Juewen Liu | University of Waterloo (original) (raw)
Papers by Juewen Liu
The Analyst, 2012
Lead is highly toxic and its detection has attracted a lot of research interests. In recent years... more Lead is highly toxic and its detection has attracted a lot of research interests. In recent years, DNA has been used for Pb(2+) recognition and many fluorescent sensors with low to sub-nM detection limits have been reported. These figures of merit were typically measured using a spectrophotometer that can detect nM DNA with a high signal-to-noise ratio. For visual detection, however, μM DNA or dye was required, making it difficult to detect low nM targets. We recently achieved a visual sensitivity of 10 nM Hg(2+) by immobilizing a DNA probe in a hydrogel. This was made possible because the gel was able to actively adsorb Hg(2+). In this work, we aim to test whether this method can be extended to the detection of Pb(2+). First, a new Pb(2+) sensor was designed based on a guanine-rich DNA and DNA binding dyes such as thiazole orange and SYBR Green I. The free DNA showed a detection limit of 8 nM Pb(2+) using 40 nM DNA. For visual detection in solution with 1 μM of the DNA probe, however, ∼300 nM Pb(2+) was required. After immobilization in a monolithic polyacrylamide hydrogel, even 20 nM Pb(2+) could be visually detected with a sample volume of 50 mL. Therefore, sensitive detection without signal amplification was achieved. Finally, we demonstrated simultaneous detection of both Hg(2+) and Pb(2+) in the same water sample with shape encoded hydrogel sensors.
Analytical chemistry, Jan 10, 2015
Mercury is a highly toxic heavy metal and detection of Hg2+ by biosensors has attracted extensive... more Mercury is a highly toxic heavy metal and detection of Hg2+ by biosensors has attracted extensive research interest in the last 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 °C and 90 °C. Various PS RNA junctions can be cleaved with a similar efficiency, but PS DNA junctions cannot be cleaved. A molecular beaco...
Nature Chemical Biology, 2007
Protein metalloenzymes use various modes for functions for which metal-dependent global conformat... more Protein metalloenzymes use various modes for functions for which metal-dependent global conformational change is required in some cases but not in others. In contrast, most ribozymes require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using singlemolecule fluorescence resonance energy transfer. Addition of Zn 2+ and Mg 2+ induced folding of the DNAzyme into a more compact structure followed by a cleavage reaction, which suggests that the DNAzyme may require metal-dependent global folding for activation. In the presence of Pb 2+ , however, the cleavage reaction occurred without a precedent folding step, which suggests that the DNAzyme may be prearranged to accept Pb 2+ for the activity. Neither ligation reaction of the cleaved substrates nor dynamic changes between folded and unfolded states was observed. These features may contribute to the unusually fast Pb 2+ -dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.
Journal of The American Chemical Society, 2007
The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of div... more The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ > Zn2+ >Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.
Proceedings of the National Academy of Sciences
Here, we report a catalytic beacon sensor for uranyl (UO2(2+)) based on an in vitro-selected UO2(... more Here, we report a catalytic beacon sensor for uranyl (UO2(2+)) based on an in vitro-selected UO2(2+)-specific DNAzyme. The sensor consists of a DNA enzyme strand with a 3' quencher and a DNA substrate with a ribonucleotide adenosine (rA) in the middle and a fluorophore and a quencher at the 5' and 3' ends, respectively. The presence of UO2(2+) causes catalytic cleavage of the DNA substrate strand at the rA position and release of the fluorophore and thus dramatic increase of fluorescence intensity. The sensor has a detection limit of 11 parts per trillion (45 pM), a dynamic range up to 400 nM, and selectivity of >1-million-fold over other metal ions. The most interfering metal ion, Th(IV), interacts with the fluorescein fluorophore, causing slightly enhanced fluorescence intensity, with an apparent dissociation constant of approximately 230 microM. This sensor rivals the most sensitive analytical instruments for uranium detection, and its application in detecting uran...
ACS applied materials & interfaces, Jan 21, 2015
Metallo-β-lactamases (MBLs) degrade a broad spectrum of antibiotics including the latest carbapen... more Metallo-β-lactamases (MBLs) degrade a broad spectrum of antibiotics including the latest carbapenems. So far, limited success was achieved in developing its inhibitors using small organic molecules. VIM-2 is one of the most studied and important MBLs. In this work, we screened ten nanomaterials, covering a diverse range of surface properties including charge, hydrophobicity and specific chemical bonding. Among these, graphene oxide and carbon nanotubes are the most potent inhibitors, while most other materials do not show much inhibition effect. The inhibition is non-competitive and is attributed to the hydrophobic interaction with the enzyme. Adsorption of VIM-2 was further probed using protein displacement assays where it cannot displace or be displaced by bovine serum albumin (BSA). This information is useful for rational design inhibitors for MBLs and more specific inhibition might be achieved by further surface modifications on these nano-carbons.
Analytical and Bioanalytical Chemistry, 2014
In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNAbas... more In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNAbased biosensors, given the DNA adsorption and fluorescence-quenching properties of GO. Adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing a fluorescence signal. In addition to this initial design, many other strategies have been reported, including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of GO, and the application of covalently linked DNA probes. The potential applications of DNAfunctionalized GO range from environmental monitoring and cell imaging to biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescencequenching mechanism. Following that, the various sensor design strategies are critically compared. Problems that must be overcome before this technology can reach its full potential are described, and a few future directions are also discussed.
Chemical communications (Cambridge, England), Jan 14, 2007
Using a Cu(2+)-dependent DNA ligation DNAzyme, a colorimetric sensor for Cu2+ has been developed ... more Using a Cu(2+)-dependent DNA ligation DNAzyme, a colorimetric sensor for Cu2+ has been developed based on directed assembly of DNA-functionalized gold nanoparticles by the ligation product, and such ligation DNAzyme-based sensors are intrinsically more sensitive than cleavage DNAzyme systems due to the lack of background.
Chemical reviews, 2009
or high toxicity. It is difficult to review all existing aptamers here, given that over one hundr... more or high toxicity. It is difficult to review all existing aptamers here, given that over one hundred aptamers were isolated for protein targets by NeXstar Pharmaceuticals Inc. and the University of Colorado alone by 1999. 16 The number of aptamers isolated by scientists throughout the world is much greater than that. Ellington and co-workers have created an online searchable aptamer database where more detailed information can be found. 17 shows a list of literature reported DNA aptamer targets, which demonstrate that aptamers can bind any analytes of choice. There are even more RNA aptamers reported in the literature and they generally have comparable binding performance to DNA aptamers.
Analytical chemistry, 2003
A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivi... more A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivity at 4 degrees C. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb(2+), the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and ...
Analytical chemistry, Jan 15, 2004
Previous work has shown that DNAzyme-directed assembly of gold nanoparticles can be utilized to m... more Previous work has shown that DNAzyme-directed assembly of gold nanoparticles can be utilized to make effective colorimetric biosensors. However, the method is restricted to analytes that are directly involved in phosphodiester cleavage. To expand the methodology to a broader range of analytes, a colorimetric adenosine biosensor based on the aptazyme-directed assembly of gold nanoparticles is reported here. The aptazyme is based on the 8-17 DNAzyme with an adenosine aptamer motif that can modulate the DNAzyme activity through allosteric interactions depending on the presence of adenosine. In the absence of adenosine, the aptazyme is inactive and the substrate strands can serve as linkers to assemble DNA-functionalized 13-nm-diameter gold nanoparticles, resulting in a blue color. However, the presence of adenosine activates the aptazyme, which cleaves the substrate strand, disrupting the formation of nanoparticle aggregates. A red color of separated gold nanoparticles is observed. Con...
Methods in molecular biology (Clifton, N.J.), 2006
Fluorescence resonance energy transfer (FRET) is a widely used technique to study the structure a... more Fluorescence resonance energy transfer (FRET) is a widely used technique to study the structure and dynamics of nucleic acids in solution. Such a technique often uses only one donor fluorophore and one acceptor fluorophore to probe the distance and its changes between the two labeled sites. To fully understand molecules with complicated structures, such as three- or four-way DNA junctions, several dual-fluorophore experiments have to be performed. Here, we describe an emerging alternative technique using multi-fluorophore FRET, in which simultaneous labeling of one molecule with several different fluorophores is performed to acquire all the distance information in a single experiment. This method decreases the number of experiments necessary to perform and increases the consistency of the results. In this chapter, FRET study of a tri-fluorophore-labeled DNAzyme serves as an example to illustrate the design of multi-fluorophore FRET experiments and the related data processing and ana...
Methods in molecular biology (Clifton, N.J.), 2006
In this chapter, methods for designing metal ion sensors using fluorophore- and quencher-labeled ... more In this chapter, methods for designing metal ion sensors using fluorophore- and quencher-labeled DNAzymes are discussed. In contrast to the classical molecular beacon method based on binding, the methods described here utilize catalytic cleavage to release the fluorophore for detection and quantification, making it possible to take advantage of catalytic turnovers for signal amplification. Unlike classical molecular beacons that detect only nucleic acids, catalytic molecular beacons can be applied to different DNAzymes to detect a broad range of analytes. The methods described are based on the finding that almost all known trans-cleaving DNAzymes share a similar structure comprised of a catalytic DNAzyme core flanked by two substrate recognition arms. Using a typical DNAzyme called the "8-17" DNAzyme as an example, the design of highly sensitive and selective Pb2+ sensors is described in detail. The initial design employs a single fluorophore-quencher pair in close proximi...
Journal of the American Chemical Society, Jan 15, 2007
Copper is a key metal ion both in environment monitoring and in biology, and exposure to high con... more Copper is a key metal ion both in environment monitoring and in biology, and exposure to high concentration of copper can cause adverse health effects. Although significant progresses have been made in designing fluorescent sensors for diamagnetic metal ions, few effective Cu ...
Analytical chemistry, Jan 16, 2014
Fluorescent aptamer probes physisorbed on graphene oxide (GO) have recently emerged as a useful s... more Fluorescent aptamer probes physisorbed on graphene oxide (GO) have recently emerged as a useful sensing platform. A signal is generated by analyte-induced probe desorption. To address nonspecific probe displacement and the false positive signal, we herein report a covalently linked aptamer probe for adenosine triphosphate (ATP) detection. A fluorophore and amino dual modified aptamer was linked to the carboxyl group on GO with a coupling efficiency of ∼50%. The linearity, specificity, stability, and regeneration of the covalent sensor were systematically studied and compared to the physisorbed probe. Both sensors have similar sensitivity, but the covalent one is more resistant to nonspecific probe displacement by proteins. The covalent sensor has a dynamic range from 0.125 to 2 mM ATP in buffer at room temperature and is resistance to DNase I. Intracellular ATP imaging was demonstrated using the covalent sensor, which generated a higher fluorescence signal than the physisorbed senso...
Journal of The American Chemical Society, 2005
In recent years, the attachment of biological molecules onto carbon nanotubes has received signif... more In recent years, the attachment of biological molecules onto carbon nanotubes has received significant interest, particularly for applications in biosensing and biorecognition. 1-3 Most of these studies have focused on the structure and activity of proteins, including enzymes, on carbon nanotubes. 4-6 Certain small singlestranded DNA fragments are also known to possess catalytic activity, with studies primarily focusing on the endonuclease activity of these DNAzymes. Interfacing these DNAzymes with nanomaterials has resulted in the development of sensors to detect metal ions 9-11 and nucleic acid, 12 and strategies for directing nanoparticle assembly. 10,13 A fundamental understanding of DNAzyme-nanomaterial interactions, however, has not been achieved. To that end, we report herein the generation and characterization of DNAzymecarbon nanotube conjugates, which may provide unique opportunities as templates for nanoscale assembly and as vectors for gene delivery.
Analytical Chemistry, 2014
Cadmium, mercury, and lead are collectively banned by many countries and regions in electronic de... more Cadmium, 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 Cd(2+), 2.0 nM Hg(2+), and 0.1 nM Pb(2+)). 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.
Chemical Communications, 2009
The temperature-dependent variability of a Pb 2+ -specific 8-17E DNAzyme catalytic beacon sensor ... more The temperature-dependent variability of a Pb 2+ -specific 8-17E DNAzyme catalytic beacon sensor has been addressed through the introduction of mismatches in the DNAzyme, and the resulting sensors resist temperature-dependent variations from 4 to 30 °C.
Journal of The American Chemical Society, 2009
Figure 4. (A) pH dependence of the fluorescence emission of the pHsensitive probe Oregon Green. (... more Figure 4. (A) pH dependence of the fluorescence emission of the pHsensitive probe Oregon Green. (B-D) Confocal projections comparing the development of pH gradients using Oregon Green in mesostructured silicaencapsulated yeast with (B) DMPC liposomes without bR but with diC 6 PC, (C) bR added with diC 6 PC only, and (D) bR added in DMPC liposomes with diC 6 PC. Decreasing fluorescence intensity corresponds to decreasing pH. Scale bars ) 3 µm.
Journal of Molecular Structure: THEOCHEM, 2002
The cations of sulfur clusters exhibit intensities of signi®cance on the mass spectra of sulfur c... more The cations of sulfur clusters exhibit intensities of signi®cance on the mass spectra of sulfur clusters generated in direct laser vaporization. We have acquired numerous isomers of cationic sulfur clusters by means of the B3LYP DFT method. According to total energies, the most stable S n 1 n 3±13 isomers are predicted. Many cationic clusters are composed of two-fold atoms; those with one-fold and three-fold atoms are higher in total energy. The most stable isomer of some cationic clusters shows a structure completely different from that of the corresponding neutral cluster. q
The Analyst, 2012
Lead is highly toxic and its detection has attracted a lot of research interests. In recent years... more Lead is highly toxic and its detection has attracted a lot of research interests. In recent years, DNA has been used for Pb(2+) recognition and many fluorescent sensors with low to sub-nM detection limits have been reported. These figures of merit were typically measured using a spectrophotometer that can detect nM DNA with a high signal-to-noise ratio. For visual detection, however, μM DNA or dye was required, making it difficult to detect low nM targets. We recently achieved a visual sensitivity of 10 nM Hg(2+) by immobilizing a DNA probe in a hydrogel. This was made possible because the gel was able to actively adsorb Hg(2+). In this work, we aim to test whether this method can be extended to the detection of Pb(2+). First, a new Pb(2+) sensor was designed based on a guanine-rich DNA and DNA binding dyes such as thiazole orange and SYBR Green I. The free DNA showed a detection limit of 8 nM Pb(2+) using 40 nM DNA. For visual detection in solution with 1 μM of the DNA probe, however, ∼300 nM Pb(2+) was required. After immobilization in a monolithic polyacrylamide hydrogel, even 20 nM Pb(2+) could be visually detected with a sample volume of 50 mL. Therefore, sensitive detection without signal amplification was achieved. Finally, we demonstrated simultaneous detection of both Hg(2+) and Pb(2+) in the same water sample with shape encoded hydrogel sensors.
Analytical chemistry, Jan 10, 2015
Mercury is a highly toxic heavy metal and detection of Hg2+ by biosensors has attracted extensive... more Mercury is a highly toxic heavy metal and detection of Hg2+ by biosensors has attracted extensive research interest in the last 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 °C and 90 °C. Various PS RNA junctions can be cleaved with a similar efficiency, but PS DNA junctions cannot be cleaved. A molecular beaco...
Nature Chemical Biology, 2007
Protein metalloenzymes use various modes for functions for which metal-dependent global conformat... more Protein metalloenzymes use various modes for functions for which metal-dependent global conformational change is required in some cases but not in others. In contrast, most ribozymes require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using singlemolecule fluorescence resonance energy transfer. Addition of Zn 2+ and Mg 2+ induced folding of the DNAzyme into a more compact structure followed by a cleavage reaction, which suggests that the DNAzyme may require metal-dependent global folding for activation. In the presence of Pb 2+ , however, the cleavage reaction occurred without a precedent folding step, which suggests that the DNAzyme may be prearranged to accept Pb 2+ for the activity. Neither ligation reaction of the cleaved substrates nor dynamic changes between folded and unfolded states was observed. These features may contribute to the unusually fast Pb 2+ -dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.
Journal of The American Chemical Society, 2007
The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of div... more The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ > Zn2+ >Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.
Proceedings of the National Academy of Sciences
Here, we report a catalytic beacon sensor for uranyl (UO2(2+)) based on an in vitro-selected UO2(... more Here, we report a catalytic beacon sensor for uranyl (UO2(2+)) based on an in vitro-selected UO2(2+)-specific DNAzyme. The sensor consists of a DNA enzyme strand with a 3' quencher and a DNA substrate with a ribonucleotide adenosine (rA) in the middle and a fluorophore and a quencher at the 5' and 3' ends, respectively. The presence of UO2(2+) causes catalytic cleavage of the DNA substrate strand at the rA position and release of the fluorophore and thus dramatic increase of fluorescence intensity. The sensor has a detection limit of 11 parts per trillion (45 pM), a dynamic range up to 400 nM, and selectivity of >1-million-fold over other metal ions. The most interfering metal ion, Th(IV), interacts with the fluorescein fluorophore, causing slightly enhanced fluorescence intensity, with an apparent dissociation constant of approximately 230 microM. This sensor rivals the most sensitive analytical instruments for uranium detection, and its application in detecting uran...
ACS applied materials & interfaces, Jan 21, 2015
Metallo-β-lactamases (MBLs) degrade a broad spectrum of antibiotics including the latest carbapen... more Metallo-β-lactamases (MBLs) degrade a broad spectrum of antibiotics including the latest carbapenems. So far, limited success was achieved in developing its inhibitors using small organic molecules. VIM-2 is one of the most studied and important MBLs. In this work, we screened ten nanomaterials, covering a diverse range of surface properties including charge, hydrophobicity and specific chemical bonding. Among these, graphene oxide and carbon nanotubes are the most potent inhibitors, while most other materials do not show much inhibition effect. The inhibition is non-competitive and is attributed to the hydrophobic interaction with the enzyme. Adsorption of VIM-2 was further probed using protein displacement assays where it cannot displace or be displaced by bovine serum albumin (BSA). This information is useful for rational design inhibitors for MBLs and more specific inhibition might be achieved by further surface modifications on these nano-carbons.
Analytical and Bioanalytical Chemistry, 2014
In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNAbas... more In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNAbased biosensors, given the DNA adsorption and fluorescence-quenching properties of GO. Adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing a fluorescence signal. In addition to this initial design, many other strategies have been reported, including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of GO, and the application of covalently linked DNA probes. The potential applications of DNAfunctionalized GO range from environmental monitoring and cell imaging to biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescencequenching mechanism. Following that, the various sensor design strategies are critically compared. Problems that must be overcome before this technology can reach its full potential are described, and a few future directions are also discussed.
Chemical communications (Cambridge, England), Jan 14, 2007
Using a Cu(2+)-dependent DNA ligation DNAzyme, a colorimetric sensor for Cu2+ has been developed ... more Using a Cu(2+)-dependent DNA ligation DNAzyme, a colorimetric sensor for Cu2+ has been developed based on directed assembly of DNA-functionalized gold nanoparticles by the ligation product, and such ligation DNAzyme-based sensors are intrinsically more sensitive than cleavage DNAzyme systems due to the lack of background.
Chemical reviews, 2009
or high toxicity. It is difficult to review all existing aptamers here, given that over one hundr... more or high toxicity. It is difficult to review all existing aptamers here, given that over one hundred aptamers were isolated for protein targets by NeXstar Pharmaceuticals Inc. and the University of Colorado alone by 1999. 16 The number of aptamers isolated by scientists throughout the world is much greater than that. Ellington and co-workers have created an online searchable aptamer database where more detailed information can be found. 17 shows a list of literature reported DNA aptamer targets, which demonstrate that aptamers can bind any analytes of choice. There are even more RNA aptamers reported in the literature and they generally have comparable binding performance to DNA aptamers.
Analytical chemistry, 2003
A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivi... more A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivity at 4 degrees C. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb(2+), the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and ...
Analytical chemistry, Jan 15, 2004
Previous work has shown that DNAzyme-directed assembly of gold nanoparticles can be utilized to m... more Previous work has shown that DNAzyme-directed assembly of gold nanoparticles can be utilized to make effective colorimetric biosensors. However, the method is restricted to analytes that are directly involved in phosphodiester cleavage. To expand the methodology to a broader range of analytes, a colorimetric adenosine biosensor based on the aptazyme-directed assembly of gold nanoparticles is reported here. The aptazyme is based on the 8-17 DNAzyme with an adenosine aptamer motif that can modulate the DNAzyme activity through allosteric interactions depending on the presence of adenosine. In the absence of adenosine, the aptazyme is inactive and the substrate strands can serve as linkers to assemble DNA-functionalized 13-nm-diameter gold nanoparticles, resulting in a blue color. However, the presence of adenosine activates the aptazyme, which cleaves the substrate strand, disrupting the formation of nanoparticle aggregates. A red color of separated gold nanoparticles is observed. Con...
Methods in molecular biology (Clifton, N.J.), 2006
Fluorescence resonance energy transfer (FRET) is a widely used technique to study the structure a... more Fluorescence resonance energy transfer (FRET) is a widely used technique to study the structure and dynamics of nucleic acids in solution. Such a technique often uses only one donor fluorophore and one acceptor fluorophore to probe the distance and its changes between the two labeled sites. To fully understand molecules with complicated structures, such as three- or four-way DNA junctions, several dual-fluorophore experiments have to be performed. Here, we describe an emerging alternative technique using multi-fluorophore FRET, in which simultaneous labeling of one molecule with several different fluorophores is performed to acquire all the distance information in a single experiment. This method decreases the number of experiments necessary to perform and increases the consistency of the results. In this chapter, FRET study of a tri-fluorophore-labeled DNAzyme serves as an example to illustrate the design of multi-fluorophore FRET experiments and the related data processing and ana...
Methods in molecular biology (Clifton, N.J.), 2006
In this chapter, methods for designing metal ion sensors using fluorophore- and quencher-labeled ... more In this chapter, methods for designing metal ion sensors using fluorophore- and quencher-labeled DNAzymes are discussed. In contrast to the classical molecular beacon method based on binding, the methods described here utilize catalytic cleavage to release the fluorophore for detection and quantification, making it possible to take advantage of catalytic turnovers for signal amplification. Unlike classical molecular beacons that detect only nucleic acids, catalytic molecular beacons can be applied to different DNAzymes to detect a broad range of analytes. The methods described are based on the finding that almost all known trans-cleaving DNAzymes share a similar structure comprised of a catalytic DNAzyme core flanked by two substrate recognition arms. Using a typical DNAzyme called the "8-17" DNAzyme as an example, the design of highly sensitive and selective Pb2+ sensors is described in detail. The initial design employs a single fluorophore-quencher pair in close proximi...
Journal of the American Chemical Society, Jan 15, 2007
Copper is a key metal ion both in environment monitoring and in biology, and exposure to high con... more Copper is a key metal ion both in environment monitoring and in biology, and exposure to high concentration of copper can cause adverse health effects. Although significant progresses have been made in designing fluorescent sensors for diamagnetic metal ions, few effective Cu ...
Analytical chemistry, Jan 16, 2014
Fluorescent aptamer probes physisorbed on graphene oxide (GO) have recently emerged as a useful s... more Fluorescent aptamer probes physisorbed on graphene oxide (GO) have recently emerged as a useful sensing platform. A signal is generated by analyte-induced probe desorption. To address nonspecific probe displacement and the false positive signal, we herein report a covalently linked aptamer probe for adenosine triphosphate (ATP) detection. A fluorophore and amino dual modified aptamer was linked to the carboxyl group on GO with a coupling efficiency of ∼50%. The linearity, specificity, stability, and regeneration of the covalent sensor were systematically studied and compared to the physisorbed probe. Both sensors have similar sensitivity, but the covalent one is more resistant to nonspecific probe displacement by proteins. The covalent sensor has a dynamic range from 0.125 to 2 mM ATP in buffer at room temperature and is resistance to DNase I. Intracellular ATP imaging was demonstrated using the covalent sensor, which generated a higher fluorescence signal than the physisorbed senso...
Journal of The American Chemical Society, 2005
In recent years, the attachment of biological molecules onto carbon nanotubes has received signif... more In recent years, the attachment of biological molecules onto carbon nanotubes has received significant interest, particularly for applications in biosensing and biorecognition. 1-3 Most of these studies have focused on the structure and activity of proteins, including enzymes, on carbon nanotubes. 4-6 Certain small singlestranded DNA fragments are also known to possess catalytic activity, with studies primarily focusing on the endonuclease activity of these DNAzymes. Interfacing these DNAzymes with nanomaterials has resulted in the development of sensors to detect metal ions 9-11 and nucleic acid, 12 and strategies for directing nanoparticle assembly. 10,13 A fundamental understanding of DNAzyme-nanomaterial interactions, however, has not been achieved. To that end, we report herein the generation and characterization of DNAzymecarbon nanotube conjugates, which may provide unique opportunities as templates for nanoscale assembly and as vectors for gene delivery.
Analytical Chemistry, 2014
Cadmium, mercury, and lead are collectively banned by many countries and regions in electronic de... more Cadmium, 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 Cd(2+), 2.0 nM Hg(2+), and 0.1 nM Pb(2+)). 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.
Chemical Communications, 2009
The temperature-dependent variability of a Pb 2+ -specific 8-17E DNAzyme catalytic beacon sensor ... more The temperature-dependent variability of a Pb 2+ -specific 8-17E DNAzyme catalytic beacon sensor has been addressed through the introduction of mismatches in the DNAzyme, and the resulting sensors resist temperature-dependent variations from 4 to 30 °C.
Journal of The American Chemical Society, 2009
Figure 4. (A) pH dependence of the fluorescence emission of the pHsensitive probe Oregon Green. (... more Figure 4. (A) pH dependence of the fluorescence emission of the pHsensitive probe Oregon Green. (B-D) Confocal projections comparing the development of pH gradients using Oregon Green in mesostructured silicaencapsulated yeast with (B) DMPC liposomes without bR but with diC 6 PC, (C) bR added with diC 6 PC only, and (D) bR added in DMPC liposomes with diC 6 PC. Decreasing fluorescence intensity corresponds to decreasing pH. Scale bars ) 3 µm.
Journal of Molecular Structure: THEOCHEM, 2002
The cations of sulfur clusters exhibit intensities of signi®cance on the mass spectra of sulfur c... more The cations of sulfur clusters exhibit intensities of signi®cance on the mass spectra of sulfur clusters generated in direct laser vaporization. We have acquired numerous isomers of cationic sulfur clusters by means of the B3LYP DFT method. According to total energies, the most stable S n 1 n 3±13 isomers are predicted. Many cationic clusters are composed of two-fold atoms; those with one-fold and three-fold atoms are higher in total energy. The most stable isomer of some cationic clusters shows a structure completely different from that of the corresponding neutral cluster. q