Entrapment of Fluorescence Signaling DNA Enzymes in Sol−Gel-Derived Materials for Metal Ion Sensing

Abstract

Three fluorescence signaling DNA enzymes (deoxyribozymes or DNAzymes) were successfully immobilized within a series of sol-gel-derived matrixes and used for sensing of various metal ions. The DNAzymes are designed such that binding of appropriate metal ions induces the formation of a catalytic site that cleaves a ribonucleotide linkage within a DNA substrate. A fluorophore (fluorescein) and a quencher (DABCYL, [4-(4-dimethylaminophenylazo)benzoic acid]) were placed on the two deoxythymidines flanking the ribonucleotide to allow the generation of fluorescence upon the catalytic cleavage at the RNA linkage. In general, all DNAzymes retained at least partial catalytic function when entrapped in either hydrophilic or hydrophobic silica-based materials, but displayed slower response times and lower overall signal changes relative to solution. Interestingly, it was determined that maximum sensitivity toward metal ions was obtained when DNAzymes were entrapped into composite materials containing approximately 40% of methyltrimethoxysilane (MTMS) and approximately 60% tetramethoxysilane (TMOS). Highly polar materials derived from sodium silicate, diglycerylsilane, or TMOS had relatively low signal enhancements, while materials with very high levels of MTMS showed significant leaching and low signal enhancements. Entrapment into the hybrid silica material also reduced signal interferences that were related to metal-induced quenching; such interferences were a significant problem for solution-based assays and for polar materials. Extension of the solid-phase DNAzyme assay toward a multiplexed assay format for metal detection is demonstrated, and shows that sol-gel technology can provide new opportunities for the development of DNAzyme-based biosensors.

In vitro selection of RNA-cleaving DNAzymes for bacterial detection

“…We term this type of DNAzyme "RNA-cleaving fluorogenic DNAzyme", or simply "RFD". Over the past decade, we have isolated many RFDs and examined their catalytic and signaling properties [38,39,[67][68][69][81][82][83][84][85][86][87][88][89][90]. More recently, we began to develop RFDs that that can be activated in the presence of a specific bacterium, such as Escherichia coli and Clostridium difficile [72,80,82,[91][92][93][94][95][96].…”

Oligonucleotide-functionalized hydrogels as stimuli responsive materials and biosensors

“…Being a polyanion, the diffusion of entrapped DNA may 7 be reduced by increasing the DNA length, gel percentage, or by using cationic monomers to form the gel. 65 The diffusion may be further reduced by linking DNA to large proteins and microparticles. Copolymerization of acrydite-modified DNA into polyacrylamide.…”

“…The authors further showed the use of such entrapped aptamer sensors to perform high throughput screening for enzyme inhibitors and for metal ion detection. 65,102 Nielsen et al employed an aptamer sensor design first reported by the Tan group ( Figure 8C). 103 In this case, the sensor is composed of only a single DNA strand which contained a fluorophore, a quencher, the ATP aptamer sequence and a polymer spacer.…”

Deoxyribozymes: useful DNA catalysts in vitro and in vivo

“…By placing a fluorophore-quencher pair directly on the RNA cleavage substrate from the outset of selection, more effective signaling deoxyribozymes have been identified [116]. Efforts have been made to entrap fluorescently signaling deoxyribozymes in sol-gel matrices, which may have certain benefits over solutionphase assays [117], and an electrochemical Pb 2+ sensor based on metal-dependent RNA cleavage has been developed [118]. The fluorophore and quencher components of the general system have also been replaced with gold nanoparticles, which change color upon cleavage-induced disassembly, allowing simple colorimetric detection of metal ions [110,119,120].…”