Scanning electrochemical microscopy coupled with intracellular standard addition method for quantification of enzyme activity in single intact cells

This paper uses scanning electrochemical microscopy (SECM) coupled with an intracellular standard addition method to quantify enzyme activity in single intact cells. In this work, peroxidase (PO) inside human neutrophils is chosen as the model system. Cells immobilized onto a silanized coverslip are perforated with digitonin to form micropores on the cell membrane. Hydroquinone (H(2)Q) and hydrogen peroxide (H(2)O(2)) as the enzyme substrates diffuse through the micropores into the cell interior. There, H(2)Q is converted into benzoquinone (BQ) by intracellular PO. BQ diffuses with a steady flux through the micropores from the cell interior onto the cell surface. The BQ near the cell surface is detected by the Au tip of SECM held at -0.3 V. When the tip is scanned laterally along the central line over the cell, a 2-D scan curve is obtained. Then, the intracellular standard addition method using ultramicroinjection with a submicrometer-sized micropipette tip is performed. After ultramicroinjection of a standard solution, another 2-D scan curve is recorded. The intercellular enzyme activity can be calculated from both peak current on two scan curves. This method to quantify PO activity in the cell environment has several obvious advantages: high sensitivity due to signal amplification via intracellular enzyme-catalyzed reaction and no sample dilution, no electrode fouling from adsorption of intracellular biological molecules, and no interference from electro-active compounds that can be directly oxidized at the SECM tip or from oxygen in the detected solution.