SPCE

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

FIGURE 5.5 Schematic representations of the two immunosensor formats (a) immunosensor based on the biotin-streptavidin interaction and (b) immunosensor based on rabbit IgG-modified SPCEs. (Reprinted from [27] with permission from Elsevier.)... 

They found that the hydrolysis products of 4-AP and 1-naphthol produced well-defined anodic responses at low potentials at a bare SPCE. However, the presence of antibody immobilized on the electrode surface slowed the diffusion of 4-AP towards the electrode surface. In addition, 4-AP may interact with polyphenols on the electrode surface, thus reducing the electroactive working area of the electrode by fouling. In contrast, diffusion of 1-naphthol to the electrode surface was not hindered by immobilized antibody. This feature, along with its low cost, ease of availability, and high solubility, resulted in 1-NP being the preferred AP substrate in their work. 

S. Centi, S. Laschi, M. Franek, and M. Mascini, A disposable immunomagnetic electrochemical sensor based on functionalized magnetic beads and carbon-based screen-printed electrodes (SPCEs) for the detection of polychlorinated biphenyls (PCBs). Anal. Chim. Acta 538, 205—212 (2005). 

Ghamouss [263] developed a screen-printed carbon electrode modified with both HRP and LOD (SPCE-HRP/LOD) to determine 1-lactate. The sensitivity of the optimized SPCE-HRP/LOD to 1-lactate was 0.84nAL jM 1 in a detection range between 10 and 180 pM. 

Boujtita et al. [11] Beer Alcohol oxidase (AOx) Screen-printed carbon electrode doped with 5% cobalt phthalocyanine (CoPC-SPCE), and coated with AOx a perm-selective membrane on the surface acts as a barrier to interferents/+400mV vs. Ag/AgCl Cobalt phthalocyanine... 

Cross-linked with BSA using glutaraldehyde Copper-plated SPCE 26.7 Peak in linear sweep voltammogram at 150 mV Kumar and Zen [31]... 

All potentials vs. screen-printed Ag/AgCl pseudo-reference, except values marked with asterisk ( ), which are vs. Ag/3M AgCl double-junction reference electrode, and values marked with dagger CfO, which are vs. saturated calomel. Abbreviations CoPC cobalt phthalocyanine, SPCE screen-printed carbon electrode, GOD glucose oxidase, MWCNT multi-walled carbon nanotubes, NAD nicotinamide adenine dinucleotide, PQQ pyrroloquinoline quinone, FIA flow injection analysis. 

Fig. 23.2. Operating principle of CoPC-modified SPCE used to detect H202 [12].

The production of H202 from this step can be monitored spectro-photometrically by the formation of a dye in the presence of peroxidase [49] disadvantages of this system include the use of carcinogenic dyes, lengthy incubation times and the need for laboratory-based equipment. As discussed in the previous section, SPCEs can be modified with mediators to produce effective H202 transducers when combined with immobilised enzymes, disposable biosensors ideal for de-centralised clinical analysis can be fabricated. The cholesterol molecule is nonpolar and thus provides an additional challenge to the development of... 

An SPCE modified with CoPC was employed as an H202 transducer in a cholesterol biosensor fabricated by the drop-coating of ChOx, followed by a cellulose acetate membrane [50]. The resulting cholesterol biosensors were operated in stirred solutions using an applied potential of +400 mV vs. screen-printed Ag/AgCl, and displayed a linear range of 0.06-5 mM. 

Table 23.3 lists recent publications concerning SPCE-based approaches towards the analysis of the naturally occurring compounds ascorbate, steroid hormones (including progesterone and estradiol) and protein hormones (hCG and insulin). 

Table 23.5 summarises the main features of papers which detail experimental approaches to the analysis of amino acids, peptides and proteins (other than hormones) using electrochemical biosensor-based methodologies involving SPCEs. 

Several different approaches have been used to monitor DNA, RNA and purines at SPCEs (Table 23.6). 

Ascorbic acid Mediated oxidation at a silica sol-gel glass coated ferricyanide-doped Tosflex-modified SPCE FIA +0.3 V 46 nM to 300 nM 46 nM Zen et al. [66]... 

L-Ascorbic acid Mn02-modified SPCE Amperometric +0.6V 50-250mg r1 0.2 mgr1 Turkusic et al. [67]... 

Progesterone in buffer Competitive immunoassay with ALP-labelled progesterone on SPCE bearing surface-immobilised antibody Chronoamperometric detection of phenol +0.7V 1 x 1(T9 to 5 x 10-6M 0.5-50 ngml-1 lx 10-9M 7ngml-1 Hart et al. [69] Pemberton et al. [70]... 

Progesterone in milk Competitive immunoassay using SPCE with immobilised prog-BSA conjugate Amperometric or DPV detection of ALP-generated p-aminophenol +350 mV or scan from +100 to +500 mV 16-256 ngml-1 3 ngml-1 Kreuzer et al. [73]... 

Estradiol (E2) in serum Competitive immunoassay with ALP-labelled E2 on SPCE bearing surface-immobilised antibody DPV of enzymegenerated 1-naphthol Scan from 0 to +0.3 V 25-500pgmr1 50pgml-1 Pemberton et al. [74]... 

Human chorionic gonadotrophin (hCG) Competitive immunoassay on Nafion-coated SPCEs in microwells DPV oxidation of accumulated cationic product of substrate hydrolysis by ALP enzyme Scan from Oto +0.7V 200-10,000 mlUmP1 100 mlU (lmg= 9825 IU) Bagel et al. [75]... 

Insulin Oxidation of tyrosine residues at pre-treated (+1.7V, 30s) microfabricated SPCEs Potentiometric stripping analysis (2 min accumulation at -0.2V) Stripping current of 3 pA 0-600 nM 20nM Wang et al. [76]... 

IgG (mouse) Sandwich immunoassay using SPCE-surface-immobilised anti-IgG antibody FLA amperometric, reduction of enzymatically generated iodine o.ov 30-700 ngmU1 3 ngmU1 Gao et al. [81]... 

IgG (mouse) Electrophoretic separation of free analyte/ferrocene-labelled anti-IgG Amperometric, oxidation of ferrocene tracer at gold-plated SPCE +0.6 V 7.5 x KT11 to 1 x lO gmr1 2.5 x lO- gmr1 Wang et al. [82]... 

IgG (rabbit) Competitive immunoassay using ALP-labelled antibody at streptavidin-modified SPCEs bearing capture antibody Cyclic voltammetry of indigo carmine generated from 3-IP substrate —0.15 V (scan from —0.3 to +0.2 V) 5 x KT11 to 1 x 1(T9M 5 x 10 nM (7-Ongmr1) Diaz-Gonzalez et al. [83]... 

IgG (mouse) Sandwich immunoassay on Os redox polymer-modified SPCEs Cyclic voltammetry of HRP-mediated electrocatalytic reduction of H2O2 +0.2 V (scan from +0.5 to -0.2 V) 4.4-440 pM 0.63 pM Gao et al. [84]... 

IgG (rabbit) Sandwich immunoassay upon Os redox hydrogel/ avidin-modified SPCE Amperometric HRP-mediated electrocatalytic reduction of H2O2 +0.2 V lpgml-1 to lOngml-1 7 pgml-1 Zhang and Heller [85]... 

Anti-DNA antibody in serum from SLE or bronchial asthma patients Suppression of enzyme activity due to formation of DNA-antibody adducts on graphite SPCEs Amperometric detection of cholinesterase or peroxidase activity +680 mV (cholinesterase) or —150 mV (peroxidase) nda nda Evtugyn et al. [86]... 

AFP) using anti-AFP antibody on Prussian blue-modified SPCEs, with antibody-GOD conjugate H2O2 produced in presence of glucose... 

AFP Inhibition of HRP activity when AFP binds to SPCE-surface-immobilised HRP-labelled AFP antibody/ chitosan Decrease in DPV current response for reduction of oxidised thionine (catalyst = H2O2)... 

Cysteine Direct electrochemical oxidation at bare SPCEs Amperometry in stirred solution... 

BSA Selective binding to anti-BSA antibody in electropolymerised polypyrrole film on SPCE surface Impedance... 

Glutathione Sol-gel graphite composite SPCEs incorporating surfactant, CoPC/ diphenylthiocarbazone Cyclic voltammetry, electrocatalytic oxidation of GSH... 

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