Browsing by Author "Liu, Juewen"

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2-Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer
(Chemistry Europe: European Chemical Societies Publishing, 2022-04-25) Lu, Chang; Huang, Po-Jung Jimmy; Zheng, Jingkai; Liu, Juewen
Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a Kd of 15.0 mM glucose, which was comparable with the 10 mM Kd previously reported using the strand displacement assay. The binding required two Na+ ions or one Mg2+ that cannot be replaced by Li+ or K+. The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucose-containing disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher Kd values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.
A DNA Aptamer for Theophylline with Ultrahigh Selectivity Reminiscent of the Classic RNA Aptamer
(American Chemical Society, 2022-08-09) Huang, Po-Jung Jimmy; Liu, Juewen
Since the report of the RNA aptamer for theophylline, theophylline has become a key molecule in chemical biology for designing RNA switches and riboswitches. In addition, theophylline is an important drug for treating airway diseases including asthma. The classic RNA aptamer with excellent selectivity for theophylline has been used to design biosensors, although DNA aptamers are more desirable for stability and cost considerations. In this work, we selected DNA aptamers for theophylline, and all the top sequences shared the same binding motifs. Binding was confirmed using isothermal titration calorimetry and a nuclease digestion assay, showing a dissociation constant (Kd) around 0.5 μM theophylline. The Theo2201 aptamer can be truncated down to 23-mer while still has a Kd of 9.8 μM. The selectivity for theophylline over caffeine is around 250,000-fold based on a strand-displacement assay, which was more than 20-fold higher compared to the classic RNA aptamer. For other tested analogs, the DNA aptamer also showed better selectivity. Using the structure-switching aptamer sensor design method, a detection limit of 17 nM theophylline was achieved in the selection buffer, and a detection limit of 31 nM was obtained in 10% serum.
Adsorption of DNA Oligonucleotides by Self-Assembled Metalloporphyrin Nanomaterials
(American Chemical Society, 2022-03-08) Wang, Jinghan; Wang, Zhen; Huang, Po-Jung Jimmy; Bai, Feng; Liu, Juewen
Porphyrin assemblies have controllable morphology, high biocompatibility, and good optical properties and were widely used in biomedical diagnosis and treatment. With the development of DNA biotechnology, combining DNA with porphyrin assemblies can broaden the biological applications of porphyrins. Porphyrin assemblies can serve as nanocarriers for DNA, although the fundamental interactions between them are not well understood. In this work, zinc meso-tetra(4-pyridyl)porphyrin (ZnTPyP) assemblies were prepared in the presence of various surfactants and at different pH values, yielding a variety of aggregation forms. Among them, the hexagonal stacking form exposes more pyridine substituents, and the hydrogen bonding force between the substituents and the DNA bases allows the DNA to be quickly adsorbed on the surface of the assemblies. The effects of DNA sequence and length were systematically tested. In particular, the adsorption of duplex DNA was less efficient compared to the adsorption of single-stranded DNA. This fundamental study is useful for the further combination of DNA and porphyrin assemblies to prepare new functional hybrid nanomaterials.
Adsorption of Linear and Spherical DNA Oligonucleotides onto Microplastics
(American Chemical Society, 2022-01-30) Zandieh, Mohamad; Patel, Kshiti; Liu, Juewen
Microplastic pollution of water and food chains can endanger human health. It has been reported that environmental DNA can be carried by microplastics and spread into the ecosystem. To better comprehend the interactions between microplastics and DNA, we herein investigated the adsorption of DNA oligonucleotides on a few important microplastics. The microplastics were prepared using common plastic objects made of polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), composite of PS/PVC, and polyethylene terephthalate (PET). The effect of environmentally abundant metal ions such as Na+, Mg2+, and Ca2+ on the adsorption was also studied. Among the microplastics, PET and PS had the highest efficiency for the adsorption of linear DNA, likely due to the interactions provided by their aromatic rings. The study of DNA desorption from PET revealed the important role of hydrogen bonding and metal-mediated adsorption, while van der Waals force and hydrophobic interactions were also involved in the adsorption mechanism. The adsorption of spherical DNA (SNA) made of a high density of DNA coated on gold nanoparticles (AuNPs) was also studied, where the adsorption affinity order was found to be PET > PS/PVC > PS. Moreover, a tighter DNA adsorption was achieved in the presence of Ca2+ and Mg2+ compared to Na+.
Adsorption of tetracycline antibiotics to gold nanoparticles and feasibility of aptamer-based label-free colorimetric detection
(Canadian Science Publishing, 2023-04-11) Song, Yuheng; von Eppinghoven, Derek H.G.; Zhou, Yang; Zhang, Hanxiao; Liu, Juewen
Tetracyclines are a group of very important antibiotics that are still in use. To extract, detect, and remove tetracyclines from the environment, various nanomaterials have been employed. Although gold nanoparticles (AuNPs) are a commonly cited material for these purposes, a fundamental understanding of these tetracycline-AuNP systems is still limited. In this work, the adsorption of tetracycline, oxytetracycline, and doxycycline to AuNPs was studied. The effect on the colloidal stability of AuNPs, adsorption kinetics, and the resulting adsorption isotherms was measured. While millimolar concentrations of the tetracyclines can cause aggregation of AuNPs, saturated monolayer adsorption was achieved with low micromolar concentrations of the tetracyclines. Adsorption was instantaneous, and adsorption to AuNPs enhanced their intrinsic fluorescence instead of quenching. With the assumption that aptamer/target complexes cannot be easily adsorbed by AuNPs compared to free aptamers, a label-free colorimetric detection method was tested. While the label-free sensor showed target-dependent aggregation of AuNPs, a nonbinding mutant aptamer showed the same trend, suggesting that the color change did not reflect aptamer adsorption but other events such as target adsorption. This study indicates the importance of the fundamental understanding of target/AuNP interactions to correctly design aptamer and AuNP-based label-free biosensors.
An aptamer array for discriminating tetracycline antibiotics based on binding-enhanced intrinsic fluorescence
(Royal Society of Chemistry, 2023-03-06) Zhao, Yichen; Gao, Biwen; Chen, Yijing; Liu, Juewen
Tetracyclines are a class of antibiotics with a similar four-ringed structure. Due to this structural similarity, they are not easily differentiated from each other. We recently selected aptamers using oxytetracycline as a target and focused on an aptamer named OTC5, which has similar affinities for oxytetracycline (OTC), tetracycline (TC), and doxycycline (DOX). Tetracyclines exhibit an intrinsic fluorescence that is enhanced upon aptamer binding, allowing convenient binding assays and label-free detection. In this study, we analyzed the top 100 sequences from the previous selection library. Three other sequences were found to differentiate between different tetracyclines (OTC, DOX, and TC) by the selective enhancement of their intrinsic fluorescence. Among them, the OTC43 aptamer was more selective for OTC with a limit of detection (LOD) of 0.7 nM OTC, OTC22 was more selective for DOX (LOD 0.4 nM), and OTC2 was more selective for TC (0.3 nM). Using these three aptamers to form a sensor array, principal component analysis was able to discriminate between the three tetracyclines from each other and from the other molecules. This group of aptamers could be useful as probes for the detection of tetracycline antibiotics.
Aptamer-Based Detection of Caffeine and Theophylline Through Utilization of Gold Nanoparticles and Graphene Oxide
(University of Waterloo, 2023-04-26) Labas, Sarah; Liu, Juewen
The caffeine and theophylline aptamers were used in tandem with gold nanoparticles and graphene oxide to study potential methods for small molecule detection. The AuNP approach is a label-free approach, and the graphene oxide-based sensor is a fluorescence based approach. Each sensor is based off previous literature reporting sensors for DNA targets; more recent literature uses both methods for small molecule detection, but fails to consider target-nanomaterial interactions. The works presented in this thesis serve to determine whether such an interaction exists, and how it may affect the accuracy of the sensors.
Binding of Au3+ Ions by Polyadenine DNA
(University of Waterloo, 2020-09-02) Zhao, Yichen; Liu, Juewen
Interactions between DNA and metal ions are important for many applications such as metal sensing, therapeutics and nanotechnology. Many studies have already been published on these topics. DNA can bind metal ions via its phosphate backbone and the nucleobases. The phosphate backbone likes to bind hard metal ions, whereas the bases tend to coordinate with softer metal ions. Gold in particular is one metal that has many interactions with DNA. Gold nanoparticles are very stable and DNA-functionalized nanoparticles have been used for biosensing, drug delivery and the design of smart materials. However, many of these studies have been carried out on gold surfaces where gold has an oxidation state of zero. The study of the interactions between gold ions (Au3+) and DNA has been limited. Au3+ is a highly soft metal ion and it may have strong interactions with DNA bases. The goal of this thesis is to study the interactions between gold ions and DNA. Fluorescein (FAM) labelled 15-mer homopolymer DNA (FAM-A15, C15, G15, and T15) were first incubated with a source of Au3+ ions (HAuCl4). The reaction products were characterized by denaturing gel electrophoresis and kinetic studies were done using fluorescence quenching assays. In the electrophoresis studies, little to no products were observed with T15 and C15 DNA. A smearing of the gel electrophoresis bands along with some fluorescence quenching was observed with both A15 and G15 DNA suggesting stable complex formation that survived the denaturing gel electrophoresis conditions. A15 DNA showed complete reaction, while G15 showed around 80% reaction yield after 1 hour. Since poly-A DNA showed the highest activity for binding to Au3+, studies of the formation of the polyadenine – Au3+ complex were then conducted as a function of pH and salt. Complex formation was favored at a lower pH (pH 4) and would not form under higher pH (pH 8) conditions. Salt was found to be required for Au3+ to react with DNA. Four different salts (NaF, NaCl, NaBr, and NaI) were tested to see the effects on binding kinetics. The addition of NaF and NaI did not allow the formation of the Au3+-poly-A complex, but NaCl and NaBr allowed the formation of this complex. Therefore, a moderate affinity ligand such as Cl- and Br- favored the reaction. A random 24mer DNA was then compared to A15 for binding to Au3+ and other metal ions. Both DNA only showed evidence of complex formation with Au3+ and not with other metals including Hg2+ and Pb2+. Fluorescence quenching assays of A15 and Au3+ were done as a function of concentration of different salts. First order kinetic rate constants were found to be 0.60 mins-1, 0.91 mins-1, 1.5 mins-1, and 1.9 mins-1 for buffers with no salt, 100 mM NaBr, 100 mM NaCl, and 10 mM NaBr respectively. This reaction was also found to be reversible as the fluorescence could be recovered using potassium cyanide (KCN) or glutathione (GSH). When added post complex formation, KCN could recover almost all the fluorescence while GSH could recover around 60%. The effects of Au3+ ions on the catalytic activity of the 17E zinc dependent DNAzyme were also studied using gel electrophoresis. Under normal conditions, 17E cleaves an RNA substrate with a cleavage percent of 71%, however with concentrations of 25 mM Au3+ and above, the cleavage of the substrate was completely inhibited. The Au3+ was found to be binding to the DNAzyme-substrate duplex in a similar fashion to A15.
Binding Studies of Cationic Conjugated Polymers and DNA for Label-Free Fluorescent Biosensors
(American Chemical Society, 2022-07-19) Zhang, Pengbo; Lu, Chang; Niu, Chenqi; Wang, Xiaoyu; Li, Zhengping; Liu, Juewen
Cationic conjugated polymers (CCPs), especially polythiophene, have been extensively used as probes for developing DNA and aptamer-based biosensors. Although many interesting applications have been achieved, a fundamental understanding of this system remains quite limited. In this work, we performed systematic binding assays to understand the interactions between poly(3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene) (PMNT) and DNA. The fluorescence of PMNT at 530 nm initially decreased and then a peak at 580 nm emerged after binding with single-stranded DNA (ssDNA). The binding force between PMNT and DNA was dominated by electrostatic interactions at first and then DNA base-mediated interactions also became important. Since the bases in double-stranded DNA (dsDNA) were shielded, their fluorescence changes were quite different. To best differentiate ssDNA and dsDNA, the optimal pH was between 6 and 8, and the optimal NaCl concentration was around 0.3 M. Moreover, by changing the sequence and length of ssDNA, poly-T had the largest fluorescence shift and poly-A had the smallest change. Under the optimized conditions, the PMNT-based biosensor had a detection limit of 1 nM DNA, which was similar to the SYBR Green I-based assay.
Capping Gold Nanoparticles to Achieve a Protein-like Surface for Loop-Mediated Isothermal Amplification Acceleration and Ultrasensitive DNA Detection
(Americal Chemical Society, 2022-06-10) Jiang, Xingxing; Yang, Minghui; Liu, Juewen
Loop-mediated isothermal amplification (LAMP) is a popular DNA amplification method. Gold nanoparticles (AuNPs) were reported to enhance the efficiency of LAMP, although the underlying mechanism remained elusive. Since AuNPs strongly adsorb a range of ligands, preadsorbed ligands cannot be easily displaced. In this work, we systematically investigated the effect of surface-modified AuNPs on LAMP by varying the order of mixing of AuNPs with each reagent in the LAMP system (Mg2+, template DNA, dNTPs, primers, and polymerase). Mixing the AuNPs with the primers delayed the LAMP based on SYBR green I fluorescence. While other orders of mixing had little effect, all accelerated the reaction. We then tested other common ligands including polymers (polyethylene glycol and polyvinylpyrrolidone), inorganic ions (Br–), proteins, glutathione (GSH), and DNA (A15) on AuNP-LAMP. The boosted AuNP performance on LAMP was most obvious when the AuNPs formed a protein-like surface. Finally, using GSH-capped AuNPs, a detection limit of around 100 copies/μL–1 of target DNA was achieved. This work has identified a ligand-capped AuNP strategy to boost LAMP and yielded a higher sensitivity in DNA sensing, which also deepens our understanding of AuNP-assisted LAMP.
Capture-SELEX of DNA Aptamers for Estradiol Specifically and Estrogenic Compounds Collectively
(American Chemical Society, 2022-11-28) Niu, Chenqi; Zhang, Chong; Liu, Juewen
Estrogenic compounds such as estrone (E1), 17β-estradiol (E2), and 17α-ethynylestradiol (EE2) are serious environmental contaminants due to their potent biological activities. At least six selections were previously reported to obtain DNA aptamers for E2, highlighting its environmental importance. A careful analysis revealed that the previous aptamers either are too long or do not bind optimally. Herein, a series of new aptamers were obtained from the capture-SELEX method with dissociation constants down to 30 nM as determined by isothermal titration calorimetry (ITC). Two aptamers were converted to structure-switching fluorescent biosensors, which achieved a limit of detection down to 3.3 and 9.1 nM E2, respectively. One aptamer showed similar binding affinities to all the three estrogens, while the other aptamer is more selective for E2. Both aptamers required Mg2+ for binding. The proposed sensors were successfully applied in the determination of E2 in wastewater. Moreover, comparisons were made with previous aptamers based on primary sequence alignment and secondary structures. Among previously reported truncated aptamers, ITC showed binding only in one of them. The newly selected aptamers have the combined advantages of small size and high affinities.
Comparison of the peroxidase activities of iron oxide nanozyme with DNAzyme and horseradish peroxidase
(Royal Society of Chemistry, 2023-04-20) Lu, Chang; Zandieh, Mohamad; Zheng, Jinkai; Liu, Juewen
Peroxidase-based assays are the most extensively used in bioanalytical sensors because of their simple colorimetric readout and high sensitivity owing to enzymatic signal amplification. To improve the stability, modification, and cost of protein-based enzymes, such as horseradish peroxidase (HRP), various enzyme mimics, such as DNAzymes and nanozymes, have emerged over the last few decades. In this study, we compared the peroxidase activities of HRP, a G-quadruplex (G4)-hemin DNAzyme, and Fe3O4 nanozymes in terms of activity and stability under different conditions. The reactions were much slower at pH 7 than at pH 4. At pH 4, the turnover rate of HRP (375 s−1) was faster than that of G4 DNAzyme (0.14 s−1) and Fe3O4 (6.1 × 10−4 s−1, calculated by surface Fe concentration). When normalized to mass concentrations, the trend was the same. Through observation of the reaction for a long time of 2 h, the changes in the color and UV-vis spectra were also different for these catalysts, indicating different reaction mechanisms among these catalysts. Moreover, different buffers and nanozyme sizes were found to influence the activity of the catalysts. Fe3O4 showed the highest stability compared to HRP and G4 DNAzyme after a catalytic reaction or incubation with H2O2 for a few hours. This study helps to understand the properties of catalysts and the development of novel catalysts with enzyme-mimicking activities for application in various fields.
Critical evaluation of aptamer binding for biosensor designs
(Elsevier, 2021-11-13) Zhao, Yichen; Yavari, Kayvan; Liu, Juewen
Over the last three decades, numerous aptamer-based biosensors have been reported. The basis of these sensors is the selective binding of target analytes by aptamers. In the last few years, a number of papers have been published questioning the binding ability of some popular aptamers such as those documented for As(III), ampicillin, chloramphenicol, isocarbophos, phorate and dopamine. In this article, these papers are reviewed, and the binding assays are described, which may provide possible reasons for obtaining false positive aptamers. Additionally, relevant aptamer selection methods and typical characterization steps are described. It is found that for small molecular targets, using an immobilized library might result in better aptamers. Furthermore, the importance of carefully designed controls to ensure the quality of binding assays is discussed, especially in the case of mutated nonbinding aptamers. Only then, with fully validated aptamers, can subsequent biosensor design bring about meaningful results.
Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines
(2023-07-11) Ding, Yuzhe; Xie, Yachen; Li, Albert Zehan; Huang, Po-Jung Jimmy; Liu, Juewen
The classical DNA aptamer for adenosine and ATP was selected twice using ATP as the target in 1995 and 2005, respectively. In 2022, this motif appeared four more times from selections using adenosine, ATP, theophylline, and caffeine as targets, suggesting that this aptamer can also bind methylxanthines. In this work, using thioflavin T fluorescence spectroscopy, this classical DNA aptamer showed Kd values for adenosine, theophylline, and caffeine of 9.5, 101, and 131 μM, respectively, and similar Kd values were obtained using isothermal titration calorimetry. Binding to the methylxanthines was also observed for the newly selected Ade1301 aptamer but not for the Ade1304 aptamer. The RNA aptamer for ATP also had no binding to the methylxanthines. Molecular dynamics simulations were performed using the classical DNA and RNA aptamers based on their NMR structures, and the simulation results were consistent with the experimental observations, explaining the selectivity profiles. This study suggests that a broader range of target analogues need to be tested for aptamers. For the detection of adenosine and ATP, the Ade1304 aptamer is a better choice due to its better selectivity.
Cytosine-Rich DNA Binding Insulin Stronger than Guanine-Rich Aptamers: Effect of Aggregation of Insulin for Its Detection
(American Chemical Society, 2023-05-30) Wu, Na; Zandieh, Mohamad; Yang, Ting; Liu, Juewen
The detection of insulin is an important analytical task. Previously, guanine-rich DNA was believed to bind insulin, and an insulin aptamer was selected based on a few guanine-rich libraries. Insulin is a unique analyte, and it forms different aggregation states as a function of its concentration and buffer conditions, which may affect the detection of insulin. Herein, using fluorescence polarization assays, three insulin preparation methods were evaluated: direct dissolution, ethylenediaminetetraacetic acid (EDTA) treatment to remove Zn2+, and dissolution in acid followed by neutralization. All the insulin samples containing Zn2+ barely bind to the aptamer DNA, whereas monomers and dimers of insulin with Zn2+ removed were able to bind. Compared to the previously reported aptamer, C-rich DNA showed stronger binding affinities and faster binding kinetics. The sigmoidal binding curves and slow binding kinetics showed that multiple DNA strands and insulin molecules gradually bind, and it took approximately 1 h to reach saturation. This insulin binding was nonspecific, and other tested proteins also can bind to C-rich and G-rich DNA with even strong affinities. These results provide important information on the detection of insulin and further insights into the binding mechanisms between oligomeric insulin and DNA.
Deployment of functional DNA-based biosensors for environmental water analysis
(Elsevier, 2022-04-14) Zhao, Yichen; Yavari, Kayvan; Wang, Yihao; Pi, Kunfu; Van Cappellen, Philippe; Liu, Juewen
Various functional DNA molecules have been used for the detection of environmental contaminants in water, but their practical applications have been limited. To address this gap, this review highlights the efforts to develop field-deployable water quality biosensors. The biosensor devices include microfluidic, lateral flow and paper-based devices, and other novel ideas such as the conversion of glucometers for the detection of environmental analytes. In addition, we also review DNA-functionalized hydrogels and their use in diffusive gradients in thin films (DGT) devices. We classify the sensors into one-step and two-step assays and discuss their practical implications. While the review is focused on works reported in the last five years, some classic early works are cited as well. Overall, most of the existing work only tested spiked water samples. Future work needs to shift to real environmental samples and the comparison of DNA-based sensors to standard analytical methods.
DNA and Hydrogels for Sensing and Binding Purine Nucleosides
(University of Waterloo, 2021-08-27) Li, Yuqing; Liu, Juewen
Purine nucleosides, such as adenosine and guanosine, are important biomolecules to regulate physiological functions, including maintain heart and brain health, exert inflammatory responses, and take part in metabolisms. Abnormal levels of purine nucleosides can lead to serious problems. Therefore, monitoring their concentrations is critical for understanding their biological roles and performing related disease diagnoses. Compared with conventional methods to detect them, such as high-performance liquid chromatography (HPLC) and mass spectrometry, DNA aptamer-based strategies are highly attractive due to their high specificity, binding affinity, low cost, and in situ detection ability. Ideally, the aptamers with any desired specific binding abilities could be isolated by systematic evolution of ligands by exponential enrichment (SELEX); however, currently, some selected aptamers cannot distinguish closely related molecules. For example, the widely used adenosine aptamer is effective in distinguishing it from other nucleosides (G, C, T and U) but not the adenine monophosphate (AMP) and adenine triphosphate (ATP), consequently aptamer-sensing platforms built by this sequence are also limited in molecular recognition. In addition, the DNA aptamers for guanosine are not isolated yet. On the other hand, to further graft aptamers on hydrogel matrix for applications, chemical modifications are also inevitable, leading to the high cost of aptamer engineering. To solve these problems, the primary focus of this thesis is to improve the specificity of aptamer-based sensors for detecting purine nucleosides, and develop a modification-free method for preparing DNA-hydrogels at reduced costs. Targeting the problem of some SELEX-derived aptamers with intrinsically limited specificity, a novel method is developed in chapter 2 to achieve highly specific recognition of adenosine. Typically, an entire adenine nucleotide was excised from the backbone of the existing adenosine aptamer (mentioned above), in which the resulting vacancy on DNA scaffold allowed highly specific re-binding of free adenosine, this way realized its molecular recognition and other cognate analytes including AMP, ATP, guanosine, cytidine, uridine, and theophylline are distinguished. This method is termed “base-excision”. To characterize the adenosine recognition, SYBR Green I (SGI) fluorescence spectroscopy and isothermal titration calorimetry (ITC) were used. The ITC demonstrated that one A-excised aptamer strand can bind to two adenosine molecules, with a Kd of 17.0 ± 1.9 µM at 10 degree, and entropy-driven binding. Since the wide-type aptamer cannot discriminate adenosine from AMP and ATP, we attribute this improved specificity to the excised site. Finally, the A-excised aptamer was tested in diluted fetal bovine serum (FBS) and showed a limit of detection of 46.7 µM adenosine. This work provides a facile, cost-effective, and non-SELEX method to engineer existing aptamers for new features and better applications. In chapter 3, the aptamer engineering strategy described in chapter 2 is further used to generate new DNA aptamers for specific recognition of guanosine. Both the Na+-binding aptamer and classical adenosine aptamer were manipulated as the base-excising scaffold. A total of seven guanosine aptamers were designed, in which a guanine-excised Na+-aptamer showed the highest binding specificity and affinity for guanosine, with an apparent Kd of 0.78 mM. Both the aptamer scaffold generality and excised-site generality were systematically studied. This work provides a few guanosine binding aptamers by non-SELEX method. It also provides deeper insights into engineering aptamers for molecular recognition. On the other hand, since the adenosine only differs deoxyadenosine by a 2´-OH and the specific recognition of adenosine from their mixture have not been realized by current methods, in chapter 4, molecularly imprinted polymers (MIPs) and aptamers as two different recognition strategies are combined. A boronic acid-containing monomer, 3-acrylamidophenylboronic acid (AAPBA), was incorporated into the MIPs to specifically target cis-diol moiety in the ribose of adenosine. ITC and SYBR Green I staining were used to measure the binding. The AAPBA-containing aptamer-MIP exhibited a 115-fold high selectivity for adenosine against deoxyadenosine at pH 6.4. The ribose in adenosine may interact with the boronic acid unit and decrease its inhibition effect to the aptamer in the MIP. Whereas for deoxyadenosine, it does not bear a cis-diol, and thus cannot rescue the aptamer. This work provides insights into the combination of aptamers with other functional groups in MIPs, which may further broaden applications in ways that free aptamers cannot achieve alone. From an application perspective, since the current preparations of DNA-hydrogels are heavily relying on acrydite-modified DNA, lowering the cost of grafting DNA on hydrogels is another issue. To this end, a modification-free method is studied in chapter 5. We show that unmodified penta-adenine (A5) can reach up to 75% conjugation efficiency in 8 h under a freezing polymerization condition in polyacrylamide hydrogels. DNA incorporation efficiency was reduced by forming duplex or other folded structures and by removing the freezing condition. By designing diblock DNA containing an A5 block, various functional DNA sequences were attached. Such hydrogels were designed for ultrasensitive DNA hybridization and Hg2+ detection, with detection limits of 50 pM and 10 nM, respectively, demonstrating the feasibility of using unmodified DNA to replace acrydite-DNA. The same method works for both gel nanoparticles and monoliths. This work reveals interesting reaction products by exploiting freezing and has provided a cost-effective way to attach DNA to hydrogels. Overall, improved molecular recognition of adenosine and guanosine has been achieved, through engineering existing aptamers for new functions or combining aptamers with other functional molecules in MIPs. To further facilitate the incorporation of DNA aptamers in hydrogel systems for various applications, the modification-free method is also developed. This thesis deepens our understandings in DNA aptamer-based molecular recognition and in nucleic acid chemistry, as well as provides opportunities for researchers to achieve more specific adenosine and guanosine recognitions in real applications for disease monitoring and diagnosis purpose.
DNA and Metal Ion Mediated Modification of Nanomaterials
(University of Waterloo, 2021-09-01) Huang, Zhicheng; Liu, Juewen; Liu, Juewen
DNA-modified nanomaterials have been applied in diverse areas such as biosensing, catalysis, drug delivery, and biomedical diagnostics. Metal ion mediated DNA conjugation is an important strategy for the construction of DNA/nanomaterials. The interactions between metal ions and DNA phosphate backbones were found critical for DNA adsorption. Most previously reported metal ion mediated DNA/nanomaterial conjugates focused on the role of metal ions for charge screening but ignored the potential formation of DNA/metal complexes, especially for multivalent ions. Since the report of DNA/Fe2+ coordination polymers (CPs), Fe2+ has become attractive in the modification of nanomaterials. One popular nanomaterial is gold nanoparticle (AuNP) which exhibits unique localized surface plasma resonance (LSPR) and enzyme-mimic catalytic activities. The primary focus of this thesis has two main parts: (a) the fundamental understandings of metal ion mediated adsorption of DNA oligonucleotides on 2D nanosheets such as graphene oxide (GO) and Ti2C MXene; (b) the exploration of Fe2+ containing complexes and their applications in designing AuNP-based colorimetric sensors.
DNA coated CoZn-ZIF metal-organic frameworks for fluorescent sensing guanosine triphosphate and discrimination of nucleoside triphosphates
(Elsevier, 2022-05-15) Wang, Zhen; Zhou, Xumei; Han, Jing; Xie, Gang; Liu, Juewen
Imidazole-based metal-organic frameworks (MOFs) are easy to prepare as well-dispersed nanoparticles, which have attracted a lot of interest in sensing. Metal substitution is an effective way to regulate the composition and performance of MOFs. Herein, by tuning the contents of Co and Zn, a series of homobimetallic CoxZn100-x-ZIF (x = 0–100) were synthesized. Using a fluorescently-labeled DNA oligonucleotide probe, guanosine triphosphate (GTP) can readily displace the adsorbed DNA from Co50Zn50-ZIF, resulting in over 30-fold fluorescence enhancement with 1 mM GTP. Co80Zn20-ZIF could specifically recognize adenosine triphosphate (ATP), whereas Co65Zn35-ZIF and Co20Zn80-ZIF responded to both ATP and GTP. For comparison, Co50Ni50-ZIF and Co50Cu50-ZIF were also prepared, but none of them were selective for any of the molecules, indicating a synergetic effect of cobalt and zinc in Co50Zn50-ZIF for the selective recognition of GTP. This system can sensitively detect GTP with a detection limit of 0.13 μM. Moreover, based on the varying binding affinities of these CoZn-ZIFs towards different nucleoside triphosphates (NTPs), a ZIF fluorescent sensor array was also designed for the discrimination of the four types of NTPs.
DNA-Directed Seeded Synthesis of Gold Nanoparticles without Changing DNA Sequence
(Asian Chemical Editorial Society (ACES), 2022-05-06) Lu, Chang; Zandieh, Mohamad; Zheng, Jinkai; Liu, Juewen
DNA has been used for directing the growth of noble metal nanoparticles into different morphologies. Most previous studies focused on the effect of DNA sequence, while the effect of DNA adsorption was not thoroughly explored. In this work, we controlled the seeded growth of AuNPs by using the same DNA sequence but under different initial adsorption conditions: room temperature and heating. DNA adsorbed by heating induced more anisotropic nanoparticle growth, and the most effect was observed with 100 nM C30 DNA, where nanoflowers were obtained for the heated sample. By measuring DNA adsorption and desorption, heating did not increase DNA adsorption density but increased the adsorption affinity. The percentage of adsorbed DNA before the growth was only about 10%, regardless of heating, while after the growth, the associated DNA reached 75% or more, indicating that the free DNA also influenced the growth. This study offers fundamental insights into the effect of DNA adsorption on seeded AuNP growth, providing a method to tune the morphology of nanoparticles without changing DNA sequence.