Comparison of Different Strategies on DNA Chip Fabrication and DNA-Sensing: Optical and Electrochemical Approaches

Abstract

International audienceNew strategies for the construction of DNA chips and the detection of DNA hybridization will be discussed in this review. The focus will be on the use of polypyrrole as a linker between a substrate and oligonucleotide probes. The modification step is based on the electrochemical copolymerization of pyrrole and oligonucleotides bearing a pyrrole group on its 5′ end. This strategy was employed for the immobilization of oligonucleotides on millimeter-sized electrodes, microelectrode arrays, as well as for the local structuring of homogeneous gold surfaces. Our approaches for the localized patterning of gold surfaces will be also discussed. Localized immobilization was achieved by using an electrospotting technique, where a micropipette served as an electrochemical cell where spot sizes with 800 μm diameters were fabricated. The use of a microcell using a Teflon covered metal needle with a cavity of 100 μm resulted in immobilized probe spots of 300 μm. Scanning electrochemical microscopy (SECM) was also used, and surface modifications of 100 μm were obtained depending on the experimental conditions. Different detection methods were employed for the reading of the hybridization event: fluorescence imaging, surface plasmon resonance imaging (SPRI), photocurrent measurements, and voltamperometric measurements using intercalators. Their advantages concerning the various immobilization strategies will also be discussed

Localized Deposition of Chitosan as Matrix for Enzyme Immobilization

“…The limited reproducibility and uniformity are attributed to the accumulation of gas bubbles (H 2 and O 2 ) beneath the UME and to the nonuniformity of the current in the interelectrode space. By the aid of a micropippet with an inserted Pt wire, localized and defined electrodeposition of polypyrrole on dry supports was reported [16]. We also tested this strategy for chitosan deposition, but similar to the documented results, the diameter of the polymer deposit cannot be reduced to less than 100 mm due to the surface tension of the polymer droplet (data not shown).…”

“…Moreover, heterocyclic aromatic pyrrole can be patterned by electropolymerizing on a conductive support using SECM coupled with pulse techniques [15]. DNA chip can be fabricated by directly electrospotting oligonucleotides-pyrrole on electrode surface [16].…”

Fabrication of Oligonucleotide Chips by Using Parallel Cantilever‐Based Electrochemical Deposition in Picoliter Volumes

“…[16] The electrodeposition process was performed on the basis of previously described work, [15][16][17][18][19] using an electrolytic aqueous solution containing sodium phosphate-buffered saline (0.05 M phosphate buffer, 0.05 M NaCl, 10 % (v/v) glycerol at pH 6.8; referred to as electrolyte A) as the supporting electrolyte, pyrrole (Py), and pyrrole-ODN at concentrations of 20 mM and 1 lM, respectively. As depicted in Scheme 2, corresponding fluorescence images were obtained after hybridization with complementary biotinylated oligonucleotides and subsequent recognition using streptavidin-phycoerythryn as a fluorescent label.…”

Scanning Electrochemical Microscopy (SECM) Based Detection of Oligonucleotide Hybridization and Simultaneous Determination of the Surface Concentration of Immobilized Oligonucleotides on Gold

“…As demonstrated first by Kranz et al [37,38], the use of a pulse sequence is critical in order to generate sufficiently high concentrations of pyrrole radical cations for local deposition of the conducting polymer. We followed this approach using a copolymerization of pyrrole/ pyrrole -ODN to link the oligonucleotides locally and irreversibly to the gold substrate [23,25]. Although the mechanisms of polypyrrole electropolymerization has been widely studied [51 -60], there is still no agreement regarding the initiation step [60].…”