersity, Wuhan, Hubei, 430030, People’s Republic of Chinab Department of Biomedical Engineering and Department of EnÕironmental Monitoring, Tongji Medical UniÕersity, Wuhan, Hubei, 430030,

People’s Republic of China

Received 30 July 1998; received in revised form 12 July 1999; accepted 29 January 2000

Abstract

wAs an alternative method for whole blood lactate monitoring in clinical and sports medicine, a needle-type lactate biosensor Z. Lin,Ž . Ž . xW. Qiao, M. Wu, Anal. Lett., 25 7 1992 1171 is designed here for in vivo detection of lactate in muscular tissues. It was constructed

Žby a stainless steel needle, with the surface modified by a polymer material for the enzyme lactate oxidase, LOD, hydrogen peroxide.oxidoreductase, HPO immobilization. The influences of pH and temperature were discussed. It can determine the substrate ranging from

4.8=10y6 to 3.2=10y3 molrl, with the response time of 3 min. The possible interference of the ascorbic acid, urea, glucose, etc., forin vivo detection was studied. The biosensor was used in the detection of the lactate in vivo in muscular tissues of the K.M. mice anddifferent concentrations of lactate were obtained for about 10 min before and after exercise. q 2000 Elsevier Science S.A. All rightsreserved.

Keywords: Lactic acid; Enzyme biosensor; In vivo

1. Introduction

Lactate is widely distributed in the blood, muscles andbody fluids. It is an important parameter for clinical moni-

w xtoring and sports medicine. In vivo 1 detection offersw xmore potential than other methods 2 . As an alternative

method for whole blood lactate monitoring, a needle-typelactate biosensor is designed for in vivo detection oflactate in muscular tissues.

2. Experimentals

2.1. Materials and apparatus

Ž . ŽPotentiometer PHS-203 from the Institute of Instru-. Žment Research, Wuhan , acupuncture needles from

. ŽShuzhou Medical Instrument , and K.M. mice from theDepartment of Animal Experiments, Tongji Medical Uni-

.versity were purchased or supplied.ŽLactate oxidase LOD, EC.1.1.3.2 Pediococcus species,

. Ž40 Urmg , hydrogen peroxide oxidoreductase HPO,.EC:1.11.1.7 Horseradish, 200 Urmg , and bovine serum

Ž .albumin BSA were purchased from Sigma, nafion fromAldrich, lactic acid, ascorbic acid, urea, periodate, glutaric

) Corresponding author. Fax: q86-27-83621424.Ž .E-mail address: martinwu87@yahoo.com M. Wu

dialdehyde, Na HPO P 12H O, and NaH PO P 2H O2 4 2 2 4 2

were from Reagent, Shanghai. Physiological phosphateŽ .buffer 0.1 molrl, pH7.4 was prepared, and NaCl was

added to adjust ion strength of buffer solution. The self-de-w xsigned polymer membrane described elsewhere 3 was

used for sensing membrane preparation. Deionized waterwas used to prepare all standard and working solutions.

2.2. Construction

Ž .The purchased acupuncture needle Øs0.20–0.22 mmwas pretreated by surface polishing and ethanol rinsing.The pretreated needles were dipped in the self-made poly-mer solution. After centrifuge, the needles were heated to1208C for an hour, then cooled for future application.

Before immobilization, the membrane was pretreatedŽ .with periodate. Both enzymes LOD and HPO were im-

mobilized by dipping the needle with the polymer mem-Ž .brane in the PBS solution of the LOD 40 Urmg , HPO

Ž .200 Urmg , and glutaric dialdehyde for a definite time atŽ .a constant temperature, then Nafion solution 5% was

coated. The sensors were preserved in the refrigerator at48C. The structure of the biosensor is indicated in Fig. 1.

2.3. Measurement

Using the newly constructed needle-type biosensor asthe working electrode, and a saturated calomel electrode

0925-4005r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved.Ž .PII: S0925-4005 00 00366-X

( )M. Wu et al.rSensors and Actuators B 66 2000 269–271270

Fig. 1. Schematic representation of lactate needle-type biosensor.

Ž .SCE as the reference electrode, the concentration oflactate was determined in the PBS solution by the potentialchanges.

2.4. Measurement in mice

K.M. mice were used throughout the experimentation.Animals were anaesthetized by intraperitional injection of

Ž .urethane 1.0 grkg . Further injection was given as re-quired. Once anaesthetized, mice were positioned in thesterotaxic frame. The working electrode was placed in themuscle of the left leg. The reference electrode was placedin a conducting cell with the tail immersed.

The exercise of the mice was performed on a wheel for10 min. The measurement is done at once.

3. Results and discussion

3.1. Immobilization of the enzyme

The lactate is converted by means of immobilized LODand HPO according to the reaction:

LODL-lactic acidqO ™ pyruateqH O2 2 2

HPO2H O ™ O q2H O2 2 2 2

Fig. 2. Temperature influence on the lactate sensor. Experiment condi-Ž . y4tion: PBS 0.1 M pHs7.4 , lactic acid: 1.5=10 molrl.

Fig. 3. pH influence on the lactate sensor. Experimental condition: lacticy4 Ž .acid: 1.5=10 molrl, PBS 0.1 molrl .

Fig. 4. The response time of the lactate sensor. Experimental condition:y4 Ž .lactic acid: 1.5=10 molrl, PBS 0.1 molrl, pH 7.4 .

HPO was used for recyclation of the LOD enzyme. Thepotential changes were recorded for quantitation.

Because the surface of the sensing needle was pre-coatedwith the functional polymer membrane, LOD and HPO canreact with the membrane to be immobilized on the needle.

In this study, the temperature is set at 308C. Theover-elongation of the immobilization reaction time mayhave larger potential change, but probably causes theswelling of the polymer membrane. The optimized reaction

Ž .time 1 h is employed in the experiment.

Table 1Ž y5The reproducibility experiments 378C, 0.1 M PBS, lactic acid: 1.5=10

.M

Ž . Ž .Needles D E mV r3min Average SD RSD %

1 21.8 21.4 20.8 20.8 18.8 20.72 1.15 5.572 20.8 21.3 19.2 17.8 18.5 19.52 1.49 7.653 17.2 19.9 21.0 20.5 21.2 19.96 1.62 8.134 18.7 19.2 20.3 21.2 19.3 19.74 1.00 5.07

( )M. Wu et al.rSensors and Actuators B 66 2000 269–271 271

Fig. 5. The in vivo experiment.

3.2. Temperature Influence

Although higher temperature may increase the activityof the enzyme, it is prone to make the enzyme be prone todeactivation. At the common physiological temperatureŽ . Ž .378C , better results were obtained Fig. 2 .

3.3. pH influence

The pH also has extraordinary influence on the responseŽ .of the enzyme reaction Fig. 3 . This influence is the

concerted result of the enzyme and the sensitive mem-brane, and the maximum was observed in the range of7.2–7.6. When the pH was greater than 7.6, the activity ofthe enzyme decreased obviously. In the present work, theexperiment was performed at pH 7.4.

3.4. Characterization

The detection limit was determined with respect tosignal-to-noise ratio of 3:1 in accordance with the require-ments of clinical analysis. The detection limit is 1.2=10y6

molrl, with linear range from 3.0=10y6 to 5.4=10y3

molrl.The preservation of the biosensor was also studied. It

was preserved at 48C in refrigerator for about a month.After every usage, the needle was deposed without furtherapplication.

The response time was determined as shown in Fig. 4.The response time is less than 3 min.

The reproducibility of the biosensor was studied asshown in Table 1. The mean relative standard error is6.6%.

3.5. In ÕiÕo experiment

The in vivo detection of lactic acid in muscle tissues ofmice was undertaken for the exercise effect on the lacticacid concentration in the muscle tissue of mice. Theapparent differences can be seen in the Fig. 5.

After a 10-min exercise, the lactic acid concentration inthe leg tissue has increased for about 15%, and a staticstage is reached in about 9 min. This is in accordance withthe sports medicine results. Through the experiment, it hasshown that the biosensor can be used in vivo for thedetection of lactic acid in the muscle after the exercise.

4. Conclusion

The biosensor based on the acupuncture needle hasgreat potential in biomedical application. It has displayed agood response for the in vivo detection of the lactic acid inmuscle tissues, and thus has great potential for the sportsmedicine, especially for the specific muscles exercises,while the whole blood lactate detection is the mean con-centration of the whole body. The characteristics of thestructure of the biosensor have made it a minimally inva-sive biosensor, which is the pre-requirement of the applica-tion in clinical detection.

Acknowledgements

This work has been supported by a grant of NationalŽ .Climbing Project of China JL93010 .

References

w x Ž .1 Z. Lin, L. Zou, M. Wu, S. Ren, Sensor World 6 1996 45.w x2 U. Spohn, D. Narasaiah, L. Gorton, D. Pfeiffer, Anal. Chim. Acta 319

Ž .1996 79.w x Ž .3 Y. Zhou, Chin. J. Biomed. Eng. 7 1997 28.

Biography

Meng Wu is an Associate Professor at the Tongji Medical University,China. He obtained his BSc degree and MSc degree in Medicine in 1987and 1995, respectively. His interests include molecular recognition andbiosensors.