Binding Aptamer Research Articles

A Multifunctional Platform for the Capture, Release, And Enumeration of Circulating Tumor Cells Based on Aptamer Binding, Nicking Endonuclease-Assisted Amplification, And Inductively Coupled Plasma Mass Spectrometry Detection.

Inductively coupled plasma mass spectrometry (ICP-MS) combined with element tags has been well designed and extensively applied in cell enumeration. It possesses superior quantitative capability, strong resistance to matrix interference, multiplex detection capability but destructive characteristic. Herein, we constructed an ICP-MS based multifunctional platform for capture, nondestructive enumeration, and release of circulating tumor cells (CTCs). Aptamer on capture probe recognized Mucin 1 (MUC1) on membrane of MCF-7 cells specifically, thus the cells were captured by probe and the Initiator originally hybridized with Aptamer was substituted by MUC1 and released into solution. Then the released Initiator was separated from the captured cells and hybridized with Tb labeled Substrate on detection probe to release a large amount of nicked Tb fragments through the nicking endonuclease assisted amplification for subsequent ICP-MS detection. Meanwhile, cells captured by probe were released by nuclease digestion for further reculture. Such a strategy effectively avoids CTCs destruction resulted from ICP-MS enumeration, increases the detection sensitivity of ICP-MS by involving nicking endonuclease assisted amplification, and realizes cell recovery for further analysis. A limit of detection of 87 MCF-7 cells and a linear range of 250-10 000 MCF-7 cells were realized for ICP-MS enumeration. A cell recovery of 52.7% (with capture and release efficiency of 63.9 and 82.5%, respectively) and a viability of 74.3% were obtained, meanwhile the released cells exhibited strong proliferation ability. This multifunctional platform for CTCs capture, enumeration, and release has great applicable potential in early diagnosis and individual treatment for cancer.

Flexible flower-like MOF of Cu2(trans-1,4-cyclohexanedicarboxylic acid)2 as the electroactive matrix material for label-free and highly sensitive sensing of thrombin

A flexible metal-organic framework (MOF) of Cu2(trans-1,4-cyclohexanedicarboxylic acid)2 (Cu2(CHDC)2) has been synthesized by a facile hydrothermal method. The physical characterization experiments show that the Cu2(CHDC)2 presents unique flower-like shape, large surface area and high electroactivity. Then gold nanoparticles (AuNPs)/Cu2(CHDC)2 modified electrode was prepared and utilized as an electroactive matrix for thrombin aptamer immobilization. The electrochemical assay shows that the AuNPs can effectively enhance the electrochemical signal of the Cu2(CHDC)2. Upon the binding of the electrode-confined aptamer with the thrombin, the electrochemical signal of the Cu2(CHDC)2 decreases obviously due to the change of the microenvironment of the sensing interface. Thus a label-free electrochemical sensing strategy for thrombin was realized. The aptasensor displays excellent performance with wide linear range, ultralow detection limit of 0.01 fM (S/N = 3), and favorable specificity. Also, the aptasensor was applied for determination of thrombin in the human serum samples, and the satisfactory results are obtained. The results imply that the proposed sensor can be potentially used for the early diagnosis of disease and clinical monitoring.

Dopamine and Melamine Binding to Gold Nanoparticles Dominates Their Aptamer-Based Label-Free Colorimetric Sensing.

Target-directed aptamer adsorption by gold nanoparticles (AuNPs) has been widely used to develop label-free colorimetric biosensors. However, the potential interactions between target molecules and AuNPs have not been considered, which may lead to misinterpretation of analytical results. In this work, the detection of dopamine, melamine, and K+ was studied as model systems to address this problem. First, dopamine and two control molecules all induced the aggregation of citrate-capped AuNPs with apparent Kd's of 5.8 μM dopamine, 51.6 μM norepinephrine, and 142 μM tyramine. Isothermal titration calorimetry measured the aptamer Kd to be 1.9 μM dopamine and 16.8 μM norepinephrine, whereas tyramine cannot bind. Surface enhanced Raman spectroscopy confirmed direct adsorption of dopamine, and the adsorbed dopamine inhibited the adsorption of DNA. Using a typical salt-induced colorimetric detection protocol, a similar color response was observed regardless of the sequence of DNA, indicating the observed color change reflected the adsorption of dopamine by the AuNPs instead of the binding of dopamine by the aptamer. For this label-free sensor to work, the interaction between the target molecule and AuNPs should be very weak, while dopamine represents an example of strong interactions. For the other two systems, the melamine detection did not reflect aptamer binding either but the K+ detection did, suggesting melamine also strongly interacted with AuNPs, whereas K+ had very weak interactions with AuNPs. Since each target molecule is different, such target/AuNP interactions need to be studied case-by-case to ensure the sensing mechanism.

Probing high-affinity aptamer binding region and development of aptasensor platform for the detection of cylindrospermopsin

Cylindrospermopsin (CYN) is one of the most concerning cyanotoxins due to its potential toxicity and spreading to various environments including drinking water. CYN has potential interferences with human and animal metabolic pathways, which influence the functions of organs including liver, kidneys, lungs, etc. CYN is involved in the inhibition of protein synthesis and detachment of ribosomes from the endoplasmic reticulum membrane. It also interacts with soluble proteins, which are associated with protein translations. It is believed that cytochrome 450 is responsible for the rapid toxicity of CYN. Researchers are urged to develop a high-throughput screening method for the detection of CYN in water. Construction of low cost, rapid, and sensitive analytical methods for the detection of CYN is challenging. Here, we used graphene oxide (GO) as the fluorescence sensing platform for probing the high affinity of the short aptamer derived from the wild-type long aptamer-CYN sensing. The biosensor construction involved two steps: first, quenching the fluorescence of fluorescent-labelled truncated aptamer using GO as a quencher and, second, fluorescence recovery in the presence of CYN by competitive binding between the target and GO. One of the truncate aptamers has a 12-fold higher affinity and enhances sensitivity compared to the long aptamer sequence. The limit of detection of the high affinity truncated aptamer is 17pM which is 6-fold lower than the long aptamer (100pM). The sensor specifically detects CYN in the presence of other potential interfering toxins. The performance of the sensor was validated using CYN spiked tap water with very good recovery percentage. A rapid and highly sensitive detection of CYN from water resources has been achieved using this method.

Study on the Method of Isolating the Aptamer from the Surface of HepG2 Cells

The hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver. It is a common malignant tumor in clinic. The main reason for its high mortality is its early latency. Therefore, how to accurately determine and test the hepatocellular carcinoma in the early stage has a very positive significance for the treatment. It is an important method for the early diagnosis of the hepatocellular carcinoma to use aptamers specifically binding to hepatocellular carcinoma cells, which has good application prospects. In order to improve the efficiency of aptamer selection of tumor cells, our group designed and developed an automated instrument for the aptamer selection. In this paper, the method to separate bound aptamers from the surface of HepG2 cells in automated selection process was studied, and the feasibility of separating binding aptamers from the HepG2 cell surface using ultrapure water and the effect of different temperature environments on its isolation were discussed. Results of the real-time fluorescent PCR and flow cytometry showed that ultrapure water could be used to isolate bound HepG2 cells and aptamers, and the concentration of the aptamers increased with the rise of the temperature between 25 and 80 degrees Celsius. This result will contribute to the improvement on the efficiency of automated selections for aptamers corresponding to HepG2 cells.

A practical aptaprobe for sulfadimethoxine residue detection in water and fish based on the fluorescence quenching of CdTe QDs by poly(diallyldimethylammonium chloride)

A practical aptaprobe for sulfadimethoxine (SDM) detection was established. Based on the electrostatic reaction, a duplex of aptamer and poly(diallyldimethylammonium chloride) (PDDA) was formed, which prevented the quenching effect of PDDA on the fluorescence of CdTe quantum dots (QDs). But the specifically binding of aptamer to SDM might result in free PDDA released and the fluorescence was quenched. Based on the fluorescence aptaprobe, SDM was quantitatively detected by measuring the fluorescence at 530 nm with a linear range of 25–300 ng mL−1, and the limit of detection was 2.24 ng mL−1 based on 3σ/K (n = 9). The aptaprobe was applied to SDM detection with recoveries of 94.2 %–113 % and 104 %–118 % for seawater and fish, respectively. The results showed that the fluorescence aptaprobe was simple and convenient for the rapid detection of SDM residue in water and fish samples.

Molecular dynamics study of the recognition of ATP by nucleic acid aptamers.

Despite their great success in recognizing small molecules in vitro, nucleic acid aptamers are rarely used in clinical settings. This is partially due to the lack of structure-based mechanistic information. In this work, atomistic molecular dynamics simulations are used to study the static and dynamic supramolecular structures relevant to the process of the wild-type (wt) nucleic acid aptamer recognition and binding of ATP. The effects brought about by mutation of key residues in the recognition site are also explored. The simulations reveal that the aptamer displays a high degree of rigidity and is structurally very little affected by the binding of ATP. Interaction energy decomposition shows that dispersion forces from π-stacking between ATP and the G6 and A23 nucleobases in the aptamer binding site plays a more important role in stabilizing the supramolecular complex, compared to hydrogen-bond interaction between ATP and G22. Moreover, metadynamics simulations show that during the association process, water molecules act as essential bridges connecting ATP with G22, which favors the dynamic stability of the complex. The calculations carried out on three mutated aptamer structures confirm the crucial role of the hydrogen bonds and π-stacking interactions for the binding affinity of the ATP nucleic acid aptamer.

Mapping the binding region of aptamer targeting small molecule: Dabigatran etexilate, an anti-coagulant

Aptamers are single-stranded DNA or RNA, which have attracted considerable scientific interest due to their characteristic of specific and selective binding to target molecules. They are evolved from the in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). This paper reports a simple experimental approach to elucidate the binding region of small targets binding aptamers. A previously isolated 60-mer aptamer for the anti-coagulant dabigatran etexilate (DBG) was used for this investigation. Complimentary sequences labelled with a fluorophore and a quencher were used for testing the binding region by change in the fluorescence signal. The full-length aptamer was truncated to multiple shorter copies including a 38 nucleotides sequence that showed 47 fold high affinity compared to the original aptamer. Circular dichroism spectroscopy (CD) measurements indicate that the 38-mer is remarkably more sensitive than the parent aptamer. The truncated 38-mer sequence was used to construct a turn-on fluorescence sensor with the detection limit of 1 nM. The performance of the sensor was examined in blood serum samples and showed excellent recovery percentages exceeding 98%. The reported screening protocol could be applied to the growing small targets aptasensors that require efficient binding aptamer sequences coupled with optimum signal transduction methods.

Phthalocyanines for G-quadruplex aptamers binding.

The G-quadruplex (G4)-forming sequence within the AS1411 derivatives with alternative nucleobases and backbones can improve the chemical and biological properties of AS1411. Zn(II) phthalocyanine (ZnPc) derivatives have potential as high-affinity G4 ligands because they have similar size and shape to the G-quartets. The interactions of four Zn(II) phthalocyanines with the G4 AS1411 aptamer and its derivatives were determined by biophysical techniques, molecular docking and gel electrophoresis. Cell viability assay was carried out to evaluate the antiproliferative effects of Zn(II) phthalocyanines and complexes. CD experiments showed structural changes after addition of ZnPc 4, consistent with multiple binding modes and conformations shown by NMR and gel electrophoresis. CD melting confirmed that ZnPc 2 and ZnPc 4, both containing eight positive charges, are able to stabilize the AT11 G4 structure (ΔTm>30°C and 18.5°C, respectively). Molecular docking studies of ZnPc 3 and ZnPc 4 suggested a preferential binding to the 3'- and 5'-end, respectively, of the AT11 G4. ZnPc 3 and its AT11 and AT11-L0 complexes revealed pronounced cytotoxic effect against cervical cancer cells and no cytotoxicity to normal human cells. Zn(II) phthalocyanines provide the basis for the development of effective therapeutic agents as G4 ligands.

(Invited) Near Infrared Imaging of Chemical Signaling between Cells Using Fluorescent Nanosensors

Cells use small molecules to exchange information. A very prominent example is neurotransmitter signaling between neuronal cells or paracrine signaling between blood cells. However, it is up to date difficult to visualize signaling processes between cells with high spatial, temporal and chemical resolution. We are interested in developing fluorescent sensors/probes that overcome these limitations and answer novel biological questions.For that purpose we use near infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs). SWCNTs fluoresce in the nIR tissue transparency window (900 nm -1700 nm) and do not bleach. They are chemically modified to render them selective for neurotransmitters such as dopamine and serotonin. In this context, we developed different chemical strategies that include screening for artificial binding motifs, aptamers and nanobodies. Using these strategies we created sensors for the neurotransmitters dopamine and serotonin that increase their fluorescence in the presence of the target molecule and detect down to single molecules. Fast parallel imaging of many of those sensors provides a nIR image (>900 nm) of the neurotransmitter concentration around cells. This technique was used to image hot spots of dopamine release from primary dopaminergic neurons. It enabled us to observe localized release sites and correlate it with the exocytosis machinery of the cells. Neurotransmitters are not only relevant in the neuronal system but as well in the periphery e.g. in blood cells. In this context, we imaged for the first time how human blood platelets release serotonin after activation and resolved the heterogeneity in release patterns. These results illustrate that tailored SWCNTs are powerful tools for quantitative chemical imaging of biological processes.

G-quadruplex-based assay combined with aptamer and gold nanoparticles for Escherichia coli K88 determination.

A colorimetric method was developed usingG-quadruplex and gold nanoparticles (AuNPs) for determination of Escherichia coli K88 (ETEC K88). It was composed of two modules: (1) an aptamer as biorecognizing element and (2) a capturing DNA (modified with AuNPs at 5') as a transducer. In the absence of target bacteria, the aptamer can form stable double strands with capturing DNA, preventing the binding of capturing DNA to the G-quadruplex. However, the double strands of capturing DNA and aptamer are untied due to the stronger binding of aptamers to bacteria in the presence of target bacteria. As a result, the G-quadruplex binds to capture DNA and leads to the aggregation and color change of AuNPs, which can be monitored by a spectrophotometer or visualization. The quantitative determination was achieved by monitoring the optical density change of AuNPs solution at 524nm after target addition. Under optimal conditions, the method has a low detection limit (1.35 × 102CFUmL-1) and a linear response in the range102 to 106CFUmL-1. Graphical abstract The manuscripts describe a colorimetric method for the detection of ETEC K88 by using intermolecular G-quadruplex to induce the agglomeration of gold nanoparticles, which can be directly used to determine the presence of bacteria with our naked eyes.

Optical Detection of Thrombin on Nanoporous Anodic Alumina with Aptamer-Modified Surface

Nanoporous anodic alumina (NAA) attracts interest in nanotechnology[1]. Its physical and chemical properties combined with a cost-effective and scalable production make it a good for nanotechnology-related applications. Its high and tuneable surface-to-volume ratio as well as its interesting optical properties[2] have been used as the basis of different biosensing schemes. Biosensing, and specifically optical biosensing is of great interest in health and environmental applications. The reflection interference spectroscopy (RIfS) method based on NAA has demonstrated its ability in detecting many kinds of molecules [3,4].Biosensors engineered with aptamers as a bioreceptor are called aptabiosensors. Thrombin Binding Aptamer (TBA) has a well-known binding process and high affinity and is one of the most studied aptamers. Thrombin is the key factor of blood coagulation, whose activity is important in wounds and in blood circulation. The free thrombin-binding aptamer remains as a random-coil state in the absence of thrombin, while in the presence of thrombin the protein attaches to the aptamer changing its conformation to quadruplex. In this study we assess the capabilities of NAA with the inner pore surfaces modified with TBA to detect specifically the thrombin protein by means of the RIfS method.We prepared NAA with the usual anodization conditions under oxalic acid electrolyte to obtain porous layers with uniform pore diameter and with a thickness that permits the measurement with RIfS in a flow-cell where the different species in solution can be introduced. The experiments consisted of monitoring the change in effective optical thickness (EOT, the quantitative characteristic parameter obtained from RIfS) in the different steps of the biosensing process. Figure 1 shows SEM pictures of the NAA platforms prepared.The NAA pore surfaces were initially functionalized with (3-aminopropyl)triethoxysilane (APTES) and then the surface mas modified in a three-step procedure monitored in real time thanks to the RIfS method. Figure 2 shows the change in EOT as a function of time during the surface functionalization of NAA: the covalent attachment of Sulfo-NHS-Biotin and subsequent streptavidin attachment (stages 1 and 2 in the figure) and the final immobilization of TBA in the pore walls of NAA (step 3 in the plot).. First, the flow of the Sulfo-NHS-Biotin solution produced a considerable increase in EOT. Then, a further increase in EOT is observed with the infiltration of streptavidin. Finally, EOT also shows clearly the immobilization of biotinylated TBA.The aptamer-functionalized NAA substrates were employed to detect human thrombin protein. For this purpose, different substrates were used in RIfS experiments with human thrombin protein at different concentrations. Figure 3 shows one example of the evolution of the EOT signal upon infiltration of a 1.35 µM thrombin solution. Results show that after obtaining a baseline with constant EOT corresponding to the flow of binding buffer, the thrombin solution is injected causing a rapid increase in EOT, at a rate of 0,05 nm/s. After this increase a stable value is reached after 3600 s, with an absolute change in EOT of ∆EOT = 28 nm. The experiment corresponding to Figure 3 has been repeated for a set of concentrations to obtain a sensitivity curve of the aptamer-functionalized NAA and to determine the lower limit of detection. With this, the dissociation constant of the aptamer-thrombin reaction is determined as Kd = 0,9 µM, the sensitivity to be m = 45.5 nm/µM and the LOD = 72 nM.Finally, in order to demonstrate the specificity of the NAA platform, experiments replacing the flow of thrombin by the flow of a different but similar protein, Bovine Serum Albumin (BSA) were conducted showing a much lower response. Furthermore, thrombin flow experiments with NAA lacking the aptamer functionalization step were also performed to demonstrate the protein does not bind non-specifically to the NAA inner pore walls.[1] Josep Ferré-Borrull, et al., Nanomaterials, vol. 7, p. 5225 (2014).[2] Josep Ferré-Borrull et al., in NANOPOROUS ALUMINA: FABRICATION, STRUCTURE, PROPERTIES AND APPLICATIONS, Springer Series in Materials Science, vol. 219, p. 185 (2015).[3] Laura Pol et al., Nanomaterials, vol. 9, p.478 (2019).[4] Laura Pol et al, Sensors, vol. 19, p. 4543 (2019). Figure 1

Effects of Physical and Chemical Stressors on DNA Conformation Investigated By Temperature-Controlled Electrochemical Microdevices.

Effects of Physical and Chemical Stressors on DNA Conformation Investigated By Temperature-Controlled Electrochemical Microdevices.

Intercalative methylene blue as an efficient signal molecule of tremella-like Bi2WO6: Toward high performance photoelectrochemical biosensing

The signaling molecule is one of the vital factors that determines the detection mechanism/strategy and the resultant performances of photoelectrochemical (PEC) bioassays. This work describes the exploration of the DNA intercalator, i.e. methylene blue (MB), as an efficient signaling molecule for high performance PEC bioanalysis. The MB can stimulate a much larger photocurrent enhancement for tremella-like Bi2WO6 than the other most often used signaling molecules, hinting its higher competence as a signaling molecule. Exemplified by carcinoembryonic antigen (CEA) as a target, we achieved homogeneous and “signal-on” PEC aptasensing, in which the target (CEA)-aptamer binding inhibited the subsequent rolling circle amplification (RCA) reaction, and thus regulating the release/intercalation of MB (in G-quadruplex motif) for signal stimuli. Different from most of the reported anodic PEC bioanalysis with immobilized biomolecules on photoelectrodes, this homogeneous detection not only avoids the cumbersome and costly immobilization/labelling procedure but also facilitates signal transduction, resulting from the well-preserved accessibility and molecular orientation of the biomolecules in solution. This work features the exploration of a DNA intercalator as a signaling molecule for exquisite anodic PEC bioanalysis, which may spark more interests in the exploration of numerous other DNA intercalators for advanced PEC bioanalysis.

Continuous Detection of Increasing Concentrations of Thrombin Employing a Label-Free Photonic Crystal Aptasensor.

Thrombin generation is a complex and finely regulated pathway that provokes dynamical changes of thrombin concentration in blood when a vascular injury occurs. In order to characterize the initiation phase of such process, when thrombin concentration is in the nM range, a label-free optical aptasensor is proposed here. This aptasensor combines a 1D photonic crystal structure consisting of a silicon corrugated waveguide with thrombin binding aptamers on its surface as bioreceptors. As a result, this aptasensor has been demonstrated to specifically detect thrombin concentrations ranging from 270 pM to 27 nM with an estimated detection limit of 33.5 pM and a response time of ~2 min. Furthermore, it has also been demonstrated that this aptasensor is able to continuously respond to consecutive increasing concentrations of thrombin and to detect binding events as they occur. All these features make this aptasensor a good candidate to continuously study how thrombin concentration progressively increases during the initiation phase of the coagulation cascade.

Identifying conformational changes of aptamer binding to theophylline: A combined biolayer interferometry, surface-enhanced Raman spectroscopy, and molecular dynamics study

Theophylline is a potent bronchodilator for the treatment of asthma, bronchitis, and emphysema. Its narrow therapeutic window (20–100 μM) demands that the blood concentration of theophylline be monitored carefully, which can be achieved by aptamer capture. Thus, an understanding of what occurs when aptamers bind to theophylline is critical for identifying a high-affinity and high-specificity aptamer, which improve the sensitivity and selectivity of theophylline detection. Consequently, there is an urgent need to develop a simple, convenient, and nondestructive method to monitor conformational changes during the binding process. Here, we report the determination of the affinity of a selected aptamer and theophylline via biolayer interferometry (BLI) experiments. Additionally, using surface-enhanced Raman spectroscopy (SERS), the conformational changes on theophylline–aptamer binding were identified from differences in the SER spectra. Finally, molecular dynamics (MD) simulations were used to identify the specific conformational changes of the aptamer during the binding process. Such a combined BLI-SERS-MD method provides an in-depth understanding of the theophylline–aptamer binding processes and a comprehensive explanation for conformational changes, which helps to select, design, and modify an aptamer with high affinity and specificity. It can also be used as a scheme for the study of other aptamer–ligand interactions, which can be applied to the detection, sensing, clinical diagnosis, and treatment of diseases.

Bridging Fe3O4@Au nanoflowers and Au@Ag nanospheres with aptamer for ultrasensitive SERS detection of aflatoxin B1

Aflatoxin B1 (AFB1) as the most toxic mycotoxin in contaminated food can greatly threaten human health, and sensitive and selective detection of AFB1 is thus highly desired. An ultrasensitive surface-enhanced Raman spectroscopy (SERS) aptasensor was developed for AFB1 detection in peanut oil samples. SH-cDNA modified Fe3O4@Au nanoflowers acted as capture probes, SH-Apt modified Au@Ag nanospheres and commercial Cy3-Apt were used as reporter probes. Strong SERS signals of reporter probes were produced due to the recognition of AFB1 aptamer and its complementary strand (SH-cDNA). With the preferred binding of AFB1 aptamer to AFB1, reporter probes were released from capture probes, causing a linear decrease in SERS intensity. Therefore an ultralow detection limit of 0.40 pg·mL−1 in a wide linear range of 0.0001–100 ng·mL−1 was obtained and the sensibility of this SERS aptasensor was higher than that of the Cy3-Apt based SERS aptasensor. In addition, an excellent selectivity in interfering toxins and satisfactory recoveries of 96.6–115% in peanut oil samples were obtained, proving this aptasensor is a promising analytical tool in AFB1 detection.

Aptamer-Functionalized Three-Dimensional Carbon Nanowebs for Ultrasensitive and Free-Standing PDGF Biosensor.

Research on flexible biosensors is mostly focused on their use in obtaining information on physical signals (such as temperature, heart rate, pH, and intraocular pressure). Consequently, there are hardly any studies on using flexible electronics for detecting biomolecules and biomarkers that cause diseases. In this study, we propose a flexible, three-dimensional carbon nanoweb (3DCNW)-based aptamer sensor to detect the platelet-induced growth factor (PDGF), which is an oncogenic biomarker. As a template for the 3D structure, poly(acrylonitrile) (PAN) nanowebs were synthesized using a facile electrospinning process. The PAN nanowebs were then subjected to chemical vapor deposition with copper powder. This was followed by Cu etching to generate carbon protrusions on the web surface. As an active site, PDGF-B binding aptamer was introduced on the 3DCNW surface to form biosensor electrodes. The 3DCNW-based aptasensor exhibited excellent sensitivity (down to 1.78 fM), with high selectivity, reversibility, and stability to PDGF-BB.

Electrochemical determination of zearalenone using alabel-free competitive aptasensor.

An electrochemical aptasensor is described for determination of the phytohormone of zearalenone (ZEA). The gold electrode was modified with ZEA via covalent attachment using cysteamine-hydrochloride and 1,4-phenylene diisocyanate linker. A truncated ZEA aptamer with a dissociation constant of 13.4 ± 2.1nM was used in an aptasensor. The electrochemical property was investigated using square wave voltammetry for monitoring the change in the electron transfer using the ferro/ferricyanide system as redox probe. Under optimal experimental conditions, the response was best measured at apotentialof 0.20V (vs. Ag/AgCl). The signals depended on the competitive mechanism between the immobilised ZEA and free ZEA for the aptamer binding site. The aptasensor works in the range0.01 to 1000ng·mL-1 ZEA concentration, with a detection limit of 0.017ng·mL-1. High degree of cross-reactivity with the other analogues of ZEA was observed, whereas none towards other mycotoxins. The aptasensor was further applied for the determination of ZEA in the extract of maize grain and showed good recovery percentages between 87 and 110%. Graphical abstract Schematic representation of the electrochemical determination of zearalenone based on indirect competitive assay. Step a Immobilisationof ZEA on the surface of gold electrode via covalent attachment, b competition for the ZEA aptamer binding site between immobilised and free ZEA, and c current signal of the binding event based on SWV technique.

UL3 Mediated Nucleolar Stress Pathway as a New Mechanism of Action of Antiproliferative G-quadruplex TBA Derivatives in Colon Cancer Cells.

The antiproliferative G-quadruplex aptamers are a promising and challenging subject in the framework of the anticancer therapeutic oligonucleotides research field. Although several antiproliferative G-quadruplex aptamers have been identified and proven to be effective on different cancer cell lines, their mechanism of action is still unexplored. We have recently described the antiproliferative activity of a heterochiral thrombin binding aptamer (TBA) derivative, namely, LQ1. Here, we investigate the molecular mechanisms of LQ1 activity and the structural and antiproliferative properties of two further TBA derivatives, differing from LQ1 only by the small loop base-compositions. We demonstrate that in p53 deleted colon cancer cells, LQ1 causes nucleolar stress, impairs ribosomal RNA processing, leading to the accumulation of pre-ribosomal RNAs, arrests cells in the G2/M phase and induces early apoptosis. Importantly, the depletion of uL3 abrogates all these effects, indicating that uL3 is a crucial player in the mechanism of action of LQ1. Taken together, our findings identify p53-independent and uL3-dependent nucleolar stress as a novel stress response pathway activated by a specific G-quadruplex TBA derivative. To the best of our knowledge, this investigation reveals, for the first time, the involvement of the nucleolar stress pathway in the mechanism of action of antiproliferative G-quadruplex aptamers.