Colorimetric Sensing by Using Allosteric‐DNAzyme‐Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe

Abstract

Target detection by the naked eye: The action of an RNA-cleaving allosteric DNAzyme in response to ligand binding was coupled to a rolling circle amplification process to generate long single-stranded DNA molecules for colorimetric sensing (see scheme). Upon hybridization of the resulting DNA with a complementary PNA sequence in the presence of a duplex-binding dye, the color of the dye changed from blue to purple.

A general approach for rational design of fluorescent DNA aptazyme sensors based on target-induced unfolding of DNA hairpins

“…In contrast to the wide applications of DNAzymes and aptamers as biosensors in multiple research fields [1][2][3][4][5][6][7][8]10,11,22,[35][36][37][38][39][40][41][42][43][44], there are considerably less studies on DNA aptazymes for similar applications [9,12,13,45]. Perhaps a major reason for this difference is that, most DNA aptazyme sensors are developed by artificial design using known DNAzymes and aptamers rather than by more efficient combinatorial techniques [46], therefore extensive trial-and-error and optimization procedures are usually required for the identification of a successful aptazyme sensor with target-dependent fluorescence switch.…”

“…The previously reported designs of aptazymes based on inserting aptamers into the DNAzymes usually involved changes in the active sites of the DNAzymes [12,13,[47][48][49][50][51][52], making the activities of the DNAzymes in the aptazymes usually difficult to predict. Therefore, one successful design by the insertion approach often required sophisticated trials and optimizations, and was very difficult to extend from using one DNAzyme to another.…”

“…This interesting class of DNA includes DNAzyme (also called deoxyribozyme or DNA enzyme) that can catalyze chemical reactions in the presence of specific cofactors [1][2][3][4][9][10][11], DNA aptamer that can bind specific targets [2,[4][5][6][7][8]11], and DNA aptazyme that is the combination of the above two [9,12,13]. Since the first discovery of a DNA aptamer and a DNAzyme in 1990's [14,15], more and more functional DNAs have been identified by combinatorial techniques such as in vitro selection and SELEX (Systematic evolution of ligands by exponential amplification) [14][15][16][17][18].…”

“…Perhaps a major reason for this difference is that, most DNA aptazyme sensors are developed by artificial design using known DNAzymes and aptamers rather than by more efficient combinatorial techniques [46], therefore extensive trial-and-error and optimization procedures are usually required for the identification of a successful aptazyme sensor with target-dependent fluorescence switch. Moreover, many of these design strategies for aptazymes often involve changes in the active sites or binding arms of functional DNAs to introduce aptamer sequences into DNAzymes [12,13,[47][48][49][50][51][52]. Such strategies have been successfully applied for target detection, but the insertion of aptamers may interfere the activity of the DNAzymes and make it M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3 difficult to apply the same design approach rationally to other DNAzyme and aptamer pairs, because of the dramatically different properties of each DNAzyme and aptamer [53].…”

Gold nanolabels and enzymatic recycling dual amplification-based electrochemical immunosensor for the highly sensitive detection of carcinoembryonic antigen

“…The AuNP label is an ideal one in biotechnological systems due to its inherent advantages, such as easy preparation and retains the biochemical activity of the labeled biomolecules, such as DNA, proteins, and so forth. The second signal amplification technique, which is based on the employment of polymerase chain reaction (PCR) [24,25] or rolling circle amplification (RCA) [26][27][28][29], significantly lower the limit of the detection for protein analysis. However, the drawbacks of this approach (strict laboratory conditions, complexity, cost, easy contamination etc.)…”