Exonuclease III-Powered Self-Propelled DNA Machine for Distinctly Amplified Detection of Nucleic Acid and Protein.

Herein, a new exonuclease III (Exo III)-powered self-propelled DNA machine was developed for the cascade multilevel signal amplification of nucleic acid and nucleic acid-related analytes. It could be easily and homogeneously operated with the use of an integral DNA hybrid probe as the recognition, amplification, and signaling element, and the Exo III cleavage as a driving force. The DNA hybrid probe was obtained by annealing two hairpin-like DNAs. The target recognition with the 3'-protruding domain of the DNA hybrid probe triggered Exo III cleavage, accompanied by target recycling and alternate generation of a large amount of target substitute and analogy. Simultaneously, the cascade bidirectional Exo III cleavage toward the DNA hybrid probe by the generated target substitute and analogy contributed for the exponential signal amplification toward target recognition event. It could be also extended for the application in protein detection with the thrombin as a protein example by introducing an additional hairpin-like aptamer switch. The proposed Exo III-powered self-propelled DNA amplification strategy showed a linear detection range for target DNA from 0.5 fM to 1 pM and for thrombin from 5 fM to 10 pM. The low detection limit toward target DNA and thrombin could reach about 0.1 fM and 5 fM, respectively, which were superior to most of reported methods. It also exhibited an excellent selectivity toward target detection. Therefore, the developed sensing system exhibits a new, simple and powerful means for amplified detection of nucleic acid and nucleic acid-related analytes, and may hold great potentials in bioanalysis, disease diagnosis and biomedicine.

Fuel strand-powered self-propelled electrochemical DNA machine for enzyme-free and distinctly amplified detection of nucleic acid with tunable performance.

In this work, a self-propelled DNA machine was proposed for enzyme-free and ultrasensitive electrochemical detection of nucleic acid, which was based on a new strategy of the fuel strand-powered target recycling and successive proximity-based inter-strand displacement for distinct signal amplification. A three-strand duplex DNA (TSD) probe was immobilized onto electrode for target DNA recognition via a fuel strand-powered strand displacement circuit, accompanied with the target recycling and the association of fuel strand onto electrode for signal amplification and readout. The associated fuel strand contained a protruding tail sequence that could be used as a target analogy to activate the neighboring TSD probe based on the proximity-based inter-strand displacement effect, inducing the self-propelled association of fuel strand onto electrode for further signal amplification. The detection performance (dynamic response range, linear interval and detection limit) of current electrochemical DNA machine could be interestingly tuned by changing the tail sequence length of fuel strand, showing the potential for different analysis requirements. The lowest detection limit of 0.1 fM could be achieved, which was lower about two orders of magnitude than that by the typical fuel strand-powered target recycling strategy. Therefore, the developed sensing system exhibits a new, simple and powerful means for amplified detection of nucleic acid and may hold great potentials in bioanalysis, disease diagnosis and biomedicine.

An Integral Recognition and Signaling for Electrochemical Assay of Protein Kinase Activity and Inhibitor by Reduced Graphene Oxide-Polydopamine-Silver Nanoparticle-Ti4+ Nanocomposite.

A novel electrochemical biosensing method for protein kinase (PKA) activity was demonstrated by using a reduced graphene oxide-polydopamine-silver nanoparticle-Ti4+ (rGO-PDA-AgNPs-Ti4+) nanocomposite. The obtained nanocomposite possessed an integral capability for phosphopeptide recognition and signal readout. The polydopamine modified reduced graphene oxide (rGO-PDA) was firstly prepared based on a self-polymerization method of dopamine. The silver ions were adsorbed onto polydopamine (PDA) layer and directly reduced into silver nanoparticles (AgNPs), which was used for electrochemical signal reporting. Then, the Ti4+ cations were attached onto the PDA layer for phosphopeptide recognition according to the strong coordination ability of PDA with Ti4+ and phosphate group. The prepared rGO-PDA-AgNPs-Ti4+ nanocomposites were characterized with different methods. The developed rGO-PDA-AgNPs-Ti4+ nanocomposites were then employed for electrochemical analysis of PKA-catalyzed kemptide phosphorylation. The sensitive detection toward PKA activity was realized with an experimental detection limit of about 0.01 U/mL. It may be also extended for the inhibitor evaluation. Thus, it provided a facile and sensitive means for electrochemical analysis of PKA activity and inhibitor screening.

Ultrasensitive Electrochemical DNA Biosensor Fabrication by Coupling an Integral Multifunctional Zirconia-Reduced Graphene Oxide-Thionine Nanocomposite and Exonuclease I-Assisted Cleavage.

In this work, a simple but sensitive electrochemical DNA biosensor for nucleic acid detection was developed by taking advantage of exonuclease (Exo) I-assisted cleavage for background reduction and zirconia-reduced graphene oxide-thionine (ZrO2-rGO-Thi) nanocomposite for integral DNA recognition, signal amplification, and reporting. The ZrO2-rGO nanocomposite was obtained by a one-step hydrothermal synthesis method. Then, thionine was adsorbed onto the rGO surface, via π-π stacking, as an excellent electrochemical probe. The biosensor fabrication is very simple, with probe DNA immobilization and hybridization recognition with the target nucleic acid. Then, the ZrO2-rGO-Thi nanocomposite was captured onto an electrode via the multicoordinative interaction of ZrO2 with the phosphate group on the DNA skeleton. The adsorbed abundant thionine molecules onto the ZrO2-rGO nanocomposite facilitated an amplified electrochemical response related with the target DNA. Since upon the interaction of the ZrO2-rGO-Thi nanocomposite with the probe DNA an immobilized electrode may also occur, an Exo I-assisted cleavage was combined to remove the unhybridized probe DNA for background reduction. With the current proposed strategy, the target DNA related with P53 gene could be sensitively assayed, with a wide linear detection range from 100 fM to 10 nM and an attractive low detection limit of 24 fM. Also, the developed DNA biosensor could differentiate the mismatched targets from complementary target DNA. Therefore, it offers a simple but effective biosensor fabrication strategy and is anticipated to show potential for applications in bioanalysis and medical diagnosis.