||An electrochemical detection method to quantify myeloperoxidase
||Institute of Biomedical Engineering
acute myocardial infarction
骨髓過氧化酶(Myeloperoxidase, MPO)是存在於嗜中性白血球和單核球中的一種酵素，其功能主要是在人體內可催化生成次氯酸(HOCl)以破壞侵入人體的病原體。過去的研究顯示，急性心肌梗塞(acute myocardial infarction, AMI)病人(>91.7 ng/ml)在血漿中MPO的含量明顯高於正常人(20~60 ng/ml)，且在動脈粥狀硬化斑形成的過程亦證實HOCl最先造成低密度脂蛋白(low density lipoprotein, LDL)氧化，進而啟動動脈粥狀硬化的產生。因此，血漿中MPO的含量可用來評估AMI發病的危險性。本研究將MPO修飾於金電極的表面，並以氧化還原活性物質四甲基聯苯胺(3,3’,5,5’-tetra-methylbenzidine, TMB)作為電子傳遞物，利用電化學分析法，量測TMB的氧化還原訊號，以討論MPO酵素與過氧化氫(hydrogen peroxide, H2O2)之間的反應。TMB於修飾MPO酵素的金電極表面，依然具有其氧化還原的特性。當溶液中含有H2O2時，可促使MPO酵素催化TMB氧化態的生成，因此，藉由TMB還原電流的變化，可以成功地推知MPO酵素與溶液中的H2O2反應之關係。相較於傳統檢測方法，我們將MPO與TMB催化反應的時間縮短至4分鐘。於本研究中，所量測的MPO線性範圍為3.3 ~ 330 ng/ml(線性為R2=0.9866)，檢測極限為3.3 ng/ml。由結果可知，我們成功利用電化學法了解MPO酵素催化反應，藉由此方法未來將有機會能夠發展出一套簡單且快速預測AMI的晶片。
Myeloperoxidase (MPO) is a protein which is present in neutrophils and monocytes; its function mainly catalyzes H2O2 and Cl− to generate hypochlorite (HOCl), destroying the pathogen in human body. Previous researches have shown that the plasma MPO concentration is significantly higher in acute myocardial infarction (AMI) patients (>91.7 ng/ml) than healthy people (20 ~ 60 ng/ml). The formation of atherosclerotic plaque also confirmed that the process of low-density lipid (LDL) oxidation is caused by HOCl first, and followed activated the production of atherosclerosis. Thus, MPO levels in plasma can be served as a marker to assess the risk of AMI onset. In this work, we modified MPO on the gold electrode surface by self-assembled monolayer. 3,3’,5,5’-tetra-methylbenzidine (TMB) was used as redox mediator to analyze the reaction between MPO and hydrogen peroxide (H2O2). It showed that the redox signal of TMB on modified MPO electrode exhibited good electrochemical response. When the solution contains H2O2, MPO can catalyze TMB to produce TMB(ox). Therefore, the change of TMB reduction current can reveal the reaction between MPO and H2O. Compared to traditional detection methods, we shortened the response time of MPO-catalyzed TMB to 4 mins. In this study, the linear regression of MPO was 3.3 ~ 330 ng/ml (R2=0.9866), and the limit of detection was 3.3 ng/ml. Based on the results, we successfully detected the MPO catalyzed reaction by electrochemical method. In the future, we can develop a simply and rapid MPO detection chip by using electrochemical method for the risk prediction of AMI.
Table List VI
Figure List VII
Chapter 1 Introduction 1
1.1 Acute Myocardial Infarction 1
1.2 Atherosclerosis 4
1.2.1 Initiation Factor 4
1.2.2 Foam Cell Formation 5
1.2.3 Stability of Plaque 6
1.3 Myeloperoxidase and Acute Myocardial Infarction 8
1.4 Myeloperoxidase 10
1.5 Myeloperoxidase Detection Method 12
1.6 3, 3’, 5, 5’-Tetramethlbenzidine 14
1.7 Aim of This Study 16
Chapter 2 Materials and Experiments 17
2.1 Equipments 17
2.2 Materials 17
2.2.1 Chemicals for Microfabrication 17
2.2.2 Chemicals for Electrochemical Analysis 17
2.3 Test Solutions 18
2.4 Chip Fabrication 18
2.5 Fabrication of the Sensing Interface 20
2.6 The Experiment of Electrochemical Characteristic 21
Chapter 3 Results and Discussion 22
3.1 The Reaction Mechanism of HRP and MPO 22
3.2 Characteristic of the Modified Au Electrode 24
3.3 The Selection of Electron Transfer Mediator 26
3.4 The Electrochemical Characteristic of TMB and H2O2 28
3.5 The Reaction of HRP/TMB/H2O2 29
3.5.1 The Incubation Time of HRP Reaction 30
3.5.2 Calibration Curve of HRP 32
3.6 The Relationship between MPO, H2O2 and TMB 34
3.7 The Effect of Incubation Time 36
3.8 Calibration of MPO Concentration 38
Chapter 4 Conclusion and Prospect 40
 V. L. Roger, A. S. Go, D. M. Lloyd-Jones, R. J. Adams, J. D. Berry, T. M. Brown, M. R. Camethon, S. Dai, G. de Simone, E. S. Ford, C. S. Fox, H. J. Fullerton, C. Gillespie, K. J. Greenlund, S. M. Hailpem, J. A. Heit, P. M. Ho, V. J. Howard, B. M. Kissela, S. J. Kittner, D. T. Lackland, J. H. Lichtman, L. D. Lisabeth, D. M. Makuc, G. M. Marcus, A. Marelli, D. B. Matchar, M. M. McDermott, J. B. Meigs, C. S. Moy, D. Mozaffarian, M. E. Mussolino, G. Nichol, N. P. Paynter, W. D. Rosamond, P. D. Sorlie, R. S. Stafford, T. N. Turan, M. B. Turner, N. D. Wong, J. Wylie-Rosett, and S. American Heart Assoc Stat Comm; Stroke Stat, "Heart disease and stroke statistics-2011 update a report from the American Heart Association," Circulation, vol. 123, pp.E18-209, 2011.
 Department of health, Executive Yuan, R.O.C. (Taiwan) http://www.doh.gov.tw/CHT2006/DisplayStatisticFile.aspx?d=80725&s=1, 2011.7.29
 E. Boersma, A. C. P. Maas, J. W. Deckers, and M. L. Simoons, "Early thrombolytic treatment in acute myocardial infarction: reappraisal of the golden hour," The Lancet, vol. 348, pp. 771-775, 1996.
 R. Bernard, E. Corday, H. Eliasch, A. Gonin, R. Hiait, L. F. Nikolaeva, V. Puddu, C. M. Oakley, M. F. Olivier, and et al., "Nomenclature and criteria for diagnosis of ischemic heart disease report of the joint international society and federation of cardiology who task force on standardization of clinical nomenclature," Circulation, vol. 59, pp. 607-609, 1979.
 C. J. Marshall, M. Nallaratnam, T. Mocatta, D. Smyth, M. Richards, J. M. Elliott, J. Blake, C. C. Winterbourn, A. J. Kettle, and D. R. McClean, "Factors influencing local and systemic levels of plasma myeloperoxidase in ST-segment elevation acute myocardial infarction," American Journal of Cardiology, vol. 106, pp. 316-322, 2010.
 D. J. Engelen, A. P. Gorgels, E. C. Cheriex, E. D. De Muinck, A. J. O. Ophuis, W. R. Dassen, J. Vainer, V. G. van Ommen, and H. J. Wellens, "Value of the electrocardiogram in localizing the occlusion site in the left anterior descending coronary artery in acute anterior myocardial infarction," Journal of the American College of Cardiology, vol. 34, pp. 389-395, 1999.
 J. V. Nable and W. Brady, "The evolution of electrocardiographic changes in ST-segment elevation myocardial infarction," The American Journal of Emergency Medicine, vol. 27, pp. 734-746, 2009.
 Y. Birnbaum and B. J. Drew, "The electrocardiogram in ST elevation acute myocardial infarction: correlation with coronary anatomy and prognosis," Postgraduate Medical Journal, vol. 79, pp. 490-504, 2003.
 A. Dominguez-Rodriguez, S. Samimi-Fard, P. Abreu-Gonzalez, M. J. Garcia-Gonzalez, and J. C. Kaski, "Prognostic value of admission myeloperoxidase levels in patients with ST-segment elevation myocardial infarction and cardiogenic shock," American Journal of Cardiology, vol. 101, pp. 1537-1540, 2008.
 P. Grande, B. F. Hansen, C. Christiansen, and J. Naestoft, "Estimation of acute myocardial infarct size in man by serum CK-MB measurements," Circulation, vol. 65, pp. 756-764, 1982.
 J. Mair, E. Artnerdworzak, A. Dienstl, P. Lechleitner, B. Morass, J. Smidt, I. Wagner, C. Wettach, and B. Puschendorf, "Early detection of acute myocardial infarction by measurement of mass concentration of creatine kinase-MB," American Journal of Cardiology, vol. 68, pp. 1545-1550, 1991.
 M. Zabel, S. H. Hohnloser, W. Koster, M. Prinz, W. Kasper, and H. Just, "Analysis of creatine-kinase, CK-MB, myoglobin, and troponin-T time-activity curves for early assessment of coronary artery reperfusion after intravenous thrombolysis," Circulation, vol. 87, pp. 1542-1550, 1993.
 R. J. Dewinter, R. W. Koster, A. Sturk, and G. T. Sanders, "Value of myoglobin, troponin-T, and CK-MB(mass) in ruling out an acute myocardial infarction in the emergency room," Circulation, vol. 92, pp. 3401-3407, 1995.
 R. J. de Winter, "Risk stratification with cardiac troponin I in acute coronary syndromes," Journal of the American College of Cardiology, vol. 36, pp. 1824-1826, 2000.
 B. Lindahl, H. Toss, A. Siegbahn, P. Venge, L. Wallentin, and F. S. Grp, "Markers of myocardial damage and inflammation in relation to long-term mortality in unstable coronary artery disease," New England Journal of Medicine, vol. 343, pp. 1139-1147, 2000.
 P. M. Ridker, C. H. Hennekens, J. E. Buring, and N. Rifai, "C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women," New England Journal of Medicine, vol. 342, pp. 836-843, 2000.
 C. J. McCann, B. M. Glover, I. B. A. Menown, M. J. Moore, J. McEneny, C. G. Owens, B. Smith, P. C. Sharpe, I. S. Young, and J. A. Adgey, "Novel biomarkers in early diagnosis of acute myocardial infarction compared with cardiac troponin T," European Heart Journal, vol. 29, pp. 2843-2850, 2008.
 M. S. Dekker, A. Mosterd, A. W. J. van't Hof, and A. W. Hoes, "Novel biochemical markers in suspected acute coronary syndrome: systematic review and critical appraisal," Heart, vol. 96, pp. 1001-1010, 2010.
 P. R. Nis I. Nissen, and J. Weis-Fogh, "Evaluation of four different serum enzymes in the diagnosis of acute myocardial infarction," British Heart Journal, vol. 27, pp. 520-526, 1965.
 C. R. R. Galen S. Wagner, Lee E. Limbird, Robert A. Rosati, and Andrew G. Wallace, "The importance of identification of the myocardial-specific isoenzyme of creatine phosphokinase (MB form) in the diagnosis of acute myocardial infarction," Circulation, vol. 47, pp. 263-269, 1973.
 S. M. Surya P. Rao, Richard Rosenbaum, and Jeffrey B. Lakier, "Cardiac troponin I and cardiac enzymes after electrophysiologic studies, ablations, and defibrillator implantations," American Journal of Cardiology, vol. 84, p. 470, 1999.
 V. R. Britta U. Goldmann, Tanja K. Rudolph, Ann-Katrin Holle, Matthias Hillebrandt, Thomas Meinertz, Stephan Baldus, "Neutrophil activation precedes myocardial injury in patients with acute myocardial infarction," Free Radical Biology and Medicine, vol. 47, pp. 79-83, 2009.
 W. Hochholzer, D. A. Morrow, and R. P. Giugliano, "Novel biomarkers in cardiovascular disease: Update 2010," American Heart Journal, vol. 160, pp. 583-594, 2010.
 T. Vainas, F. R. M. Stassen, R. de Graaf, E. L. L. Twiss, S. B. Herngreen, R. J. T. J. Welten, L. H. J. M. van den Akker, M. P. van Dieijen-Visser, C. A. Bruggeman, and P. J. E. H. M. Kitslaar, "C-reactive protein in peripheral arterial disease: Relation to severity of the disease and to future cardiovascular events," Journal of Vascular Surgery, vol. 42, pp. 243-251, 2005.
 G. M. Chisolm and D. Steinberg, "The oxidative modification hypothesis of atherogenesis: An overview," Free Radical Biology and Medicine, vol. 28, pp. 1815-1826, 2000.
 E. A. Podrez, H. M. Abu-Soud, and S. L. Hazen, "Myeloperoxidase generated oxidants and atherosclerosis," Free Radical Biology and Medicine, vol. 28, pp. 1717-1725, 2000.
 P. Libby, "Inflammation in atherosclerosis," Nature, vol. 420, pp. 868-874, 2002.
 A. J. Lusis, "Atherosclerosis," Nature, vol. 407, pp. 233-241, 2000.
 P. Libby, "Inflammation and atherosclerosis," Circulation, vol. 105, pp. 1135-1143, 2002.
 S. Lorkowski and P. Cullen, "Atherosclerosis: Pathogenesis, clinical features and treatment," 2007.
 H. D. White and D. P. Chew, "Acute myocardial infarction," The Lancet, vol. 372, pp. 570-584, 2008.
 N. G. Frangogiannis, C. W. Smith, and M. L. Entman, "The inflammatory response in myocardial infarction," Cardiovascular Research, vol. 53, pp. 31-47, 2002.
 G. S. Getz, "Immune function in atherogenesis," Journal of Lipid Research, vol. 46, pp. 1-10, 2005.
 G. K. Hansson, "Mechanisms of disease - inflammation, atherosclerosis, and coronary artery disease," New England Journal of Medicine, vol. 352, pp. 1685-1695, 2005.
 W. B. Kannel, "Overview of hemostatic factors involved in atherosclerotic cardiovascular disease," Lipids, vol. 40, pp. 1215-1220, 2005.
 V. C. Mehra, V. S. Ramgolam, and J. R. Bender, "Inflammatory mechanisms in vascular disease," Drug Discovery Today: Disease Mechanisms, vol. 2, pp. 77-84, 2005.
 R. Virmani, A. P. Burke, A. Farb, and F. D. Kolodgie, "Pathology of the vulnerable plaque," Journal of the American College of Cardiology, vol. 47, pp. C13-C18, 2006.
 K. J. Moore and I. Tabas, "Macrophages in the pathogenesis of atherosclerosis," Cell, vol. 145, pp. 341-355, 2011.
 R. Ross, "Mechanisms of disease - atherosclerosis - an inflammatory disease," New England Journal of Medicine, vol. 340, pp. 115-126, 1999.
 M. I. Cybulsky and M. A. Gimbrone, "Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis," Science, vol. 251, pp. 788-791, 1991.
 O. M. Panasenko, S. A. Evgina, R. K. Aidyraliev, V. I. Sergienko, and Y. A. Vladimirov, "Peroxidation of human blood lipoproteins induced by exogenous hypochlorite or hypochlorite generated in the system of myeloperoxidase+H2O2+Cl−," Free Radical Biology and Medicine, vol. 16, pp. 143-148, 1994.
 A. Daugherty, J. L. Dunn, D. L. Rateri, and J. W. Heinecke, "Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions," Journal of Clinical Investigation, vol. 94, pp. 437-444, 1994.
 R. Zhang, "Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation," Journal of Biological Chemistry, vol. 277, pp. 46116-46122, 2002.
 R. K. Schindhelm, L. P. van der Zwan, T. Teerlink, and P. G. Scheffer, "Myeloperoxidase: A useful biomarker for cardiovascular disease risk stratification?," Clinical Chemistry, vol. 55, pp. 1462-1470, 2009.
 S. J. Nicholls and S. L. Hazen, "Myeloperoxidase and cardiovascular disease," Arteriosclerosis Thrombosis and Vascular Biology, vol. 25, pp. 1102-1111, 2005.
 D. Lau and S. Baldus, "Myeloperoxidase and its contributory role in inflammatory vascular disease," Pharmacology & Therapeutics, vol. 111, pp. 16-26, 2006.
 E. Cavusoglu, C. Ruwende, C. Eng, V. Chopra, S. Yanamadala, L. T. Clark, D. J. Pinsky, and J. D. Marmur, "Usefulness of baseline plasma myeloperoxidase levels as an independent predictor of myocardial infarction at two years in patients presenting with acute coronary syndrome," American Journal of Cardiology, vol. 99, pp. 1364-1368, 2007.
 M. C. Meuwese, E. S. G. Stroes, S. L. Hazen, J. N. van Miert, J. A. Kuivenhoven, R. G. Schaub, N. J. Wareham, R. Luben, J. J. P. Kastelein, and K.-T. Khaw, "Serum myeloperoxidase levels are associated with the future risk of coronary artery disease in apparently healthy individuals-the EPIC-Norfolk prospective population study," Journal of the American College of Cardiology, vol. 50, pp. 159-165, 2007.
 M. Cheng, C. Chen, P. Gu, H. Ho, and D. Chiu, "Elevated levels of myeloperoxidase, white blood cell count and 3-chlorotyrosine in Taiwanese patients with acute myocardial infarction," Clinical Biochemistry, vol. 41, pp. 554-560, 2008.
 G. Ndrepepa, S. Braun, J. Mehilli, N. Von Beckerath, A. Schömig, and A. Kastrati, "Myeloperoxidase level in patients with stable coronary artery disease and acute coronary syndromes," European Journal of Clinical Investigation, vol. 38, pp. 90-96, 2008.
 R. Zhang, "Association between myeloperoxidase levels and risk of coronary artery disease," The Journal of the American Medical Association, vol. 286, pp. 2136-2142, 2001.
 S. Baldus, "Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes," Circulation, vol. 108, pp. 1440-1445, 2003.
 T. J. Mocatta, A. P. Pilbrow, V. A. Cameron, R. Senthilmohan, C. M. Frampton, A. M. Richards, and C. C. Winterbourn, "Plasma concentrations of myeloperoxidase predict mortality after myocardial infarction," Journal of the American College of Cardiology, vol. 49, pp. 1993-2000, 2007.
 G. Brevetti, V. Schiano, E. Laurenzano, G. Giugliano, M. Petretta, F. Scopacasa, and M. Chiariello, "Myeloperoxidase, but not C-reactive protein, predicts cardiovascular risk in peripheral arterial disease," European Heart Journal, vol. 29, pp. 224-230, 2008.
 A. Dominguezrodriguez, S. Samimifard, P. Abreugonzalez, M. Garciagonzalez, and J. Kaski, "Prognostic value of admission myeloperoxidase levels in patients with st-segment elevation myocardial infarction and cardiogenic shock," American Journal of Cardiology, vol. 101, pp. 1537-1540, 2008.
 L. Kubala, G. Lu, S. Baldus, L. Berglund, and J. Eiserich, "Plasma levels of myeloperoxidase are not elevated in patients with stable coronary artery disease," Clinica Chimica Acta, vol. 394, pp. 59-62, 2008.
 A. Hoy, D. Tregouet, B. Leininger-Muller, O. Poirier, M. Maurice, C. Sass, G. Siest, L. Tiret, and S. Visvikis, "Serum myeloperoxidase concentration in a healthy population: biological variations, familial resemblance and new genetic polymorphisms," European Journal of Human Genetics, vol. 9, pp. 780-786, 2001.
 S. W. S. Fred S. Apple, Lesly A. Pearce, Karen M. Schulz, Ranka Ler, and MaryAnn M. Murakami, "Myeloperoxidase improves risk stratification in catients with Ischemia and Normal cardiac troponin I concentrations," Clinical Chemistry, vol. 57, pp. 603-608, 2011.
 C. L. Heslop, J. J. Frohlich, and J. S. Hill, "Myeloperoxidase and C-reactive protein have combined utility for long-term prediction of cardiovascular mortality after coronary angiography," Journal of the American College of Cardiology, vol. 55, pp. 1102-1109, 2010.
 S. J. Klebanoff, "Myeloperoxidase: friend and foe," Journal of Leukocyte Biology, vol. 77, pp. 598-625, 2005.
 M. B. Hampton, A. J. Kettle, and C. C. Winterbourn, "Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing," Blood, vol. 92, pp. 3007-3017, 1998.
 M. Brennan, M. S. Penn, F. Van Lente, V. Nambi, M. H. Shishehbor, R. J. Aviles, M. Goormastic, M. L. Pepoy, E. S. McErlean, E. J. Topol, S. E. Nissen, and S. L. Hazen, "Prognostic value of myeloperoxidase in patients with chest pain," New England Journal of Medicine, vol. 349, pp. 1595-1604, 2003.
 C. C. Winterbourn, M. C. M. Vissers, and A. J. Kettle, "Myeloperoxidase," Current Opinion in Hematology, vol. 7, pp. 53-58, 2000.
 A. Mayer-Scholl, P. Averhoff, and A. Zychlinsky, "How do neutrophils and pathogens interact?," Current opinion in microbiology, vol. 7, pp. 62-6, 2004.
 I. OXIS Health Products, "BIOXYTECH® MPO-EIA™ Assay Kit," ed, 2004.
 N. L. S. Specialties, "NWLSSTM Myeloperoxidase Activity Assay," ed.
 P. Fanjul-Bolado, M. B. González-García, and A. Costa-García, "Amperometric detection in TMB/HRP-based assays," Analytical and Bioanalytical Chemistry, vol. 382, pp. 297-302, 2005.
 B. L. Li, Y. Du, T. Li, and S. J. Dong, "Investigation of 3,3 ',5,5 '-tetramethylbenzidine as colorimetric substrate for a peroxidatic DNAzyme," Analytica Chimica Acta, vol. 651, pp. 234-240, Oct 2009.
 P. D. Josephy, T. Eling, and R. P. Mason, "The horseradish peroxidase-catalyzed oxidation of 3,5,3',5'-tetramethylbenzidine - free radical and charge transfer complex intermediates," Journal of Biological Chemistry, vol. 257, pp. 3669-3675, 1982.
 N. Zhang and D. H. Appella, "Colorimetric detection of anthrax DNA with a peptide nucleic acid sandwich-hybridization assay," Journal of the American Chemical Society, vol. 129, pp. 8424-8425, 2007.
 D. M. Kong, J. Wu, Y. E. Ma, and H. X. Shen, "A new method for the study of G-quadruplex ligands," The Analyst, vol. 133, pp. 1158-60, 2008.
 M. De Backer and F. X. Sauvage, "In situ FTIR spectroelectrochemistry and spectral simulations using DFT: Efficient complementary tools to elucidate complex electrochemical mechanisms," Journal of Electroanalytical Chemistry, vol. 602, pp. 131-137, 2007.
 J. Lin, L. Zhang, and S. Zhang, "Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium–tin oxide electrode," Analytical Biochemistry, vol. 370, pp. 180-185, 2007.
 M. Liu, Y. Zhang, Y. Chen, Q. Xie, and S. Yao, "EQCM and in situ FTIR spectroelectrochemistry study on the electrochemical oxidation of TMB and the effect of large-sized anions," Journal of Electroanalytical Chemistry, vol. 622, pp. 184-192, 2008.
 G. K. Ahirwal and C. K. Mitra, "Gold nanoparticles based sandwich electrochemical immunosensor," Biosensors and Bioelectronics, vol. 25, pp. 2016-2020, 2010.
 V. R. Holland, B. C. Saunders, F. L. Rose, and A. L. Walpole, "Safer substitute for benzidine in detection of blood," Tetrahedron, vol. 30, pp. 3299-3302, 1974.
 T. Ruzgas, L. Gorton, J. Emnéus, and G. Marko-Varga, "Kinetic models of horseradish peroxidase action on a graphite electrode," Journal of Electroanalytical Chemistry, vol. 391, pp. 41-49, 1995.
 N. C. Veitch, "Horseradish peroxidase: a modern view of a classic enzyme," Phytochemistry, vol. 65, pp. 249-259, 2004.
 A. J. Kettle and C. C. Winterbourn, "A kinetic analysis of the catalase activity of myeloperoxidase," Biochemistry, vol. 40, pp. 10204-10212, 2001.
 B. Y. Chang and S. M. Park, "Electrochemical impedance spectroscopy," Annual review of analytical chemistry, vol. 3, pp. 207-29, 2010.
 S. D. Collyer, F. Davis, A. Lucke, C. J. M. Stirling, and S. P. J. Higson, "The electrochemistry of the ferri/ferrocyanide couple at a calix 4 resorcinarenetetrathiol-modified gold electrode as a study of novel electrode modifying coatings for use within electro-analytical sensors," Journal of Electroanalytical Chemistry, vol. 549, pp. 119-127, 2003.
 Y. C. Liu, H. Y. Liu, J. H. Qian, J. Q. Deng, and T. Y. Yu, "Feature of an amperometric ferrocyanide-mediating H2O2 sensor for organic-phase assay based on regenerated silk fibroin as immobilization matrix for peroxidase," Electrochimica Acta, vol. 41, pp. 77-82, 1996.
 J. Biscay, E. C. Rama, M. B. G. Garcia, J. M. P. Carrazon, and A. C. Garcia, "Enzymatic sensor using mediator-screen-printed carbon electrodes," Electroanalysis, vol. 23, pp. 209-214, 2011.
 B. Li, Y. Du, T. Li, and S. Dong, "Investigation of 3,3’,5,5’-tetramethylbenzidine as colorimetric substrate for a peroxidatic DNAzyme," Analytica Chimica Acta, vol. 651, pp. 234-240, 2009.