Designs of Enterobacteriaceae Lac Z Gene DNA Gold Screen Printed Biosensors

Abstract

This paper describes specific electrochemical enterobacteriaceae lac Z gene DNA sensors based on immobilization of a thiolated 25 base single stranded probe onto disposable screen printed gold electrodes (gold SPEs). Two configurations have been evaluated. In the first one, the capture probe was attached to the electrode surface through its ÀSH moiety, while mercaptohexanol (MCH) was used as spacer for the displacement of nonspecifically adsorbed oligonucleotide molecules. The hybridization event between the probe and target DNA sequences was detected at À 0.20 V by square-wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. The second genosensor configuration involved modification of gold high temperature SPEs with a 3,3'-dithiodipropionic acid di(N-succinimidyl ester) (DTSP) self-assembled monolayer (SAM). Moreover, 2-aminoethanol was used as blocking agent, and further modification with avidin allowed binding of the biotinylated enterobacteriaceae lac Z gene DNA probe. An enzyme amplified detection scheme was applied, based on the coupling of streptavidin-peroxidase to the biotinylated complementary target, after the hybridization process, and immobilization of tetrathiafulvalene (TTF) as redox mediator atop the modified electrode. The amperometric response obtained at À 0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. Experimental variables concerning sensors composition and electrochemical transduction were evaluated in both cases. A better precision and reproducibility in the fabrication process, as well as a higher sensitivity were achieved using the biotinylated probe-based sensor configuration. A limit of detection of 0.002 ng/mL was obtained without any preconcentration step.

Ternary Monolayer Interfaces for Ultrasensitive and Direct Bioelectronic Detection of Nucleic Acids in Complex Matrices

“…In contrast, sensors prepared using 1 mM of SHCP showed no decrease in the S/N characteristics even after 2 months. These results suggest that increasing the level of SHCP of the SHCP/HDT + MCH to the micromolar concentration, commonly used with the binary SHCP + MCH SAM [32,36], can lead to an enhanced storage stability of the resulting SAM [32,36]. This can be attributed to a slower dynamic competition between the SHCP and MCH at higher concentrations of capture probe.…”

“…In particular, control of the surface chemistry and coverage of the electrode transducer are essential for maximizing the hybridization efficiency and minimizing of nonspecific adsorption events, as desired for ultrasensitive measurements in complex biological fluids. Several strategies have been employed for attaching nucleic acid probes onto the surface of electrode transducers and for controlling the surface chemistry [25][26][27][28][29][30][31][32]. Alkanethiol SAM methods have been particularly attractive for designing reproducible probe-modified interfaces on different gold transducers with high hybridization activity [33].…”

Disposable Biosensors for Clinical Diagnosis

“…113 Oligonucleotide molecules were adsorbed onto the screen-printed electrodes using mercaptohexanol as a spacer in the detection of enterobacteriaceae lac Z, which is an indicator of faecal contamination. 114 A disposable magnetic genosensor was also proposed by the same group of researchers for the analysis of Escherichia coli DNA fragments by enzyme-amplified coupling of streptavidin-peroxidase to immobilized biotinylated lacZ gene target sequences. 115 Loaiza et al modified the above architecture using a magnetic field on the surface of a home-made carbon screenprinted electrode, as shown in Figure 5 Figure 6.…”

Electrochemical detection of a Vibrio parahaemolyticus sequence-specific gene based on a gold electrode modified with a single stranded probe

“…Electrochemical DNA sensors [8][9][10] significantly shorten the time without extensive pretreatment steps, frequently utilized for the detection of bacteria [11,12]. However, studies of electrochemical DNA sensors for detection of VP have rarely been reported.…”

“…Among these electrodes, gold electrodes have specific advantages, including a wide potential window at the cathode region and a smooth surface. The strong affinity of the Au-S bond makes use of thiol based self assembled mono-layers (SAMs) [14,15] for the immobilization of a single-stranded DNA probe on a gold electrode [16][17][18]11]. The electrode surface is polishable and renewable.…”