Label-Free Direct Detection

Guanine as a purine base is the most easily oxidized base in DNA, and its irreversible peak has been found at the 1.0 V vs. Ag/AgCl reference electrode [27]. This peak height 

Electrochemical detection methods are based on the electroactivity of the nucleotide bases [28]. Redox processes of NAs mostly involve the reduction and oxidation of bases. NA oxidation has been analyzed at different surfaces, such as carbon, platinum, gold, silver, and so on, and the NA reduction has been observed only at mercury electrodes. For G being more advantageous to study the oxidation of its reduction product (7,8-dihydroguanine), for example, when the mercury electrode is exposed to highly negative potentials (— 1.8 V at neutral pH), its residues in an adsorbed NA molecule are reduced. G-reduction product remains at the electrode and is reoxidized back to G in an anodic scan, obtaining an oxidation peak of —0.3 V [25]. 

DNA oxidation at carbon electrodes is associated with the irreversible oxidation of guanine and adenine [33], For example, the G oxidation signal observed at -i-1.0 V, without external labels, has been used to monitor telomerase activity by using a carbon graphite electrode (CGE) as an electrochemical transducer [5], Telomerase activity has been detected in cell extracts containing as low as 100 ng pl of protein. This label-free assay is practical in the quantitative determination of telomerase activity providing a cheap and simple detection protocol for the diagnosis of cancer that can also be extended to the analysis of food related to DNA. 

A novel label-free electrochemical genosensor for the detection of catechol-o-methyltransferase (COMT) Val 108/158Met polymorphism based on the guanine signal change by using a disposable CGE in connection with differential pulse voltammetry 

Carbon nanotubes (CNTs) are used as carriers. CNTs serve as a part of the transducer that carries DNA probes and, at the same time, promotes electron transfer. Thus, CNTs have brought further advancements into the rapidly evolving field of DNA sensors. The excellent conductivity of CNTs allows their use as highly sensitive DNA sensors [36,30]. Also, the inorganic oxide nanoparticles (i.e., Ce02, Fe203, etc.) are used as immobilizing carriers of the ssDNA probe [17, 37]. 

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C.A. Marquette, M.F. Lawrence and L.J. Blum, DNA covalent immobilization onto screen-printed electrode networks for direct label-free hybridization detection of p53 sequences, Anal. Chem., 78 (2006) 959-964. J.-M. Zen, Y.-Y. Lai, H.-H. Yang and A.S. Kumar, Multianalyte sensor for the simultaneous determination of hypoxanthine, xanthine and uric acid based on a preanodized nontronite-coated screen-printed electrode, Sens. Actuators B Chem., 84 (2002) 237-244. 

Fig. 1. General schematic of biosensors (a) direct detection biosensors where the recognition element is label free (b) indirect detection biosensors using a sandwich assay where the analyte is detected by labeled molecule. Direct detection biosensors are simpler and faster but typically yield a higher limit of detection compared with indirect detection...

Direct detection biosensors utilize direct measurement of the biological interaction. Such detectors typically measure physical changes (e.g., changes in optical, mechanical, or electrical properties) induced by the biological interaction, and they do not require labeling (i.e., label free) for detection. Direct biosensors can also be used in an indirect mode, typically to increase their sensitivity. Direct detection systems include optical-based systems (most common being surface plasmon resonance) and mechanical systems such as quartz crystal resonators. 

Osakai, T., Yuguchi, Y., Gohara, E., Katano, H. Direct label-free electrochemical detection of proteins using the polarized oil/water interface. Langmuir 26, 11530-11537 (2010)... 

Kerman et al, 2004 also described the label-free electrochemical detection of DNA based on the direct attachment of adenine probes to the sidewall and end of functionalized MWCNTs. The MWCNTs were attached onto the carbon paste electrode surface modified with thymine probes by hybridization between adenine and thymine. The combination of sidewall and end functionalization of MWNT showed enhancement of the guanine oxidation signal in the direct measurement compared to the ones from only end-modified MWCNT. 

Kerman, K Morita, Y. Takamura, Y. Ozsoz, M. Tamiya, E. (2004). DNA-Directed Attachment of Carbon Nanotubes for Enhanced Label-Free Electrochemical Detection of DNA Hybridization. Electroanalysis, 16,1667-1672. 

K. Kerman, Y. Morita, Y. Takamura, M. Ozsoz, and E. Tamiya, DNA-directed attachment of carbon nanotubes for the enhanced electrochemical label-free detection of DNA hybridization. Electroanalysis 16,1667-1672 (2004). 

F. F., Direct detection of tuberculosis infection in blood serum using three optical label free approaches, Sens. Actuators B Chem. 2008, 129, 934 940... 

SPR biosensors are label-free detection devices - binding between the biomolecular recognition element and analyte can be observed directly without the use of radioactive or fluorescent labels. In addition, the binding event can be observed in real-time. SPR affinity biosensors can, in principle, detect any analyte for which an appropriate biomolecular recognition element is available. Moreover, analyte molecules do not have to exhibit any special properties such as fluorescence or characteristic absorption or scattering bands. 

Schmerr and Jenny established a CE-based immunoassay for the detection of prion protein (24). In this competitive assay, peptides derived from the prion protein and labeled with fluorescein were used. This allowed them to distinguish scrapie-infected brain preparations from noninfected. For identification, the ratio between the peaks resulting from the free and the com-plexed peptide with a specific antibody was used. The results were in agreement with other data on the brain preparations achieved by Western blot analysis. The CE-based assay provides the advantage of direct detection of the scrapie protein in blood and tissue preparations with high sensitivity. Furthermore, due to the small sample amount needed for analysis, the CE-based assay is applicable to the putative diagnostics of prion protein in body fluids. 

The biosensors can be divided into nonlabelled or label-free types, which are based on the direct measurement of a phenomenon occurring during the biochemical reactions on a transducer surface and labelled, which relies on the detection of a specific label. Research into label-free biosensors continues to grow [13] however labelled ones are more common and are extremely successful in a multitude of platforms. 

Increased attention has been given to new indicator-free electrochemical detection schemes that greatly simplify the sensing protocol. Such direct, label-... 

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