93 research outputs found
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 |
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 |
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 |
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 |
DNA Adsorption, Desorption, and Fluorescence Quenching by Graphene Oxide and Related Analytical Application
Graphene is a single layer of graphite with many unique mechanical, electrical, and optical properties. In addition, graphene is also known to adsorb wide range of biomolecules including single-stranded DNA. On the other hand, the adsorption of double-stranded DNA was much weaker. To properly disperse in water, graphene oxide (GO) is often used due to its oxygen-containing groups on the surface. Recently, it was discovered that it could efficiently quench the fluorescence of fluorophores that were adsorbed. With these properties, it is possible to prepare DNA-based optical sensors using GO. Majority of the DNA/GO-based fluorescent sensors reported so far were relied on the complete desorption of DNA probes. Even though all these reports demonstrated the sensitivity and selectivity of the system, the fundamentals of binding between DNA and GO were hardly addressed.
Understanding and controlling binding between biomolecules and inorganic materials is very important in biosensor development. In this thesis, adsorption and desorption of DNA on the GO surface under different buffer conditions including ionic strength, pH, and temperature were systematically evaluated. For instance, adsorption is favored in a lower pH and a higher ionic strength buffer. It was found that once a DNA was adsorbed on the surface, little desorption occurred even in low salt buffers. Even with high pH or temperature, only small percentage of adsorbed DNA can be desorbed. To completely desorb the DNA, complementary DNA is required. The energies and activation energies associated with DNA adsorption/desorption were measured and molecular pictures of these processes were obtained. With the fundamental understanding of the DNA/GO interaction, we demonstrated that it is possible to achieve sensor regeneration without covalent immobilization. In addition, we also achieved the separation of double-stranded DNAs from single-stranded ones without using gel electrophoresis.
We also studied the fluorescence property of DNA near the GO surface using covalently attached DNA probes. It was found that the fluorophore quantum yield and lifetime changed as a function of DNA length. This study is important for rational design of covalently linked DNA sensors. This study confirmed that fluorescence quenching by GO occurs in a distance-dependent manner. Energy transfer occurred between the fluorophore and GO to result in reduced quantum yield, shorter lifetime, and lower fluorescence intensity. Although fluorescent sensors based on covalently attached DNA probes on GO have not yet been reported, the study presented here clearly supported its feasibility
In Vitro Selection of New DNAzymes as Metal Biosensors
In vitro selection of metal-specific DNAzymes has been shown to be a powerful method to obtain biosensors for metal ion detection. With 14 independent in vitro selection experiments using each of the trivalent lanthanide as the metal cofactor, five DNAzymes with distinct activity patterns cross the lanthanide series were identified and characterized. In addition, these DNAzyme beacon sensors were developed by using fluorophore/quencher modified pairs. Each of these DNAzymes showed low nM lanthanide detection limit with minimal interference from other metals. A sensor array was prepared to achieve pattern-recognition-based detection using linear discriminant analysis (LDA), where most of the 14 lanthanides can be well separated. In addition, a selection using a phosphorothioate modified DNA library was carried out and a new DNAzyme with high sensitivity and specificity for Cd2+ was obtained
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 |
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 |
- …