Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection

Abstract

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.

Intrant ELISA: A Novel Approach to Fabrication of Electrospun Fiber Mat-Assisted Biosensor Platforms and Their Integration within Standard Analytical Well Plates

“…In addition, electrospinning technique could be optimized to produce sub-micron polymer fibers [49], which could increase the level of sensitivity of fibrous platform. Although there are several different techniques for regeneration of the analytical platforms such as treatment with strong oxidizer, acid/base components and/or concentrated salts [50,51], reusability of the developed platforms is not debated. Since i-ELISA can be fabricated through an inexpensive and straightforward method of molding, the device can be considered as a disposable analytical platform.…”

Thermodynamics and Kinetics of the Complexation Reaction of Lead by Calix‐DANS4

“…Recently, a microfluidic sensor for lead detection has been developed by using a lead-specific DNAzyme. [11] However, in this elegant work, besides the sophisticated technology needed to elaborate a Pb 2 + -specific DNAzyme, the optical detection was made by fluores-The thermodynamics and kinetics of the complexation reaction between lead ions and the fluorescent sensor Calix-DANS4 are determined to optimize the geometry of the microreactor used for the flow-injection analysis of lead and to tune the working conditions of this microdevice. Under our experimental conditions (pH 3.2, low concentration of Calix-DANS4) the 1:1 Pb 2 + -Calix-DANS4 complex is predominantly formed with a high stability constant (log K 1:1 = 6.82) and a slow second-order rate constant (k = 9.4 10 4 L mol À1 s…”

A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

“…Of these, [8][9][10][11][12][13][14][15][16][17] DNAzyme has attracted much more attention. They have been extensively explored for Pb 2+ detection via fluorescence [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29], fluorescence anisotropy [30], colorimetry [31][32][33][34], electrochemistry [35][36][37][38], electrochemiluminescence [39], dynamic light scattering [40,41], and surface enhanced Raman scattering [42].…”