Disposable Magnetic DNA Sensors for the Determination at the Attomolar Level of a Specific Enterobacteriaceae Family Gene

Abstract

Disposable magnetic DNA sensors using an enzyme-amplified strategy for the specific detection of a gene related to the Enterobacteriaceae bacterial family, based on the coupling of streptavidin-peroxidase to biotinylated lacZ gene target sequences, has been developed. A biotinylated 25-mer capture probe was attached to streptavidin-modified magnetic beads and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxidase polymer was attached to the biotinylated target, and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene (TTF) modified gold screen-printed electrodes (Au/SPEs). The amperometric response obtained at -0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. In order to improve the sensitivity of the determination and reduce the assay time, different variables of the assay protocol were optimized. A low detection limit (5.7 fmol) with good stability (RSD = 7.1%, n = 10) was obtained. The DNA nonspecific adsorption at the magnetic beads was negligible, the obtained results thus demonstrating the possibility to detect the hybridization event with great specificity and sensitivity. The developed method was used for the analysis of Escherichia coli DNA fragments (326 bases) in polymerase chain reaction (PCR) amplicons extracted from a cell culture. As low as 2.5 aM asymmetric PCR product could be detected with the developed methodology.

Magnetic solids in analytical chemistry: A review

“…One of the objectives of these devices is to decrease the limits of detection so that they are useful for analyzing real samples [193,194]. The use of modified magnetic particles in sensing is targeted at developing more specific, stable and reproducible devices [195].…”

Quantum‐Dot‐Functionalized Poly(styrene‐co‐acrylic acid) Microbeads: Step‐Wise Self‐Assembly, Characterization, and Applications for Sub‐femtomolar Electrochemical Detection of DNA Hybridization

“…Compared to other electrochemical methods [42][43][44][45] without the signal amplification, this proposed nanoparticle-based method showed a much-lower limit of detection, and was competitive with the highly sensitive detection of DNA by amplifying the sample with PCR technique. [46] In addition, this method was superior to some amplification techniques, such as liposome (0.75 amol), [28] magnetic particles (5.7 fmol), [47] bio-barcodes (2.5 fmol L À1 ) [48] and Au-NP-based amplification (10 zmol). [36] The selectivity of the streptavidin/CdTe-tagged polybead-based amplification was studied using three kinds of DNA sequence including perfectly complementary targets, one-base mismatched strands and non-complementary strands, at concentrations of 100 fmol L À1 .…”

Disposable Biosensors for Clinical Diagnosis

“…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. DNA binding induced by Hoechst 33258 molecule, along with changes in the anodic current peak, were used to detect the modified cry9c gene variety with high efficiency.…”