Serum cytochrome c detection using a cytochrome c oxidase biosensor

Abstract

Within the last ten years it has emerged that the release of cytochrome c plays a critical role in the important process of programmed cell death. It has also been shown that this protein is released into the circulating blood following MIs (myocardial infarctions). Methods for the detection of this protein have therefore become important. The enzyme cytochrome c oxidase is specific for cytochrome c. Bovine cytochrome c oxidase was successfully immobilized in a didodecyldimethylammonium bromide vesicular system on to gold electrodes and its interaction with cytochrome c was studied. Square-wave-voltammetric analysis of the biosensor showed two redox couples with midpoint potential, E(0)', values of +182 and +414 mV compared with Ag/AgCl. The redox couple with E(0)' of +414 mV showed a cathodic sensitivity to the presence of cytochrome c in both buffer solution and human serum. Responses of the cytochrome c oxidase biosensor to oxidized cytochrome c followed hyperbolic electrochemical Michaelis-Menten kinetics with a K(m) of 1.57 microM and maximum current (I(max)) of 1.38 x 10(-6) muA. The detection limit of the biosensor in human serum was 0.2 microM, which is well below the lowest physiological concentration of 0.8 muM previously reported for MIs [Alleyne, Joseph and Sampson (2001) Appl. Biochem. Biotechnol. 90, 97-105]. These results indicate that the cytochrome c oxidase biosensors could be used to determine variations in cytochrome c concentration and thus have potential to be used as a diagnostic tool in the detection of MIs and possibly also in the study of programmed cell death.

Electrocatalysis by Heme Enzymes—Applications in Biosensing

“…To that end, the few existing reports have mainly focused on detection of cyt c in biological samples, since its presence in blood can be indicative of several pathologies related to damaged mitochondria, such as apoptotic cell death, myocardial infarctions, and a number of neurological diseases [224]. The reported cyt c sensing applications include those that rely on (i) immobilization of CcO onto Au electrode-supported lipid bilayer membranes; the oxidase-modified electrode operates as an amperometric biosensor capable of detection of reduced cyt c with detection limit of 0.1 µM [127], (ii) casting of CcO-didodecyldimethylammonium bromide (DDAB) vesicle films onto Au electrodes for detection of ferric cyt c in human blood serum [126], and (iii) CcO deposited over Au electrodes modified with NiONPs, CNTs and a conducting PANI polymer (NiONPs/cMWCNT/PANI hybrid) [225]. The amperometric biosensors for cyt c based on these hybrid films were investigated by CV, electrochemical impedance spectroscopy (EIS), SEM, and FTIR [225], revealing a linear range between 1 × 10 −6 and 0.1 µM, a superior detection limit in different serum samples (5 × 10 −6 µM) and sensitivity of 3.7 × 10 6 A M −1 cm −2 [225].…”

Recent advances in cytochrome c biosensing technologies

“…9). Ashe et al, have designed a CcO based biosensor for the detection of cyt c levels in serum (Ashe et al, 2007). For example, a study by Cullison and Hawkridge showed that CcO can measure either both oxidized or reduced cyt c when an appropriate voltage is applied (Cullison et al, 1994).…”

“…However, the rate of reduction of oxidized form of cyt c by CcO was less than the oxidation of reduced form of cyt c. Ashe et al, also used to detect oxidized form of cyt c in serum samples using CcO to evaluate its possible application in detecting early myocardial infarctions (Ashe et al, 2007).…”

“…The detection limit of the sensor is 2 nM with a dynamic range spanning to 1 µM. (Zhao et al, 2008) sBLM/GNP/PC/LA/GCE LSV 0.1 µM -3.2 µM 50 nM (Liu and Wei, 2008) CcO/NiONPs/MWCNT/PANI/Au CV 5 pM -0.5 µM 5 pM (Batra et al, 2013) CcO/DDAB/Au SWV 0.2 µM -800 µM 0.2 µM (Ashe et al, 2007) CcR/SAM/GNP/PPy/Pt CV 5 µM -600 µM 2 µM (Pandiaraj et al, 2013b) CcR/CNT/PPy/Pt CV 1 µM -1000 µM 0.5 µM (Pandiaraj et al, 2013b) CcR/CNT/PPy/SPE CV 50 nM -500 µM 50 nM (Pandiaraj et al, 2014a) anti-cyt c/SAM/GNP/PPy/SPE CV 2 nM -150 µM 2 nM (Pandiaraj et al, 2014b) anti-cyt c/CNT/PPy/SPE CV 10 nM -50 µM 10 nM (Pandiaraj et al, 2014b) PEG/Aptamer/Epoxy-Graphite EIS 50 pM -50 nM 63.2 pM (Ocaña et al, 2014) Aptamer/EDC-NHS/MUA/Gold SPR 80 nM -80 pM ≥50 pM (Loo et al, 2014) DNA-Cellulose/CPE DPV 0.1 mM -1µM 0.5 µM (Lee et al, 2004) Single-strand DNA (ssDNA), Supported bilayer lipid membrane (sBLM), Soybean Phosphatidylcholine, (PC), Lauric acid (LA), Glassy carbon electrode (GCE), Nickel oxide nanoparticles (NiONPs), Polyaniline (PANI), didodecyldimethylammonium bromide (DDAB), Polyethylene glycol (PEG), 11-mercaptoundecanoic acid (MUA), Carbon paste electrode (CPE).…”

A cost-effective volume miniaturized and microcontroller based cytochrome c assay

“…Over the past two decades, electrochemical biosensors have gained considerable attention and evolved as an alternative technique for the measurement of cyt c [11][12][13]. These biosensors either based on electrostatic interaction or cyt c oxidase, lacking specificity or measure only the non-apoptotic form of cyt c (Fe(II)) and hence are not suitable for the measurement of apoptosis.…”