CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 37, Issue 4, April 2009 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2009, 37(4), 624–628.
RESEARCH PAPER
Development of Amperometric Lactate Biosensor Modified with Pt-black Nanoparticles for Rapid Assay LIU Chun-Xiu1,2, LIU Hong-Min1,2, YANG Qing-De1,2, TIAN Qing1,2, CAI Xin-Xia1,2,* 1 2
State Key Lab of Transducer Technology, Institute of Electronics, Chinese Academy of Sciences, Beijing 100080, China Graduate School of Chinese Academy of Sciences, Beijing 100080, China
Abstract: For point-of-care determination, a disposable biosensor for the analysis of L-lactate in athletes’ serum samples was developed and optimized. The planar electrochemical biosensor was fabricated with gold thin-film two-electrode system and was modified with platinum-black nanoparticles and ferricyanide mediator. Platinum-black nanoparticles were deposited on the electrodes to improve the sensitivity and stability. Lactate oxidase (LOD, E.C.1.1.3.2) was immobilized on working electrodes with ferricyanide as mediator to improve the electron transfer ability by lowering the work potential to +0.2 V. The optimizations of the deposition of platinum-black nanoparticles, concentration of LOD, and concentration of ferricyanide in this article have resulted in the development of a lactate electrode with a wide linear range of 1–20 mM lactate, a high sensitivity of 1.43 μA mM–1, a fast detection time of 50 s, and a coefficient variation (CV) of 0.0549. The activity of biosensor held above 90% after one year storage at room temperature. The biosensor was successfully applied to the determination of L-lactate in serum samples without dilution. This disposable biosensor combined with portable meter is promising for rapid determination of point-of-care lactate. Key Words: Biosensor; Lactate oxidase; Mediator; Pt-black particles; Modified electrode
1
Introduction
The pattern of change or the trend towards an increase of blood lactate is a sensitive indicator of survival. Accurate and rapid point-of-care determination of L-lactate is important in medicine and sports for point-of care testing[1–3]. The serum values of lactate in healthy and diseased persons were in the range 0.5í2.9 mM and 5.0í15.0 mM, respectively[4]. Especially, the blood lactate of athletes can reach 20 mM after anaerobic training, indicating the oxygenation state of tissues[5]. Detection of lactate by optical methods needs exact instruments and should be carried in special laboratory, which cannot be satisfied with the requirement of fieldwork. Portable electrochemical lactate biosensors attract people’s research interest. Modifications of the lactate biosensor electrode were currently focused on the method of enzyme immobilization[6,7], mediators, and a change in materials[8–10] to improve the
sensitivity. These biosensors have good response characteristics, yet cannot satisfy the broad detection demand from 0.5 to 20.0 mM lactate of athletes serum. This study represents a novel lactate biosensor with a broad detections range, a high sensitivity, and a rapid assay time with enhanced electron transfer between the redox center of the enzyme and the electrodes surface based on LOD/Ferri-Pt black-coated Au electrodes. Pt-black nanoparticles were chosen as modification layer in this study because of its powerful signal magnification function and catalytic capabilities as a noble metal[11]. Ferricyanide was used as mediator owing to the excellent electron transfer capability at low potential (0.2 V) and a high degree of reversibility[12]. The optimization of components and testing conditions of the disposable lactate biosensor were performed in this work, and the optimized biosensors were used to determinate lactate in athletes’ serum samples with satisfied results.
Received 28 August 2008; accepted 11 November 2008 * Corresponding author. Email:
[email protected] This work was supported by the Hi-Tech R. & D. Program of China (No. 2007AA03Z428), the NSFC (No. 60576049), the CAS Program (No. KGCX2-YW-111 -2) and IECAS Program (Nos. 08CX790151, 07QNCX-9240). Copyright © 2009, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(08)60099-7
LIU Chun-Xiu et al. / Chinese Journal of Analytical Chemistry, 2009, 37(4): 624–628
2 2.1
Experimental
Preparation of L-lactate biosensor
The arrays contained two-electrode system thin film electrodes that were fabricated with the vacuum sputtering technology where gold was sprayed on polycarbonate (PC) plastic substrate material[13]. The nanoporous platinum-black layer was deposited on the gold working and counter electrodes. Four microliters of the reagent (0.8 U μl–1 lactate oxidase, 100 mM potassium ferricyanide, 6% BSA, 0.2% CMC, 0.1% triton) was added on the channel region of the electrodes and dried at 25 ºC. The biosensor arrays were split into single biosensors (Fig.1), which were packed into container with sufficient desiccant and stored at room temperature. 2.3
Platinizing condition was optimized on the plating potential from –2.4 V to –1.8 V and the deposition time (20–500 s) with CHI workstation system. When the plating potential was higher than –1.8 V, the Pt-black nanoparticles layer was grey and thin. With the decrease of potential, the Pt-black nanoparticles layer became blacker and thicker. Yet when the potential was lower than –2.4 V, the Pt-black nanoparticles layer was easy to shell. Hence, the plating potential of⧏2.0 V was chosen. Pt-black nanoparticles layer became thicker with the increase of plating time (as shown in Fig.2). Because the layer was too thin or too thick to be firmly deposited on the electrodes shell, the plating time of 3 min was adopted. The Pt-black nanoparticles formed many micropockets[14], which help enzyme adsorption, sensitivity, and storage stability of biosensors (Fig.2c). The sensitivity of the biosensor modified with pt-black particles was 100 times higher than that of biosensor with bare-Au electrode when both the biosensors were not modified with ferricyanide mediator[15]. The porous structure of Pt-black nanoparticles produced a huge increase in the microscopic area of the electrode surface, which improved the sensitivity prodigiously. The stability of the biosensor modified by Pt-black particles was improved markedly. The sensitivity of the biosensor remained at 90% after one year of storage at room temperature. The activity of biosensor with bare-Au dropped to 70% after 7 d storage at room temperature and dropped to 40% after 7 d storage at 37 ºC. The porous micropocket structure prevents the loss of activity of the enzyme and restrains the staining of the electrodes.
Test methods
During the experiments, the lactate biosensors were inserted into the electrode faucet of the CHI 660 workstation or the YT 2005-1 lactate detector. The test condition of the biosensor was optimized on the CHI 660 system. The performance of biosensor was tested by portable meter YT2005-1. Three microliters of serum was added on the biosensor with capillary effect. Then, the testing current was displayed and recorded.
3
Plating condition and effect of Pt-black nanoparticles to biosensor
Reagents and materials
The CHI 660 electrochemical workstation and the plasma cleaner were purchased from CH Instruments Inc. USA and Harrick Scientific Corp. USA, respectively. A blast desiccator was purchased from Yiheng Technology Co., China. YT2005-1 lactate detector was homemade. The standard serum samples mensurated by YSI-1500 Sport meter as the criterion were supplied by Biology Center of China Institute of Sport Science. Lactate oxidase (EC 1.1.3.2 from Aerococcus species) with a specific activity of 42 U mg–1 was obtained from Genyme (Kent, UK). Lactic acid, potassium ferricyanide, and carboxymethylcellulose (CMC) were purchased from Sigma (St. Louis, MO, USA). All the other chemicals were analytical reagent grade. All solutions were prepared with deionized (DI) water. 2.2
3.1
Results and discussion
Fig.1 Schematic structure of lactate biosensor 1. Plastic substrate; 2. electrode leader/line; 3. reaction region of electrode modified with Pt-black; 4. dry reagent layer; 5. insulated double adhesive tape; 6. plastic enveloper
Fig.2 SEM micrographs (15.0 kV) of electrode surface: (a) Bare Au, (b) modified with Pt-black nanoparticles for 20 s deposition time and (c) 180 s deposition time, (d) Pt-Black-LOD coated
LIU Chun-Xiu et al. / Chinese Journal of Analytical Chemistry, 2009, 37(4): 624–628
3.2
Effect of ferricyanide as mediator and influence of work potential on response current
Ferricyanide redox system has high electron transfer ability[16,17] and it can increase the current signal over 10 times than without mediator. The effect of work potential on biosensors with ferricyanide and without mediator was studied from +0.1 V to +0.8 V in 0.1 V steps. The response current of biosensor with bare Au electrode could not be observed obviously until the work potential was increased to +0.5 V, at which the potential interferants could be oxided and increased with the work potential until +0.8 V (line a in Fig.3). The detection current of the biosensor modified with Pt-black nanoparticles responded to lactate at +0.2 V and enhanced obviously with the increase of potential (line b in Fig.3). Yet the response current of the biosensor modified with Pt-black particles and mediator was dissimilar, and it had maximum value at +0.2 V (line c in Fig.3). The background current increased much with the increase of potential, so the signal decreased with the increase of potential in the range of 0.4–0.8 V. Such phenomena supported further the excellent electron transfer ability of ferricyanide as mediator. The response sensitivity and linear range of the L-lactate biosensor strongly depends on the mediator and the concentration of mediator. Several mediators[3,4] have high electron conducing ability and low background in the specific range, yet it cannot reach broad test range. Potassium ferricyanide was chosen as mediator of lactate biosensor because of high electron conducing ability during broad range. The effect of concentration of ferricyanide was investigated in 20–200 mM. With the increase of ferricyanide concentration, the sensitivity and the background current both increased, yet the background current increased abundantly when the ferricyanide concentration was over 100 mM (Fig.4). To obtain the high sensitivity and low background current of biosensor, 100 mM ferricyanide was chosen. 3.3
Effect of lactate oxidase concentration
In this study, the effect of lactate oxidase concentration on response to sensitivity of the biosensor was investigated over the enzymatic activity range of 0.05–1.2 U ȝl–1. Figure 5 shows the relationship between response sensitivity and lactate oxidase concentration. The sensitivity increased with the increase of the enzyme loading activity and then leveled off in the range of 0.4–0.8 U ȝl–1. This indicates that the response signal of the biosensor was controlled by the enzyme activity as the limit of determination (LOD) was below 0.4 U ȝl–1. It is well known that enzyme denaturation often causes a considerable reduction in a loss of biosensor response. Biosensors with higher enzyme concentration had better stability on sensitivity to high lactate concentration. Considering both the production cost of biosensor and the
sensitivity of biosensor, finally 0.8 U ȝl–1 enzyme concentration was used on the biosensor with 4 ȝl every strip.
Fig.3 Signal of 10 mM lactate on different modified electrodes a. Bare Au, b. Pt-black particles, c. Pt-black particles and ferricyanide to 10 mM L-lactate at different potential
Fig.4 Effect of ferricyanide concentration on background current (a) and sensitivity of biosensor (b) during 1–20 mM L-lactate
Fig.5 Effect of lactate oxidase concentration on the sensitivity of biosensor during 1–20 mM L-lactate
3.4
Response behavior of L-lactate biosensor
The real-time response of biosensor to lactate was carried by CHI 660 at 0.2 V. It showed that the biosensor gave a well-defined response to lactate in serum. The lactate calibration curve was linear up to 20 mM. The standard serum samples were mensurated by YSI-1500 Sport meter as the criterion. The biosensors combining with YT2005-1 meter were used to determinate lactate in serum samples in triplicate over the wide detection range of 1–20 mM. The incubation time was 20 s, and the response time was 30 s at a 0.2 V of working potential. The biosensor performed high sensitivity of 1.43 mA Mí1, good linear correlation of r =0.9935, and the regression equation y (μA) = 1.4336x (mM) + 4.5364. The
LIU Chun-Xiu et al. / Chinese Journal of Analytical Chemistry, 2009, 37(4): 624–628
LOD for the L-lactate sensors was 0.1 mM. The wide linearity of the biosensors can be explained by use of excessive high concentration of ferricyanide, which may overcome the relatively slow kinetics of LOD oxidation by ferricyanide[18]. Moreover, the enhancement of sensitivity for LOD/Ferri-Pt black-coated Au electrodes can be probably attributed to the denser electroactive redox species, the more uniform distribution of ferricyanide on the electrode surface, and the magnification of effectual area of electrode modified by Pt-black nanoparticles.
Table 2 Precision of biosensors between batches (20 mM lactate serum samples) Detection No.
Detection current (ȝA)
1
36.62
2
40.28
3 4 5 6
39.87 36.05 33.81 35.52
Average current (ȝA)
RSD (%)
37.03
0.0549
Table 3 Stability of biosensors (20 mM lactate serum samples)
3.5
Precision of biosensor
The precision of the biosensor in batch and between batches was investigated with 20 mM serum samples. The relative standard deviation (RSD) in batch was less than 5% (n = 8, Table 1). Six biosensors from 6 batches were used one-off with detection time 50 s to study the precision of biosensor. The RSD was 5.49% (Table 2). Thus, the biosensors exhibited excellent electrode-to-electrode and batch-to-batch reproducibility. 3.6
Storage stability of biosensor
The storage stability of the biosensors was evaluated by measuring the response current to 1–20 mM L-lactate solutions at room temperature three times every two month for one year. If the sensors contained 90% of the initial signals, the sensors were considered to be available[19]. The response signal to 20 mM lactate remained approximately 90% of its initial value and the activity to 10 mM lactate remained 95% over 1 year at room temperature. The background current was slightly increased after long time storage despite it did not effect the detect result. Hence, the biosensor showed excellent storage stability. 3.7
Interference of substances for determination of lactate
The interference study of the biosensors was investigated with 10 mM lactate and possible interferents: 5 mM glucose, 0.05 mM cysteine, 0.1 mM ascorbic acid, and 0.5 mM uric acid. Table 1 Precision of biosensors in batch (20 mM lactate serum samples) Detection No. 1 2 3 4 5 6 7 8
Detection current (ȝA) 37.10 36.58 37.03 38.24 38.16 35.98 36.85 38.56
Average current (ȝA)
Relative standard deviation (RSD, %)
Storage time (month) Average detection current (ȝA) Decrease of signal (%)
0
2
4
6
8
10
12
38.23
39.33
36.25
37.20
35.24
34.55
34.76
0
–2.9
5.2
2.7
7.8
9.6
9.1
It is found that glucose and cysteine did not result in significant interference on the response of the L-lactate biosensor. However, slightly improved signals and small positive bias (< 3%) were produced by ascorbic acid and uric acid. In conclusion, the substances in serum samples at normal concentration do not exhibit significant interference on the determination of L-lactate using the biosensor.
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