Picomolar peroxide detection using a chemically activated redox mediator and square wave voltammetry.
A method for low-level, low-potential electrochemical detection of hydrogen peroxide using a chemically activated redox mediator is presented. This method is unique in that it utilizes a mediator, Amplex Red, which is only redox-active when chemically oxidized by H2O2 in the presence of the enzyme horseradish peroxidase (HRP). When employed in concert with microelectrode square wave voltammetry to optimize sensing at ultralow concentrations (<1 microM), this method exhibits marked improvements in analytical sensitivity and detection limits (limit of detection as low as 8 pM) over existing protocols. Sensing schemes incorporating both freely diffusing and immobilized HRP are evaluated, and the resulting analytical sensitivities are 1.22 +/- 0.04 and (2.1 +/- 0.6) x 10(-1) microA/(microM mm2), respectively, for peroxide concentrations in the high picomolar to low micromolar range. A second linear region exists for lower peroxide concentrations. Furthermore, quantitative enzyme kinetics analysis using Michaelis-Menten parameters is possible through interpretation of data collected in this scheme. Km values for soluble and immobilized HRP were 84 +/- 13 and 504 +/- 19 microM, respectively. This method is amenable to any biological detection scheme that generates hydrogen peroxide as a reactive product.
Lyon
Jennifer L
JL
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station, MC A5300, Austin, Texas 78712, USA.
Stevenson
Keith J
KJ
D015374:Biosensing Techniques; D004566:Electrodes; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis*; D007700:Kinetics; D010084:Oxidation-Reduction; D011199:Potentiometry; D012680:Sensitivity and Specificity; D012834:Silver / Q000737:chemistry; D018030:Silver Compounds / Q000737:chemistry
D018030:Silver Compounds; D012834:Silver; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; C037548:silver chloride