Browsing by Author "Zhao, Yichen"

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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.
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.
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.
Label-free and Dye-free Fluorescent Sensing of Tetracyclines Using a Capture-Selected DNA Aptamer
(American Chemical Society, 2022-07-01) Zhao, Yichen; Ong, Steven; Chen, Yijing; Huang, Po-Jung Jimmy; Liu, Juewen
Tetracyclines are a group of important antibiotics with a common four-ring scaffold. While most tetracyclines are currently used only in animals, their leaching into the environment and residues in food have caused health concerns. Aptamers are an attractive way to detect tetracyclines, and all previously reported aptamers for tetracyclines were obtained by immobilizing target molecules. In this work, we selected a few DNA aptamers by immobilizing the DNA library using oxytetracycline as the target. We obtained new aptamers with no overlapping sequences compared to the previously reported ones, and a representative sequence named OTC5 had a dissociation constant of 147 nM measured by isothermal titration calorimetry. Similar binding affinities were also observed with tetracycline and doxycycline. Because tetracyclines are fluorescent and their fluorescence intensity was enhanced by binding to the aptamers, a label-free and dye-free fluorescent biosensor was developed with a detection limit of 25 nM oxytetracycline. The sensor was able to detect targets in milk after extraction. Fluorescence polarization measurement showed that this aptamer is insensitive to sodium concentration but requires magnesium. Finally, a strand-displacement biosensor was designed, and it has a detection limit of 1.2 μM oxytetracycline.
Light-up split aptamers: binding thermodynamics and kinetics for sensing
(Royal Society of Chemistry, 2023-09-22) Zhao, Yichen; Patel, Nikesh; Sun, Peihuan; Faulds, Karen; Graham, Duncan; Liu, Juewen
Due to their programmable structures, many aptamers can be readily split into two halves while still retaining their target binding function. While split aptamers are prevalent in the biosensor field, fundamental studies of their binding are still lacking. In this work, we took advantage of the fluorescence enhancement property of a new aptamer named OTC5 that can bind to tetracycline antibiotics to compare various split aptamers with the full-length aptamer. The split aptamers were designed to have different stem lengths. Longer stem length aptamers showed similar dissociation constants (Kd) to the full-length aptamer, while a shorter stem construct showed an 85-fold increase in Kd. Temperature-dependent fluorescence measurements confirmed the lower thermostability of split aptamers. Isothermal titration calorimetry indicated that split aptamer binding can release more heat but have an even larger entropy loss. Finally, a colorimetric biosensor using gold nanoparticles was designed by pre-assembling two thiolated aptamer halves, which can then link gold nanoparticles to give a red-to-blue color change.
Selection and Characterization of DNA Aptamers for the Detection of Antibiotics
(University of Waterloo, 2024-08-28) Zhao, Yichen; Liu, Juewen
DNA is a naturally occurring biomacromolecule that plays many roles in living organisms. While the majority of natural DNA is double stranded, chemical synthesis allows the production of single-stranded DNA oligonucleotides that can carry chemical functions for molecular recognition, and these are known as DNA aptamers. There is wide range of targets that DNA can bind, from metabolites, drugs, toxins, and other small molecules to proteins and even cells and tissues. Aptamers are generally selected through an iterative process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). After this process is completed, the newly selected aptamers can then be subject to binding assays for characterization before finally becoming useful for sensing. Compared to the traditional methods of sensing such as HPLC, mass spectroscopy, or antibodies, aptamers are cheaper, easier to transport and use, have longer shelf lives, and can have a wider range of targets. This focus of this thesis is to use a method called capture-SELEX to isolate new aptamers for a few important antibiotics. While aptamers have been reported for them, they were mostly obtained by the immobilization of target molecules. In capture-SELEX, the DNA library is immobilization allowing the use of free target molecules. In Chapter 1, background information is given about nucleic acid structure, DNA, and the current state of aptamers. The SELEX process is also discussed in detail as well as some characterization methods and sensor applications. In Chapter 2, a new DNA aptamer for the family of tetracycline antibiotics was selected using capture-SELEX and oxytetracycline (OTC) as the target. This new aptamer was called OTC5 and had a dissociation constant (Kd) of 150 nM OTC measured using ITC. This aptamer could also enhance the intrinsic fluorescence of the tetracycline antibiotics and this property could be exploited for label free and dye free sensing. Follow-up studies were done on the OTC5 aptamer. vii It was found that metal ions (specifically Mg2+) had an effect on the binding of OTC5 to the tetracyclines. pH also affected binding with pH 6 promoting binding more than higher pH values. Studies were also done on splitting the OTC5 aptamer. The split aptamer retained its binding with doxycycline. In Chapter 3, from the remaining SELEX pool of the selection in Chapter 2, 10 other aptamers were identified to have binding with the tetracyclines. Some of these could distinguish between tetracycline, doxycycline, and oxytetracycline which led to the development of an aptamer sensor array that could differentiate these antibiotics with statistical significance. In Chapter 4, a new aptamer called CAP1 was selected for chloramphenicol (CAP) using capture- SELEX. Previous aptamers for CAP were selected by target immobilization which omitted a portion of the CAP molecule. When subjected to ITC, this previous aptamer did not show any indications of binding to the full CAP molecule. The newly selected CAP1 showed a fitted Kd of 9.8 μM using ITC. A sensor was also developed using Thioflavin-T fluorescence and had a limit of detection of 1.8 μM in lake water and 3.8 μM in wastewater. In Chapter 5, the effect of pH on aptamer selection was studied by a parallel selection at pH 6 and pH 8 for the aminoglycoside antibiotic kanamycin A. The selection at pH 6 showed better convergence than pH 8 and yielded an aptamer (KAN6-1). This aptamer had a Kd of around 300 nM from ITC. pH and salt studies were done using Thioflavin-T fluorescence assays and the optimal condition for binding was at pH 6 with no added salts. When KAN6-1 was tried with thioflavin-T under pH 8 selection conditions, there was no evidence of any binding. A sensor was then designed using KAN6-1 and had a limit of detection of 0.1 μM in lake water.