Quantitative Determination of Enzyme Activity in Single Cells by

Dec 24, 2006 - and a nitrocellulose film with micropores. A single cell perforated by digitonin was injected into the microwell. After the perforated ...
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Anal. Chem. 2007, 79, 1256-1261

Quantitative Determination of Enzyme Activity in Single Cells by Scanning Microelectrode Coupled with a Nitrocellulose Film-Covered Microreactor by Means of a Scanning Electrochemical Microscope Xiaoli Zhang, Fuchan Sun, Xuewei Peng, and Wenrui Jin*

School of Chemistry and Chemical Engineering, Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, Shandong University, Jinan 250100, China

An electrochemical method for quantitative determination of enzyme activity in single cells was developed by scanning a microelectrode (ME) over a nitrocellulose filmcovered microreactor with micropores by means of a scanning electrochemical microscope (SECM). Peroxidase (PO) in neutrophils was chosen as the model system. The microreactor consisted of a microwell with a solution and a nitrocellulose film with micropores. A single cell perforated by digitonin was injected into the microwell. After the perforated cell was lysed and allowed to dry, physiological buffer saline (PBS) containing hydroquinone (H2Q) and H2O2 as substrates of the enzyme-catalyzed reaction was added in the microwell. The microwell containing the extract of the lysed cell and the enzyme substrates was covered with Parafilm to prevent evaporation. The solution in the microwell was incubated for 20 min. In this case, the released PO from the cell converted H2Q into benzoquinone (BQ). Then, the Parafilm was replaced by a nitrocellulose film with micropores to fabricate the microreactor. The microreactor was placed in an electrochemical cell containing PBS, H2Q, and H2O2. After a 10-µm-radius Au ME was inserted into the electrochemical cell and approached down to the microreactor, the ME was scanned along the central line across the microreactor by means of a SECM. The scan curve with a peak was obtained by detecting BQ that diffused out from the microreactor through the micropores on the nitrocellulose film. PO activity could be quantified on the basis of the peak current on the scan curve using a calibration curve. This method had two obvious advantages: no electrode fouling and no oxygen interference. Electrochemical detection is a useful tool for determining biochemical components in single cells.1-21 When a working * To whom correspondence should be addressed. E-mail: [email protected]. Fax +86-531-8856-5167. (1) Meulemans, A.; Poulain, B.; Baux, G.; Tauc, L.; Henzel, D. Anal. Chem. 1986, 58, 2088-2091. (2) Meulemans, A.; Poulain, B.; Baux, G.; Tauc, L. Brain Res. 1987, 414, 158162. (3) Chien, J. B.; Wallingford, R. A.; Ewing, A. G. J. Neurochem. 1990, 54, 633638.

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electrode is implanted into the cell, electrode fouling from biological macromolecules in a single cell is a thorny problem.8 Different methods have been used to overcome the difficulty for quantitative analysis of intracellular components.7,8 Recently, we also reported a voltammetry for determination of peroxidase (PO) activity in single cells using a microcell coupled with a positionable dual electrode.22 In this approach, the single-cell extract has to be diluted to a very low concentration, to reduce the concentration of biological macromolecules that can foul the electrode. However, if the concentration of the macromolecules in the diluted singlecell extract is high, electrode fouling still exists. To solve the problem, the best way is to completely separate the electrode from the single-cell extract. In addition, oxygen in the diluted singlecell extract is also an important interference factor for voltammetric measurements at a negative potential range. The high (4) Leszczyszyn, D. J.; Jankowski, J. A.; Viveros, O. H.; Diliberto, E. J., Jr.; Near, J. A.; Wightman, R. M. J. Biol. Chem. 1990, 265, 14736-14737. (5) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, E. J., Jr.; Viveros, O. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10754-10758. (6) Abe, T.; Lau, Y. Y.; Ewing, A. G. J. Am. Chem. Soc. 1991, 113, 7421-7423. (7) Lau, Y. Y.; Chien, J. B.; Wong, D. K. Y.; Ewing, A. G. Electroanalysis 1991, 3, 87-95. (8) Chen, T. K.; Lau, Y. Y.; Wong, D. K. Y.; Ewing, A. G. Anal. Chem. 1992, 64, 1264-1268. (9) Kennedy, R. T.; Huang, L.; Atkinson, M. A.; Dush, P. Anal. Chem. 1993, 65, 1882-1887. (10) Chen, T. K.; Luo, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (11) Zhou, R.; Luo, G.; Ewing, A. G. J. Neurosci. 1994, 14, 2402-2407. (12) Garris, P. A.; Ciolkowski, E. L.; Pastore, P.; Wightman, R. M. J. Neurosci. 1994, 14, 6084-6093. (13) Pihel, K.; Hsieh, S.; Jorgenson, J. W.; Wightman, R. M. Anal. Chem. 1995, 67, 4514-4521. (14) Chen, G.; Gavin, P. F.; Luo, G.; Ewing, A. G. J. Neurosci. 1995, 15, 77477755. (15) Kennedy, R. T.; Huang, L.; Aspinwall, C. A. J. Am. Chem. Soc. 1996, 118, 1795-1796. (16) Xin, Q.; Wightman, R. M. Anal. Chem. 1998, 70, 1677-1681. (17) Bunin, M. A.; Wightman, R. M. J. Neurosci. 1998, 18, 4854-4860. (18) Hochstetler, S. E.; Puopolo, M.; Gustincich, S.; Raviola, E.; Wightman, R. M. Anal. Chem. 2000, 72, 489-496. (19) Xue, J.; Ying, X.; Chen, J.; Xian, Y.; Jin, L. Anal. Chem. 2000, 72, 53135321. (20) Huang, W. H.; Cheng, W.; Zhang, Z.; Pang, D. W.; Wang, Z. L.; Cheng, J. K.; Cui, D. F. Anal. Chem. 2004, 76, 483-488. (21) Wu, W. Z.; Huang, W. H.; Wang, W.; Wang, Z. L.; Cheng, J. K.; Xu, T.; Zhang, R. Y.; Chen, Y.; Liu, J. J. Am. Chem. Soc. 2005, 127, 8914-8915. (22) Gao, N.; Zhao, M.; Zhang, X.; Jin, W. Anal. Chem. 2006, 78, 231-238. 10.1021/ac061450y CCC: $37.00

© 2007 American Chemical Society Published on Web 12/24/2006

Figure 1. Schematic diagram showing the process of electrochemical measurment of PO activity in single cells by scanning a microelectrode coupled with a microreactor.

oxygen reduction current that changes with potential affects the limit of detection of the analyte. In this work, we developed a new electrochemical method for quantitative analysis of PO activity in single neutrophils without electrode fouling and oxygen interference. In this method, to lyse cells more easily, neutrophils were first perforated with digitonin, which binds to cholesterol on the cell membrane to form micropores (Figure 1B). A single perforated cell was transferred into a microwell fabricated on the Plexiglas plate (Figure 1C). After the cell was lysed, the physiological buffer saline (PBS) containing enzyme substrates (hydroquinone (H2Q) and H2O2) was added to the microwell (Figure 1D). The microwell was covered with Parafilm to prevent solution evaporation. The solution in the microwell was incubated for a certain time, to allow released PO from the cell to convert H2Q into benzoquinone (BQ) according to the enzyme-catalyzed reaction expressed in reaction

1 (Figure 1E). Then, the Parafilm was removed, and a dilute nitrocellulose solution with ether and ethanol as solvents was spread on the Plexiglas plate, to form a film on the microwell. After ether and ethanol in the nitrocellulose film were evaporated, micropores were formed on the film and the microreactor was fabricated (Figure 1F). The Plexiglas plate with the microreactor was positioned in an electrochemical cell. PBS containing H2Q and H2O2 was added into the electrochemical cell over the Plexiglas plate, but not over the microreactor (Figure 1G). An Au microelectrode (ME) held at the stand of a scanning electrochemical microscope (SECM), an Ag/AgCl reference electrode, and a Pt auxiliary electrode were inserted into the solution in the electrochemical cell. The ME tip was moved down to the Plexiglas plate (Figure 1H) and then laterally toward the microreactor (Figure 1I) by means of SECM. Then, the microreactor was covered with the solution by stirring the solution in the electroAnalytical Chemistry, Vol. 79, No. 3, February 1, 2007

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chemical cell (Figure 1J). In this case, only small molecules such as BQ could diffuse out from the microreactor interior through the micropores on the nitrocellulose film to the solution on top of the microreactor, while the large molecule PO remained inside the microreactor due to the small micropore size. (Figure 1K). The BQ amount above the microreactor reflected the PO activity of the single cell in the microreactor. The ME tip held at a negative potential was moved over the microreactor and scanned along the central line across the microreactor. During the scan of the ME, BQ was electrochemically reduced at the ME (Figure 1L) according to reaction 2. The scan curve with a peak was recorded. The peak current on the scan curve was proportional to the PO activity of the single cell. The PO activity in the single cell could be obtained on the basis of the calibration curve.

H2Q + H2O2 y\ z BQ + 2H2O PO

(1)

BQ + 2H+ + 2e- f H2Q

(2)

EXPERIMENTAL SECTION Chemicals. Nitrocellulose solution (4% nitrocellulose, 20% ether, and 76% ethanol; chemical grade) was purchased from a standard reagent supplier. Before use, the nitrocellulose solution was diluted with the same volume of ethanol. Other chemicals and solution preparation were the same as in our previous work.22 Several steps described in ref 23 were taken to minimize contamination. Apparatus. A CHI 900 SECM (CH Instruments, Austin, TX) was employed to accomplish the electrochemical experiments. All experiments were carried out with a three-electrode system that consisted of a 10-µm-radius Au ME as the working electrode, an Ag/AgCl electrode (1 mol/L KCl) as the reference electrode, and a Pt wire as the auxiliary electrode. A Petri dish as the electrochemical cell with the three electrodes was housed in a Faraday cage to minimize the noise from external sources. The fabrication of 10-µm-radius Au MEs was similar to our previous work.22 Fabrication of Microreactor. First, the microwell was constructed by punching a hole on a Plexiglas plate of ∼2-mm thickness using a drill. The microwells with different diameters and different depths could be constructed by using the drills with different diameters and controlling the punching depth. The depth of the microwells was measured according to our previous work.22 The microwell with a volume of ∼17 nL was used in the work. The Plexiglas surface was ground with emery paper. The Plexiglas plate with the hole was ultrasonicated for 5 min in water, to remove the Plexiglas powder and dust. After the plate was immersed in a chromic acid mixture containing K2Cr2O7 and H2SO4 for 30 min, it was rinsed with tap water and then doubly distilled water. The microwell was allowed to dry for use. The microreactor consisted of the microwell with a solution containing a HRP standard solution (or a single-cell extract), PBS, H2Q as well as H2O2, and a nitrocellulose film with micropores. A drop of diluted nitrocellulose solution containing 2% nitrocellulose, 10% ether, and 88% ethanol was deposited onto the side of the Plexiglas plate with the microwell. The plate was inclined at a ∼30° angle. A microscope slide was held by the tips of the index finger and the (23) Sun, X.; Jin, W. Anal. Chem. 2003, 75, 6050-6055.

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thumb with its placed edge on the drop of the nitrocellulose solution. The cellulose solution was distributed along the edge of the slide. Then, the slide was pushed along the surface of the plate to introduce the nitrocellulose solution across the microwell. In this case, the microwell was covered with the nitrocellulose film. The manufacturing process of the microreactor is illustrated in the Supporting Information. After the solvents (ether and ethanol) in the nitrocellulose film were evaporated, micropores were formed on the film with a thickness of 2.7-4.1 µm. Then, the electrochemical measurement was performed. After each electrochemical measurement, the nitrocellulose film on the Plexiglas plate was removed with a tissue. Then, the plate was immersed in 2% NaOH over night. Next, the plate was immersed in 5% HCl for 30 min to neutralize NaOH. Finally, the plate was rinsed with tap water, followed by doubly distilled water. After drying the microwell was used for the next experiment. Preparation of Cells. Human neutrophils were isolated and perforated as described previously.22 Measurement of HRP Activity in the Standard Solution. Since the activity of all peroxidases is defined and determined with the same method,24 horseradish peroxidase (HRP) served as a standard to quantify PO activity and the same activity unit for both HRP and PO was used in this work. For the detection of the HRP activity in the standard solution, 15 nL of the HRP solution was added into the ∼17-nL microwell using a laboratorymade microinjector.22 The HRP standard solution in the microwell was allowed to evaporate for 2 min. Then, 15 nL of PBS containing 2.0 × 10-3 mol/L H2Q and 2.0 × 10-3 mol/L H2O2 was added into the microwell. The microwell was covered with a Parafilm to prevent evaporation. After the solution in the microwell was incubated for 20 min, the Parafilm was removed and the microwell was covered with a nitrocellulose film according to the fabrication procedure of the microreactor described above. The Plexiglas plate with the microreactor was placed on the bottom of a Petri dish as the electrochemical cell. PBS containing 2.0 × 10-3 mol/L H2Q and 2.0 × 10-3 mol/L H2O2 was added into the electrochemical cell over the Plexiglas plate, but not over the microreactor. A 10-µm-radius Au ME, an Ag/AgCl reference electrode, and a Pt auxiliary electrode were inserted into the solution in the electrochemical cell. The Au ME was held at 0.8 V versus Ag/AgCl to detect the oxidization current of H2Q. The ME was approached slowly and vertically down to the Plexiglas plate with the aid of SECM. The oxidization current decreased with the distance due to negative feedback as the ME was close to the plate. When the current was 80% of the steady-state current, the ME was stopped. In this case, the distance between the ME and the plate surface was ∼20 µm. The ME was laterally moved toward the microreactor and stopped when the ME was close to the microreactor. Then, the microreactor was covered with the solution in the electrochemical cell by stirring the solution. The potential of the ME was switched to -0.3 V to detect the BQ from the microreactor through the micropores on the nitrocellulose film. The ME was scanned at the x-y plane over the microreactor at a constant height and the 3-D (i-x-y) image was recorded. To record the 2-D (i-y) scan curve, the central line of the microreactor was (24) Stellmach, B. Bestimmungsmethoden Enzyme (fu ¨ r Phamazie, Lebensmittelchenie, Technik, Biochemie, Biologie, Medizin); Steinkopff Verlag: Darmatadt, Germany, 1988; Chapter 26.

found by moving the ME held at -0.3 V at a constant height according to ref 22. Then, the ME was scanned along the central line across the microreactor, and the scan curve was recorded. Determination of PO Activity in Single Neutrophils. To transfer a single cell into the microwell, a microscope slide was placed on the inversed biological microscope with a magnification of 400×. A 25-µm-i.d. cone-shaped capillary etched by HF was placed on the microscope slide (Caution: HF can cause severe lesions! Care should be taken to avoid skin contact). A 10-µL aliquot of the PBS suspension of the perforated neutrophil was placed on the tip of the capillary. When PBS with a single perforated cell entered the capillary due to capillary tension, the capillary with the introduced cell was quickly removed from the cell suspension and inserted into the needle of a 5-mL syringe, the tip of which had been cut off partly, through a silicon washer, which was previously inserted into the fitting sleeve of the syringe needle. The cell in the capillary was immediately expelled into the ∼17-nL microwell by depressing the syringe plunger. The manufacturing process of injecting a perforated cell into the microwell is illustrated in the Supporting Information. In order to obtain the single-cell extract containing intracellular substances involving PO, the perforated cell in the microwell was lysed using the freeze-thawing method described in ref 22. The single-cell extract in the microwell was allowed to evaporate for ∼2 min. Then, 15 nL of PBS containing 2.0 × 10-3 mol/L H2Q and 2.0 × 10-3 mol/L H2O2 was added using the laboratory-made microinjector to redissolve the intracellular substances. The microwell was covered with Parafilm, and the solution in the microwell was incubated. At a time of 20 min later, the Parafilm was replaced by the nitrocellulose film with micropores indicated earlier, and the electrochemical scan curve along the central line of the microreactor was recorded according to the procedure for measurement of the HRP activity in the standard solutions described above. The peak current on the scan curve was used for quantification PO activity in single neutrophils. RESULTS AND DISCUSSION Fabrication of Microreactor. The microreactor consisted of a microwell with a solution and a nitrocellulose film with micropores. To fabricate the microreactor, several factors should be considered. One of the key factors was that the film should attach compactly on the Plexiglas substrate, on which the microreactor was fabricated. It was found that nitrocellulose film could adhere to the slightly rough Plexiglas substrate. The commercial nitrocellulose solution containing 4% nitrocellulose, 20% ether, and 76% ethanol could not be directly used to fabricate the films because its high viscidity led to inhomogeneity and rupture of the films. When the commercial nitrocellulose solution was diluted with the same volume of ethanol, homogeneous films could be obtained. More dilute nitrocellulose solutions could result in solution leakage from the microreactor. The micropores were formed by evaporating the solvents (ether and ethanol). Using the approach, the micropore-film with a thickness of 2.7-4.1 µm was obtained. The micropore size on the film was an important factor for the determination of PO activity. Micropores on the nitrocellulose film should hold back PO molecules while letting small molecules H2Q, H2O2, and BQ through. To know whether PO molecules remained inside the nitrocellulose film-covered microreactor, the peroxidase staining experiment was adopted.

Peroxidases can catalyze the oxidation of the substrate 3,3,5,5tetramethylbenzidine to its blue oxidized form in the presence of H2O2. This reaction is very sensitive and is widely used for detecting peroxidases in ELISA.25 In the present experiment, several drops of water were added on the nitrocellulose film of the microreactor containing the HRP solution or the cell extract placed in a constant-humidity chamber. At a time 30 min later, no blue color was monitored in the water on the nitrocellulose film by using the peroxidase staining, implying no HRP or PO leakage from the microreactor through the nitrocellulose film. To obtain 3-D images of BQ from the microreactors, the HRP solution with a high activity concentration (0.188 unit/mL) and its substrates were added into the microwells with different diameters. After the microwells were covered with the nitrocellulose film, 3-D images could be obtained. Figure 2 shows the 3-D images and the scan curves along the central line of the microreactors with 130, 200, and 350 µm in diameter. When the diameters of the microreactors were smaller than 200 µm, symmetrical peakshaped images were obtained. When the diameter of the microreactor was 350 µm, a concave image was observed because the nitrocellulose film on the microreactor was caved in (Figure 2C). In addition, when the diameters of the microwell were larger than 350 µm, sometimes the microwell could not be sealed completely with the nitrocellulose film and the solution leakage could occur. In our experiments, the 200-µm microreactor was used. To test the reproducibility of the microreactor, we have recorded scan curves along the central line of the microreactors fabricated with the same microwell but different batches of the films. The relative standard deviation (RSD) of the peak current on the scan curves for six nitrocellulose films was 3.9%, which concludes the reproducibility of the nitrocellulose film and electrochemical measurement. Measurement of HRP Activity in the Standard Solution. Solution evaporation must be considered when the HRP solution were incubated with H2Q and H2O2 in the microwell. To prevent quick evaporation of the solution in the microwell, after all solutions were added the microwell was covered immediately with Parafilm. Since both PO and HRP are peroxidase, both of them can catalyze the same substrates. Therefore, HRP could be used to investigate the optimal conditions for detection of PO activity. In our previous work for voltammetric measurement of both HRP and PO,22 it was found that PBS containing 2.0 × 10-3 mol/L H2Q and 2.0 × 10-3 mol/L H2O2 was suitable for the detection of both HRP and PO. The solution was also used in the present work. The effect of the detection potential was also tested. When -0.2 V was applied, the peak current of the scan curve (ip) was lower than that with use of -0.3 or -0.4 V. ip was almost the same for the detection potentials of -0.3 and -0.4 V. The potential of -0.3 V was used in subsequent measurements. Since the detected signal was proportional to the BQ concentration generated by the enzyme-catalyzed reaction in the microreactor, ip depended on the reaction time of the enzyme-catalyzed reaction including the incubation time (tinc) from adding the solutions into the microwell to fabricating the microreactor by sealing the microwell with a nitrocellulose film, and the preparation time (tpre) from placing the microreactor into the electrochemical cell to (25) Frey, A.; Meckelein, B.; Externest, D.; Schmidt, M. A. J. Immunol. Methods 2000, 233, 47-56.

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Figure 2. HRP gray-scale (right) and 3-D (center) images, as well as 2-D scan curves along the central line of the microreactor (left) for the microreactors with (A) 130, (B) 200, and (C) 350 µm in diameter. Solution, PBS containing 0.188 unit/mL HRP, 2.00 × 10-3 mol/L H2Q, and 2.00 × 10-3 mol/L H2O2 (pH 7.4); incubation time, 20 min; ME, 10-µm-radius Au disk; potential, -0.3 V (vs Ag/AgCl); distance of tip and microreactor, 20 µm; scan rate, 600 µm/s for the 3-D images and 50 µm/s for 2-D scan curves.

recording the scan curve. ip increased with tinc (see Supporting Information). A tinc ) 20 min was used for the determination of PO activity in single cells. In our experiments, tpre took less than 10 min. To ensure the same reaction time of the enzyme-catalyzed reaction in the microreactor for each measurement, a tpre ) 10 min was used. Thus, the total reaction time including tinc and tpre was 30 min in all measurements. It was found that, for the same reaction time, ip changed with the detection time (tdet), at which the ME started scanning. The relationship between ip and tdet was different for high enzyme concentrations and low enzyme concentrations. Figure 3A shows the relationship between ip and tdet for a high HRP concentration (0.125 unit/mL). ip increased with tdet before 6 min and then changed little until 18 min. This was because the enzyme amount in the microreactor was so high that the BQ concentration in the microreactor almost did not change and the flux of BQ from the microreactor to the outside of the nitrocellulose film reached a steady-state value between 6 and 18 min. Therefore, the 3-D images could be obtained for high HRP concentrations as shown in Figure 2. The steady-state duration shortened with decreasing HRP concentration. Figure 3B shows 1260 Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

the relationship between ip and tdet for a low HRP concentration (8.00 × 10-3 unit/mL). ip decreased with tdet. In this case, the enzyme amount in the microreactor had decreased before recording the first scan curve, because a portion of BQ had diffused out from the microreactor and left the microreactor surface while looking for the central line of the microreactor. Similar phenomena were also observed by using BQ instead of HRP in the microreactor. Intermediate concentrations between both situations were 5 to 7 × 10-2 unit/mL. To obtain the highest peak current for quantification, the first scan curve was used. Under the selected conditions, ip could be used to quantify enzyme activity. The linear range of HRP activity (i.e., PO activity) in 15 nL was 7.5 × 10-9 to 5.6 × 10-7 unit with a correlation coefficient of 0.997, a slope of 2.6 × 10-4 A/unit, and an intercept of 3.5 × 10-12 A. The limit of detection of activity was 3.7 × 10-9 unit, when the signal-noise ratio was 3. The RSD of ip for 2.5 × 10-7 unit was 4.2% (n ) 3). Since the BQ that diffused out from the microreactor through the nitrocellulose film was detected, the signal detected using the method should be lower than that directly detected in the microwell without the film using linear scan voltammetry reported

Figure 3. Relationships between ip and tdet for (A) 0.125 and (B) 8.00 × 10-3 unit/mL HRP. Microreactor diameter, 200-µm; scan rate, 50 µm/s; other conditions, the same as in Figure 2.

Figure 4. Typical scan curve of a single neutrophil. Conditions are the same as in Figure 2.

previously.22 For a HRP activity of 7.5 × 10-9 unit in 15 nL, an ip value of 4.8 pA was obtained in the present method. For the same HRP activity, a steady-state current of 139 pA was measured by using the voltammetry. However, the sensitivity of the current method was enough for the detection of the PO activity in single neutrophils. Determination of PO Activity in Single Neutrophils. In the present method, there are two significant advantages over the voltammetry reported previously.22 First, in the voltammetric measurement, the electrode was directly inserted into the singlecell extract in the microcell. The biological macromolecules in the extract could foul the electrode. To solve the problem, the extract was diluted to a very low concentration. However, when the concentration of the biological macromolecules in the diluted single-cell extract was high, the electrode was still fouled. In the present method, the electrode and the single-cell extract was (26) Gao, N.; Wang, W.; Zhang, X.; Jin, W.; Yin, X.; Fang, Z. Anal. Chem. 2006, 78, 3213-3220.

separated completely by the nitrocellulose film. Thus, no electrode fouling existed. Second, in the voltammetric measurement, when the potential was scanned at a negative potential range, oxygen in the diluted single-cell extract produced a high reduction current. The high oxygen reduction current limited the detection of the analyte of interest. In the present work, a constant negative potential was applied at the electrode and the electrode was moved on the microreactor at a constant height to detect BQ from the microreactor. In this case, the oxygen reduction current that formed the baseline of the scan curve with a peak corresponding to BQ was constant. When the peak current on the scan curve was used for quantification, the constant oxygen reduction current did not interfere with the determination of BQ corresponding to PO activity in the single cells. When the nitrocellulose film-covered microreactor with a volume of 15 nL and the total reaction time of 30 min including tinc and tpre were used, the PO activity in single neutrophils could be measured. A typical scan curve of a single neutrophil is shown in Figure 4. The PO activities determined for five individual neutrophils were 1.78 × 10-8, 2.54 × 10-8, 1.48 × 10-8, 1.40 × 10-8, and 2.12 × 10-8 unit, respectively. The PO activities have the same order of magnitude compared with the values ((0.112.8) × 10-8 unit) of other samples measured by other electrochemical methods,22,26 implying that the results are reliable. CONCLUSION The combination of electrochemical detection using scanning ME and a nitrocellulose film-covered microreactor with micropores is capable of analyzing enzyme activity in single cells. The method has two obvious advantages. First, there is no electrode fouling. The ME as the working electrode and the sample solution are completely separated by the nitrocellulose film with micropores. The micropores are so small that only small molecules that do not foul the ME can diffuse out from the microreactor and contact ME. Therefore, the ME is very clean during measurement. Second, oxygen in the solution does not interfere with the determination of enzyme activity, which often limits electrochemical detection at a negative potential, because a constant baseline corresponding to the oxygen reduction current appears on the scan curve corresponding to the enzyme activity in the microreactor. The method described here can also be applied to measure other enzymes in single cells, as long as suitable enzyme substrates, the products of which are electroactive, are chosen. ACKNOWLEDGMENT This project was supported by the National Natural Science Foundation of China (Grant Nos. 20235010, 20475033, and 20675047), the Natural Science Foundation of Shandong Province (Grant Y2003B04) in China and the State Key Laboratory of Electroanalytical Chemistry, Changchun, Institute of Applied Chemistry, Chinese Academy of Science. SUPPORTING INFORMATION AVAILABLE Three additional figures. This material is available free of charge via the Internet at http://pubs.acs.org Received for review August 4, 2006. Accepted November 20, 2006. AC061450Y Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

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