Tetracycline Aptamer Research Articles

Highly sensitive tetracycline detection using a triple amplification aptasensor based on graphene-carbon nanotube, Exonuclease I, and gold nanoprobes

Enhancing the sensitivity of targeted substance detection is crucial, yet prior research seldom incorporates more than two signal amplification methods. In this study, we introduced a novel triple signal amplification strategy for tetracycline detection using an aptasensor. This strategy integrates a graphene and multi-walled carbon nanotubes composite (GO-MWCNTs), Exonuclease I (Exo I), and a hybrid DNA-gold nanoparticle (AuNPs)-horseradish peroxidase (HRP) system. The GO-MWCNTs serve as a conductive carrier, boosting electron transfer for initial signal amplification. Exo I, targeting single-stranded DNA, facilitates target recovery and secondary signal amplification. The gold nanoprobe, through specific base pairing, binds to the tetracycline aptamer’s complementary chains on the electrode surface. Horseradish peroxidase’s catalytic action then generates a robust electrochemical signal, culminating in three-stage signal amplification. This optimized approach achieved a low detection limit of 3.3 × 10−4 ng mL−1, with a range from 1 × 10−3 ng mL−1 to 1 × 103 ng mL−1. Notably, the aptasensor demonstrated high selectivity, repeatability, stability, and reliability. These findings offer a promising reference for developing effective aptasensors in antibiotic detection.

Electrochemical Determination of Tetracycline Using the Synergy of a Molybdenum (IV) Sulfide-Thionine Nanocomposite with an Aptamer by Differential Pulse Voltammetry (DPV)

An electrochemical aptasensor was established for the fast and sensitive determination of tetracycline based on the synergistic effect of MoS2-thionine nanocomposite and aptamer. The glassy carbon electrode was coated with MoS2 nanosheets of thionine to immobilize the tetracycline aptamer. In the absence of tetracycline, large quantities of thionin were loaded on the surface of the MoS2 nanosheet to serve as the electrochemical probe. After tetracycline was added to the surface of the electrode, an aptamer analyte complex was produced on the modified electrode. Through intercalation and electrostatic interactions, the complex was substantially adsorbed onto thionine, and the peak currents of the redox signal of thionine decreased. Therefore, the concentration of tetracycline was monitored by the change in signal intensity. Under optimized conditions, the developed electrochemical strategy with MoS2-thionine composite and aptamer exhibited an excellent linear detection range of 1.0 nM–1.0 μΜ and a low detection limit of 0.6 nM with suitable selectivity and stability. Therefore, the fabricated platform offers great potential for food safety, medical detection, and drug analysis.

Electrochemically synthesized polyanine@Cu-BTC MOF as a bifunctional matrix for aptasensing of tetracycline in aquatic products

The development of new method for the rapid and facile screening of antibiotics residues in food is of great importance in food safety. Herein, a nanocompisite of polyaniline@copper-1,3,5-benzenetricarboxylic acid (PAN@Cu-BTC) with excellent electrochemical activity was directly prepared on the screen-printed electrode surface by a two-step electrochemical synthesis method. Then the tetracycline aptamer (TC-Apt) was assembled onto the electrode surface through the coordination between 5′-PO43- on the aptamer and Cu2+ on the MOF, achieving a novel electrochemical aptasensor for tetracycline (TC). The PAN@Cu-BTC has two functions in the aptasensors, namely the immobilization matrix of the aptamer probe and the internal signal source for target recognition. The electrochemical sensing assays showed that the capture of TC-Apt to the target on the electrode surface could cause the decrease of the electrochemical signal of PAN@Cu-BTC. Based on this, the TC can be analyzed in a wide range from 10 pM to 1 µM, with a low detection limit of 0.32 pM. The recoveries of the aptasensor for the detection of TC from Ctenopharyngodon idella meat were 90.0 %∼102.0 %, indicating that the constructed aptasensor is applicable for the detection of TC residues in fresh aquatic products.

Octahedral Cu2O nanomaterials as electrochemical aptasensor for sensitive detection of tetracycline in milk

In this work, A novel label-free electrochemical biosensor based on octahedral Cu2O @ Au nanocomposites was developed for the detection of tetracycline. The gold nanoparticles (AuNPs) on the surface of Cu2O nanomaterials not only improve the electrochemical performance, but also can be used as a binding site for thiol-modified tetracycline aptamers, which can specifically bind to tetracycline. Cu2O @ Au nanocomposites provide a synergistic effect of electrochemical signal amplification and tetracycline recognition strategy. Under the optimal conditions, the proposed biosensor exhibited different electrochemical reactions for different concentrations of tetracycline, with a linear range of 1 nM-1000 μM and a detection limit of 0.16 nM. This work provides a new idea for rapid and sensitive detection of tetracycline by using a simple and economical tetracycline aptamer combined with Cu2O @ Au biosensor platform.

Magnesium Ion-Driven Folding and Conformational Switching Kinetics of Tetracycline Binding Aptamer: Implications for in vivo Riboswitch Engineering

Engineering in vitro selected RNA aptamers into in vivo functional riboswitches represents a long-standing challenge in molecular biology. The highly specific aptamer domain of the riboswitch undergoes a conformational adjustment in response to ligand sensing, which in turn exerts the regulatory function. Besides essential factors like structural complexity and ligand binding kinetics, the active role of magnesium ions in stabilizing RNA tertiary structures and assisting in ligand binding can be a vital criterion. We present spectroscopic studies on the magnesium ion-driven folding of the Tetracycline binding aptamer. Using fluorescent labels, the aptamer pre-folding and subsequent ligand binding is monitored by magnesium titration experiments and time-resolved stopped-flow measurements. A minimum concentration of 0.5 mM magnesium is required to fold into a magnesium ion-stabilized binding-competent state with a preformed binding pocket. Tetracycline binding causes a pronounced conformational change that results in the establishment of the triple helix core motif, and that further propagates towards the closing stem. By a dynamic acquisition of magnesium ions, a kink motif is formed at the intersection of the triple helix and closing stem regions. This ultimately entails a stabilization of the closing stem which is discussed as a key element in the regulatory function of the Tetracycline aptamer.

Aptamer tuned nanozyme activity of nickel-metal–organic framework for sensitive electrochemical aptasensing of tetracycline residue

It is urgently needed to develop high-performance method for tetracycline (TC) analysis to meet the growing concerns about food safety. Herein, a MOF of Ni2+-2,3,6,7,10,11-hexahydroxytriphenylene (Ni-HHTP) with peroxidase activity has been prepared and coated on a screen printing electrode, followed by non-covalent adsorption of tetracycline aptamer (TC-Apt) through the π-stacking. The spectroscopic and electrochemical assays show that TC-Apt can effectively enhance the nanozyme activity of Ni-HHTP using 3,3′,5,5′-tetramethylbenzidine (TMB)/H2O2 as the probe. Upon binding with TC, the configuration of TC-Apt is changed and desorbs from the Ni-HHTP, resulting in the decrease of the nanozyme activity of aptasensor. Based on this principle, the target TC can be analyzed in concentrations ranging from 10 pM to 1.0 μM, with a detection limit of 1.9 pM. The aptsensor is also applicable for TC analysis in fresh Ctenopharyngodon idella meat and milk, which provides a new approach for TC residue monitoring in food.

A novel metal–organic frameworks composite-based label-free point-of-care quartz crystal microbalance aptasensing platform for tetracycline detection

A novel label-free point-of-care quartz crystal microbalance (QCM) aptasensing platform based on metal–organic frameworks (MOFs) and gold nanoparticles (AuNPs) was fabricated for tetracycline (TC) detection. MOFs (HKUST-1) and AuNPs were modified onto the sensing interface of QCM sensor to enhance the sensing performance of the QCM aptasensor. TC aptamer with sulfhydryl group was fixed through Au-S bond. The recognition performance of the aptasensor was predicted and verified by the computer simulation. At the optimal conditions, the frequency change of the sensor was adopted for quantitative detection of TC. The prepared QCM aptasensor exhibited a wide linear range from 1 × 10−10 g mL−1 to 1 × 10−5 g mL−1 with low limit of detection (0.8 × 10−11 g mL−1). High sensitivity, good selectivity, acceptable recoveries (87.6–91.4%) in real samples were obtained. For the first time, MOFs were utilized in the construction of QCM aptasensing platform, providing a promising application way of MOFs in the QCM sensing.

An upconversion nanosensor for rapid and sensitive detection of tetracycline in food based on magnetic-field-assisted separation

Tetracycline, a broad-spectrum antibiotic, has been widely used in disease treatment and other fields. However, due to the unreasonable use, its residue remains in food which eventually harms human health. Here described an upconversion nanosensor for tetracycline detection based on magnetic separation and electrostatic adsorption. To identify tetracycline, tetracycline aptamer, and europium ions (Eu3+) were introduced in the system. According to the electrostatic adsorption principle, Eu3+ exposed core–shell UCNPs were bound to negative complex of magnetic nanoparticles (MNPs) and aptamer. In the presence of tetracycline, UCNPs separated with MNPs-aptamer and remained in the supernatant by an external magnetic field. Under optimal conditions, the linear detection range of tetracycline was 0.5–1000 ng·mL−1, and the detection limit was 0.17 ng·mL−1. It has been successfully applied to detect tetracycline in food samples. The constructed method provided broad prospects for tetracycline detection with the merits of simple operation, high sensitivity, excellent repeatability, and selectivity.

Aptamer-Pendant DNA Tetrahedron Nanostructure Probe for Ultrasensitive Detection of Tetracycline by Coupling Target-Triggered Rolling Circle Amplification.

Tetracycline (TET) is a broad-spectrum antibiotic, which is frequently used in the prevention and treatment of animal diseases, feed additives, and so on. However, its residue and accumulation in animal-derived foods could cause several side effects to the human body. Herein, we fabricated TET aptamer-pendant DNA tetrahedral nanostructure-functionalized magnetic beads (Apt-tet MBs) as a probe to detect TET. In the presence of target TET, DNA primer was released from Apt-tet MBs since the TET aptamer could specifically bind TET. Next, the separated DNA primer could effectively initiate rolling circle amplification (RCA) reaction and generate a long tandem single-stranded sequence. Finally, with SYBR Green I as the fluorescence dye, the fluorescence signal could be detected by detection probes through hybridizing the RCA product. Under optimal conditions, the fluorescent signal increased with the increasing target TET concentration within the 5 orders of magnitude dynamic range from 0.001 to 10 ng mL-1. The detection limit was calculated to be 0.724 pg mL-1 and the method showed high selectivity toward TET among different antibiotics. More impressively, this method was employed for TET determination in fish and honey samples. The as-obtained results were consistent with those of ELISA kits, holding great potential in the field of food analysis.

Selection of High-Affinity RNA Aptamers That Distinguish between Doxycycline and Tetracycline.

Oligonucleotide aptamers are found in prokaryotes and eukaryotes, and they can be selected from large synthetic libraries to bind protein or small-molecule ligands with high affinities and specificities. Aptamers can function as biosensors, as protein recognition elements, and as components of riboswitches allowing ligand-dependent control of gene expression. One of the best studied laboratory-selected aptamers binds the antibiotic tetracycline, but it binds with a much lower affinity to the closely related but more bioavailable antibiotic doxycycline. Here we report enrichment of doxycycline binding aptamers from a selectively randomized library of tetracycline aptamer variants over four selection rounds. Selected aptamers distinguish between doxycycline, which they bind with dissociation constants of approximately 7 nM, and tetracycline, which they bind undetectably. They thus function as orthogonal complements to the original tetracycline aptamer. Unexpectedly, doxycycline aptamers adopt a conformation distinct from that of the tetracycline aptamer and depend on constant regions originally installed as primer binding sites. We show that the fluorescence emission intensity of doxycycline increases upon aptamer binding, permitting their use as biosensors. This new class of aptamers can be used in multiple contexts where doxycycline detection, or doxycycline-mediated regulation, is necessary.

Cyclodextrin subject-object recognition-based aptamer sensor for sensitive and selective detection of tetracycline

Based on a β-cyclodextrin (β-CD) subject-object competition model, a simple and sensitive electrochemical aptamer sensor for the determination of tetracycline (TET) was fabricated. First, the TET aptamer modified with ferrocene (Fc) as a signal molecule was captured by β-CD loaded onto the gold electrode. Subsequently, TET was added to the detection system, causing a binding event between the target and aptamer with strong affinity during which process the aptamer configuration was changed from the original upright linear state to an agglomeration structure, resulting in its departure from the electrode surface. Consequently, an “off-signal” was turned along with the presence of the target. The results indicated that the TET concentration had a linear response to the signal ranging from 0.01 to 100 nM and the accurate detection limit could reach as low as 0.008 nM (3δ). The fabricated TET biosensor also showed outstanding detection specificity. Moreover, the suitability of the developed method was demonstrated in the determination of TET concentrations in different samples comprising water, milk, and bacteria culture medium, achieving acceptable recoveries for spiked samples ranging from 96.0 to 104.4%. This detection system was simple, economical, time-saving but remarkably sensitive, selective, and efficient, potentially rendering services in food safety screening and medical testing.

A nanocomposite prepared from bifunctionalized ionic liquid, chitosan, graphene oxide and magnetic nanoparticles for aptamer-based assay of tetracycline by chemiluminescence.

A nanocomposite was prepared from a bifunctionalized ionic liquid, chitosan on magnetic nanoparticle-modified graphene oxide (IL/Chit@MGO). It was used in a chemiluminescencc (CL) assay for tetracycline. The materials were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray powder diffraction, nitrogen adsorption-desorption isotherm, vibrating sample magnetometry and zeta potentials. Subsequently, a tetracycline-binding aptamer (TC-Apt) acting as a recognition element, and G-quadruplex DNAzyme (G-DNAzyme) acting as a signal amplification component were modified on IL/Chit@MGO. So, the bifunctional G-DNAzyme/TC-Apt/IL/Chit@MGO was prepared. The IL/Chit@MGO is found to possess excellent loading capability for TC-Apt. This is attributed to the large specific surface and abundant charge on the surface of IL/Chit@MGO. The composite was used to construct a CL assay for tetracycline. Tetracycline binds to TC-Apt and causes the release of the G-DNAzyme. The latter catalyzes the CL of luminol-H2O2 CL system at pH7.4. Under optimized conditions, the blue CL at the emission wavelength of 425nm increases linearly in the 0.16 pM to 2.0nM concentration range, and the detection limit is 21 fM (at 3σ). The assay is selective, reproducible and stable. The assay was applied to tetracycline detection in practical samples. The apparent recoveries are 98.0% to 101.3% for the milk sample and 97.0% to 102.2% for the water sample. Graphical abstractG-quadruplex DNAzyme (G-DNAzyme) and tetracycline aptamer (TC-Apt) bifunctionalized ionic liquid/chitosan@magnetic graphene oxide (IL/Chit@MGO) was prepared. The nanocomposite was used to construct a chemiluminescence (CL) assay for tetracycline.

Highly selective and sensitive streptomycin chemiluminescence sensor based on aptamer and G-quadruplex DNAzyme modified three-dimensional graphene composite

In our work, a highly selective and sensitive streptomycin (STR) chemiluminescence (CL) sensor was successfully prepared based on aptamer and G-quadruplex DNAzyme (G-DNAzyme) modified three-dimensional graphene composite. Initially, β-cyclodextrins and ionic liquids functionalized graphene oxide aerogel (β-CD/IL@GOGA) was successfully prepared and characterized, in which graphene oxide aerogel (GOGA) was used as a skeleton material and provided large specific surface area, β-CD rich in hydroxyl groups and IL containing lots of stable ions increased the biocompatibility and stability of the composite, respectively. Then, tetracycline aptamer (S-Apt) and G-DNAzyme was immobilized on the surface of β-CD/IL@GOGA, consecutively. Finally, G-DNAzyme/S-Apt/β-CD/IL@GOGA was used to construct the CL sensor for STR detection. S-Apt with specific recognition ability to its target and G-DNAzyme as a catalyst of luminol-H2O2 CL system improved the selectivity and sensitivity of the sensor, respectively. When STR existed, G-DNAzyme was released from the surface of S-Apt/β-CD/IL@GOGA due to the specific recognition and binding ability between STR and S-Apt, catalyzing the CL reaction. The CL sensor showed the linear range of 1.4 × 10−12 to 2.8 × 10−9 mol/L and detection limit of 9.2 × 10-14 mol/L (3δ). It was successfully used for STR detection in cucumber and milk samples.

Conditional Regulation of Gene Expression by Ligand-Induced Occlusion of a MicroRNA Target Sequence.

RNA switches that modulate gene expression with small molecules have a number of scientific and clinical applications. Here, we describe a novel class of small regulatory on switches based on the ability of a ligand-bound aptamer to promote stem formation between a microRNA target sequence (miR-T) and a complementary competing strand. Two on switch architectures employing this basic concept were evaluated, differing in the location of a tetracycline aptamer and the region of a miR-21 target sequence (miR-21-T) masked by its competing strand. Further optimizations of miR-21-T and its competing strand resulted in tetracycline-regulated on switches that induced luciferase expression by 19-fold in HeLa cells. A similar switch design based on miR-122-T afforded 7-fold regulation when placed in tandem, indicating that this approach can be extended to additional miR-T. Optimized on switches introduced into adeno-associated virus (AAV) vectors afforded 10-fold regulation of two antiviral proteins in AAV-transduced cells. Our data demonstrate that small-molecule-induced occlusion of a miR-T can be used to conditionally regulate gene expression in mammalian cells and suggest that regulatory switches built on this principle can be used to dose expression of an AAV transgene.

A small, portable RNA device for the control of exon skipping in mammalian cells.

Splicing is an essential and highly regulated process in mammalian cells. We developed a synthetic riboswitch that efficiently controls alternative splicing of a cassette exon in response to the small molecule ligand tetracycline. The riboswitch was designed to control the accessibility of the 3′ splice site by placing the latter inside the closing stem of a conformationally controlled tetracycline aptamer. In the presence of tetracycline, the cassette exon is skipped, whereas it is included in the ligand's absence. The design allows for an easy, context-independent integration of the regulatory device into any gene of interest. Portability of the device was shown through its functionality in four different systems: a synthetic minigene, a reporter gene and two endogenous genes. Furthermore, riboswitch functionality to control cellular signaling cascades was demonstrated by using it to specifically induce cell death through the conditionally controlled expression of CD20, which is a target in cancer therapy.

A Label-Free Electrochemical Aptasensor for the Rapid Detection of Tetracycline Based on Ordered Mesoporous Carbon–Fe3O4

A novel aptasensor based on a tetracycline (TET) aptamer immobilized by physical adsorption on an ordered mesoporous carbon–Fe3O4 (OMC-Fe3O4)-modified screen-printed electrode surface was successfully fabricated. OMC-Fe3O4 was characterized by scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The modification procedure of the aptasensor was characterized by cyclic voltammetry. Interaction between the TET aptamer and target was determined by differential pulse voltammetry. Under optimal conditions, the proposed aptasensor exhibited good electrochemical sensitivity to TET in a concentration range of 5 nM to 10 μM, with a detection limit of 0.8 nM (S/N = 3). This aptasensor exhibited satisfactory specificity, reproducibility, and stability.

Electrochemical aptasensor based on a novel flower-like TiO2 nanocomposite for the detection of tetracycline

Abstract Herein, we developed a sensitive sensing platform for tetracycline detection by combining the specific DNA aptamers with a novel TiO2 nanocomposite. In this composite, cylinder-shaped TiO2 nanorods were synthesized on MoS2 nanospheres by a facile hydrothermal method to produce a MoS2-TiO2 composite, which was then aminated. Following that, gold nanoparticles (Au) were deposited on the aminated MoS2-TiO2 to form MoS2-TiO2@Au composite, on which thiolated DNA aptamers were applied to construct an electrochemical aptasensor. The tetracycline aptamer could well hybridize to its complementary biotin-DNA oligonucleotide (bio-cDNA), and then avidin-horseradish peroxidase (avidin-HRP) mediated by H2O2 was introduced to produce amplified electrochemical response. The competition between tetracycline and bio-cDNA for binding of the aptamer would decrease the electrochemical signal, which was used for quantitative detection of tetracycline. The prepared aptasensor showed a wide linear range from 1.5 × 10−10 to 6.0 × 10−6 M, and a low detection limit of 5 × 10−11 M.

An interdigital array microelectrode aptasensor based on multi-walled carbon nanotubes for detection of tetracycline.

In this study an impedance aptasensor was designed for sensitive, selective, and fast detection of tetracycline (TET) based on an interdigital array microelectrode (IDAM). The IDAM was integrated with impedance detection to miniaturize the conventional electrodes, enhance the sensitivity, shorten the detection time, and minimize interfering effects of non-target analytes in the solution. Due to their excellent conductivity, good biocompatibility, the multi-walled carbon nanotubes (MWCNTs) were used to modify the IDAM to immobilize TET aptamer effectively. The proposed aptasensor produced a sensitive impedance change which was characterized by the electrochemical impedance spectroscopy (EIS). With the addition of TET, the formation of TET-aptamer complex on the surface of MWCNTs modified electrode resulted in an increase of electron transfer resistance (R et). The change of R et depends on the concentration of TET, which is applied for the quantification of TET. A wide linear range was obtained from 10-9 to 10-3M. The linear regression equation was y(ΔR)=21.310×x(LogC)(M)+217.25. It was successfully applied to detect TET in real milk samples.

Use of the U1A Protein to Facilitate Crystallization and Structure Determination of Large RNAs.

The preparation of well-ordered crystals of RNAs with complex three-dimensional architecture can be facilitated by engineering a binding site for the spliceosomal protein U1A into a functionally and structurally dispensable stem-loop of the RNA of interest. Once suitable crystals are obtained, the U1A protein, of known structure, can be employed to facilitate preparation of heavy atom or anomalously scattering atom derivatives, or as a source of partial model phases for the molecular replacement method. Here, we describe the methods for making U1A preparations suitable for cocrystallization with RNA. As an example, the cocrystallization of the tetracycline aptamer with U1A is also described.

Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

A protein depletion by promoter shutoff or protein destabilization is an important tool in investigation of functions of essential genes. Various approaches using different repressible promoters, inducible degrons, or their combinations were developed. While successful, the current techniques have a drawback in that they require fusion of a large degradation tag to the target protein and/or a change in growth conditions to repress the promoter. We describe efficient protein depletion using the combination of a metabolically inert tetracycline repressible promoter with tetracycline aptamer and constitutive target protein destabilization by means of ubiquitin fusion. The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems. A depletion time of <40 min was sufficient to achieve a robust phenotype.