Two Pb2+-specific DNAzymes with opposite trends in split-site-dependent activity

By splitting the catalytic core of DNAzymes into two halves, two Pb2+-specific DNAzymes retain partial activity, while they show opposite trends of activity as a function of the split site, revealing important nucleotides for catalysis and metal binding.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ministry of Research and Innovation of Ontario |

Immobilization of DNA on Magnetic Microparticles for Mercury Enrichment and Detection with Flow Cytometry

This is the peer reviewed version of the following article: Huang, P.-J. J., & Liu, J. (2011). Immobilization of DNA on Magnetic Microparticles for Mercury Enrichment and Detection with Flow Cytometry. Chemistry - A European Journal, 17(18), 5004–5010, which has been published in final form at https://doi.org/10.1002/chem.201002934. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Mercury detection in water has attracted a lot of research interest due to its highly toxic nature and adverse environmental impact. In particular, the recent discovery of specific binding of HgII to thymine-rich (T-rich) DNA resulting in T-HgII-T base pairs has led to the development of a number of sensors with different signaling mechanisms. However, the majority of such sensors were non-immobilized. Immobilization, on the other hand, allows active mercury adsorption, signal amplification, and sensor regeneration. In this work, we immobilized a thymine-rich DNA on a magnetic microparticle (MMP) surface through biotin–streptavidin interactions. In the presence of HgII, the DNA changes from a random coil structure into a hairpin, upon which SYBR Green I binds to emit green fluorescence. Detection was carried out by using flow cytometry where the fluorescence intensity increased ≈9-fold in the presence of mercury and the binding of mercury reached equilibrium in less than 2 min. The sensor showed a unique sample-volume-dependent fluorescence signal change where a higher fluorescence was obtained with a larger sample volume, suggesting that the particles can actively adsorb HgII. Detection limits of 5 nM (1 ppb) and 14 nM (2.8 ppb) were achieved in pure buffer and in mercury-spiked Lake Ontario water samples, respectively.University of Waterloo || Natural Sciences and Engineering Research Council |

Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Huang, P.-J. J., & Liu, J. (2010). Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor. Analytical Chemistry, 82(10), 4020–4026. https://doi.org/10.1021/ac9028505Aptamers are single-stranded nucleic acids that can selectively bind to essentially any molecule of choice. Because of their high stability, low cost, ease of modification, and availability through selection, aptamers hold great promise in addressing key challenges in bioanalytical chemistry. In the past 15 years, many highly sensitive fluorescent aptamer sensors have been reported. However, few such sensors showed high performance in serum samples. Further challenges related to practical applications include detection in a very small sample volume and a low dependence of sensor performance on ionic strength. We report the immobilization of an aptamer sensor on a magnetic microparticle and the use of flow cytometry for detection. Flow cytometry allows the detection of individual particles in a capillary and can effectively reduce the light scattering effect of serum. Since DNA immobilization generated a highly negatively charged surface and caused an enrichment of counterions, the sensor performance showed a lower salt dependence. The detection limits for adenosine are determined to be 178 μM in buffer and 167 μM in 30% serum. Finally, we demonstrated that the detection can be carried out in 10 μL of 90% human blood serum.University of Waterloo || Natural Sciences and Engineering Research Council (NSERC) of Canad

DNA-Length-Dependent Fluorescence Signaling on Graphene Oxide Surface

This is the peer reviewed version of the following article: Huang, P.-J. J., & Liu, J. (2012). DNA-Length-Dependent Fluorescence Signaling on Graphene Oxide Surface. Small, 8(7), 977–983, which has been published in final form at https://doi.org/10.1002/smll.201102156. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Fluorescence energy transfer to graphene oxide is studied using covalently linked DNA probes ranging from 4 to 70 base pairs. The characteristic distance and mechanism of energy transfer are reported.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation |

Sensing Parts-per-Trillion Cd2+, Hg2+ and Pb2+ Collectively and Individually Using Phosphorothioate DNAzymes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Huang, P.-J. J., & Liu, J. (2014). Sensing Parts-per-Trillion Cd 2+ , Hg 2+ , and Pb 2+ Collectively and Individually Using Phosphorothioate DNAzymes. Analytical Chemistry, 86(12), 5999–6005. https://doi.org/10.1021/ac501070aCadmium, mercury, and lead are collectively banned by many countries and regions in electronic devices due to their extremely high toxicity. To date, no sensing method can detect them as a group and also individually with sufficient sensitivity and selectivity. An RNA-cleaving DNAzyme (Ce13d) was recently reported to be active with trivalent lanthanides, which are hard Lewis acids. In this work, phosphorothioate (PS) modifications were systematically made on Ce13d. A single PS modification at the substrate cleavage site shifts the activity from being dependent on lanthanides to soft thiophilic metals. By incorporating the PS modification to another DNAzyme, a sensor array was prepared to detect each metal. Individual sensors have excellent sensitivity (limit of detection = 4.8 nM Cd2+, 2.0 nM Hg2+, and 0.1 nM Pb2+). This study provides a new route to obtain metal-specific DNAzymes by atomic replacement and also offers important mechanistic insights into metal binding and DNAzyme catalysis.University of Waterloo || Ontario Ministry of Research & Innovation || Natural Sciences and Engineering Research Council |

Molecular Beacon Lighting up on Graphene Oxide

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Huang, P.-J. J., & Liu, J. (2012). Molecular Beacon Lighting up on Graphene Oxide. Analytical Chemistry, 84(9), 4192–4198. https://doi.org/10.1021/ac300778sA molecular beacon (MB) is comprised of a fluorophore and a quencher linked by a DNA hairpin. MBs have been widely used for homogeneous DNA detection. In addition to molecular quenchers, many nanomaterials such as graphene oxide (GO) also possess excellent quenching efficiency. Most reported fluorescent sensors relied on DNA probes physisorbed by GO, which may suffer from nonspecific probe displacement and false positive signal. In this work, we report the preparation and characterization of a MB using graphene oxide (GO) as quencher, where an amino and FAM (6-carboxyfluorescein) dual labeled DNA was covalently attached to GO via an amide linkage. A major challenge was to remove noncovalently attached probes due to strong DNA adsorption by GO. While DNA desorption was favored at low salt, high pH, high temperature, and by using organic solvents, the cDNA was required to achieve complete desorption of noncovalently linked DNA probes. The DNA adsorption energy was measured using isothermal titration calorimetry, revealing the heterogeneous nature of GO. The covalent probe has a detection limit of 2.2 nM using a sample volume of 0.05 mL. With a 2 mL sample, the detection limit can reach 150 pM. The covalent probe is highly resistant to nonspecific probe displacement and will find applications in serum and cellular samples where high probe stability is demanded.University of Waterloo || Canada Foundation for Innovation || Ontario Ministry of Research and Innovation || Natural Sciences and Engineering Research Council |

Synergistic pH effect for reversible shuttling aptamer-based biosensors between graphene oxide and target molecules

DNA aptamers are known to desorb from graphene oxide (GO) surface in the presence of target molecules. We demonstrate herein that the binding equilibrium can be shifted by simply tuning the solution pH. At lower pH, the aptamer/GO binding is enhanced while aptamer/target binding is weakened, making this system a regenerable biosensor without covalent conjugation.University of Waterloo || Natural Sciences and Engineering Research Council |

Citrate inhibition of cisplatin reaction with DNA studied using fluorescently labeled oligonucleotides: implication for selectivity towards guanine

The reaction between cisplatin and DNA is conveniently studied using fluorescently labeled oligonucleotides and gel electrophoresis; as an example of application, the inhibition of this reaction by citrate is demonstrated, which might increase selectivity of cisplatin towards guanine over adenine.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research, Innovation and Science |

Desulfurization Activated Phosphorothioate DNAzyme for the Detection of Thallium

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Huang, P.-J. J., Vazin, M., & Liu, J. (2015). Desulfurization Activated Phosphorothioate DNAzyme for the Detection of Thallium. Analytical Chemistry, 87(20), 10443–10449. https://doi.org/10.1021/acs.analchem.5b02568Thallium (Tl) is a highly toxic heavy metal situated between mercury and lead in the periodic table. While its neighbors have been thoroughly studied for DNA-based sensing, little is known about thallium detection. In this work, in vitro selection of RNA-cleaving DNAzymes is carried out using Tl3+ as the target metal cofactor. Both normal DNA and phosphorothioate (PS)-modified DNA are tested for this purpose. While no Tl3+-dependent DNAzymes are obtained, a DNA oligonucleotide containing a single PS-modified RNA nucleotide is found to cleave by ∼7% by Tl3+ at the RNA position. The remaining 93% are desulfurized. By hybridization of this PS-modified oligonucleotide with the Tm7 DNAzyme, the cleavage yield increases to ∼40% in the presence of Tl3+ and Er3+. Tm7 is an Er3+-dependent RNA-cleaving DNAzyme. It cleaves only the normal substrate but is completely inactive using the PS-modified substrate. Tl3+ desulfurizes the PS substrate to the normal substrate to be cleaved by Tm7 and Er3+. This system is engineered into a catalytic beacon for Tl3+ with a detection limit of 1.5 nM, which is below its maximal contamination limit defined by the U.S. Environmental Protection Agency (10 nM).Natural Sciences and Engineering Research Council ||Discovery Grant: 386326 and Strategic Project Grant: STPGP-447472-2013 05576

A Cleavable Molecular Beacon for Hg2+ Detection based on Phosphorothioate RNA Modifications

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/ 10.1021/acs.analchem.5b01362Mercury is a highly toxic heavy metal, and detection of Hg2+ by biosensors has attracted extensive research interest in the past decade. In particular, a number of DNA-based sensing strategies have been developed. Well-known examples include thymine-Hg2+ interactions and Hg2+-activated DNAzymes. However, these mechanisms are highly dependent on buffer conditions or require hybridization with another DNA strand. Herein, we report a new mechanism based on Hg2+-induced cleavage of phosphorothioate (PS) modified RNA. Among the various metal ions tested, Hg2+ induced the most significant cleavage (∼16%), while other metals cleaved less than 2% of the same substrate. The uncleaved substrate undergoes desulfurization in the presence of Hg2+. This cleavage reaction yields a similar amount of product from pH 3.5 to 7 and in the temperature range between 20 and 90 °C. Various PS RNA junctions can be cleaved with a similar efficiency, but PS DNA junctions cannot be cleaved. A molecular beacon containing three PS RNA modifications is designed, detecting Hg2+ down to 1.7 nM with excellent selectivity. This sensor can also detect Hg2+ in the Lake Ontario water sample, although its response is significantly masked by fish tissues.Ontario Ministry of Research & Innovation || Natural Sciences and Engineering Research Council |