Measurement of Enzyme Activity in Single Cells by Voltammetry Using a Microcell with a Positionable Dual Electrode
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- 作者：Ning Gao ; Minghui Zhao ; Xiaoli Zhang ; and Wenrui Jin
- 刊名：Analytical Chemistry
- 出版年：2006
- 出版时间：January 1, 2006
- 年：2006
- 卷：78
- 期：1
- 页码：231 - 238
- 全文大小：226K
- 年卷期：v.78,no.1(January 1, 2006)
- ISSN：1520-6882
文摘
The electrochemical single-cell analysis for enzyme activitywas developed using microcells on a microcell arraycoupled with a positionable dual microelectrode. Themicrocell array with the nanoliter-scale microcells wasconstructed using simple chemical etching without photolithographic techniques. The positionable dual microelectrodes consisted of the nanometer-to-micrometer-radiusAu disk working electrode and a ~80-
m-radius Ag/AgClreference electrode. Peroxidase was chosen as the modelenzyme. Factors that concern electrochemical single-cellanalysis in microcells such as solution evaporation,interference of soluble oxygen, electrode size, solutionvolume, and electrode fouling were investigated anddiscussed. The 20 or 100 nL of detection volume wasfound to be suitable for peroxidase determination in singleneutrophils or single acute promyelocytic leukemia cellswithout interference from intracellular macromoleculesand electrode fouling, when the dual electrode with a 10-m-radius Au disk working electrode was used. Cells wereperforated with digitonin before transferring them into themicrocells, to lyse cells easily. The perforated cells weretransferred into the microcells by pushing a microscopeslide on a drop of the cell suspension on the microcellarray. After a single cell in the microcell was lysed usinga freeze-thawing technique and allowed to dry, physiological buffer saline containing 2.0 × 10-3 mol/L hydroquinone and 2.0 × 10-3 mol/L H2O2 as the substrates ofthe enzyme-catalyzed reaction was added. The microcellarray was positioned in a constant-humidity chamber toprevent evaporation. Then the dual electrode was insertedinto the microcell by means of a scanning electrochemicalmicroscope and the product benzoquinone of the enzyme-catalyzed reaction was voltammetrically detected. Peroxidase activity could be quantified using the steady-statecurrent on the voltammogram after subtracting the blankand using the calibration curve.
m-radius Ag/AgClreference electrode. Peroxidase was chosen as the modelenzyme. Factors that concern electrochemical single-cellanalysis in microcells such as solution evaporation,interference of soluble oxygen, electrode size, solutionvolume, and electrode fouling were investigated anddiscussed. The 20 or 100 nL of detection volume wasfound to be suitable for peroxidase determination in singleneutrophils or single acute promyelocytic leukemia cellswithout interference from intracellular macromoleculesand electrode fouling, when the dual electrode with a 10-m-radius Au disk working electrode was used. Cells wereperforated with digitonin before transferring them into themicrocells, to lyse cells easily. The perforated cells weretransferred into the microcells by pushing a microscopeslide on a drop of the cell suspension on the microcellarray. After a single cell in the microcell was lysed usinga freeze-thawing technique and allowed to dry, physiological buffer saline containing 2.0 × 10-3 mol/L hydroquinone and 2.0 × 10-3 mol/L H2O2 as the substrates ofthe enzyme-catalyzed reaction was added. The microcellarray was positioned in a constant-humidity chamber toprevent evaporation. Then the dual electrode was insertedinto the microcell by means of a scanning electrochemicalmicroscope and the product benzoquinone of the enzyme-catalyzed reaction was voltammetrically detected. Peroxidase activity could be quantified using the steady-statecurrent on the voltammogram after subtracting the blankand using the calibration curve.