ssDNA Aptamer Research Articles

Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP.

SummaryA poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study.ResultsWe have selected a ssDNA aptamer population against a recombinant 6xHIS–LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes.ConclusionResults presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.

Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects.

BackgroundSoft-shelled turtle iridovirus (STIV) causes severe systemic disease in farmed soft-shelled turtles (Trionyx sinensis). More efficient methods of controlling and detecting STIV infections are urgently needed. MethodsIn this study, we generated eight single-stranded DNA (ssDNA) aptamers against STIV using systematic evolution of ligands by exponential enrichment (SELEX).ResultsThe aptamers formed representative stem-loop secondary structures. Electrophoretic mobility shift assays and fluorescent localization showed that the selected aptamers had high binding affinity for STIV. Aptamer QA-36 had the highest calculated binding affinity (Kd) of 53.8 nM. Flow cytometry and fluorescence microscopy of cell-aptamer interactions demonstrated that QA-12 was able to recognize both STIV-infected cells and tissues with a high level of specificity. Moreover, the selected aptamers inhibited STIV infection in vitro and in vivo, with aptamer QA-36 demonstrating the greatest protective effect against STIV and inhibiting STIV infection in a dose-dependent manner.DiscussionWe generated DNA aptamers that bound STIV with a high level of specificity, providing an alternative means for investigating STIV pathogenesis, drug development, and medical therapies for STIV infection.ConclusionsThese DNA aptamers may thus be suitable antiviral candidates for the control of STIV infections.

Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin.

Ortho-Stabilized (18) F-Azido Click Agents and their Application in PET Imaging with Single-Stranded DNA Aptamers.

Azido (18) F-arenes are important and versatile building blocks for the radiolabeling of biomolecules via Huisgen cycloaddition ("click chemistry") for positron emission tomography (PET). However, routine access to such clickable agents is challenged by inefficient and/or poorly defined multistep radiochemical approaches. A high-yielding direct radiofluorination for azido (18) F-arenes was achieved through the development of an ortho-oxygen-stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido (18) F-arene clickable agent for bioconjugation reactions. A ssDNA aptamer was radiolabeled with this agent and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates that this OID approach is a convenient and highly efficient way of labeling and tracking biomolecules.

Ortho‐Stabilized 18F‐Azido Click Agents and their Application in PET Imaging with Single‐Stranded DNA Aptamers

AbstractAzido 18F‐arenes are important and versatile building blocks for the radiolabeling of biomolecules via Huisgen cycloaddition (“click chemistry”) for positron emission tomography (PET). However, routine access to such clickable agents is challenged by inefficient and/or poorly defined multistep radiochemical approaches. A high‐yielding direct radiofluorination for azido 18F‐arenes was achieved through the development of an ortho‐oxygen‐stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido 18F‐arene clickable agent for bioconjugation reactions. A ssDNA aptamer was radiolabeled with this agent and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates that this OID approach is a convenient and highly efficient way of labeling and tracking biomolecules.

Abstract POSTER-THER-1429: Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing

Abstract Humanized antibodies have been extensively investigated as therapeutic as well as diagnostic agents. While the antigen specificity offered by antibodies makes them very attractive for such theranostic applications, their large-scale synthesis can be challenging and expensive. We are therefore investigating alternate strategies to develop agents that can be used for in vivo monitoring as well as for treatment of epithelial ovarian tumors. One approach is to develop Single Stranded DNA aptamers that selectively bind to ovarian cancer cells. The ssDNA aptamers can be synthesized using template-driven or de novo chemical synthetic approaches to manufacture agents at a large scale and low cost. The challenge however, is to develop aptamers that are specific to ovarian cancer cells. In the current study, we report a streamlined approach that incorporates the cell-based Systematic Evolution of Ligands by Exponential Enrichment (cell-SELEX) with DNASeq technology to select aptamers that recognize ovarian cancer cells. An ssDNA aptamer library composed of ~1015 sequences was subjected to ten iterative rounds of selection against the ovarian cancer cell line OVCAR-3. Aptamers from each round were amplified by asymmetric PCR and subjected to high-throughput sequencing. Eight ssDNA aptamers enriched through the selection process were identified by DNASeq and subsequent bioinformatics analysis and their selectivity and affinity for OVCAR-3 cells was determined by flow cytometry. Two of these aptamers (Apt-1 and Apt-8) showed significant binding to OVCAR-3 cells with Kd of 24 and 28 nM, respectively. Secondary structure analysis using mfold indicated that Apt-1 and Apt-8 had defined secondary structures resulting from ordered base pairing of the ssDNA. The inclusion of high-throughput sequencing techniques has therefore allowed rapid identification of theranostic aptamers from an large randomized library of ssDNA sequences. Our ongoing experiments are focused on coupling of the ovarian cancer cell-specific ssDNA aptamers to contrast agents or cytotoxic drugs. The ssDNA aptamers coupled to contrast agents are specifically being investigated for in vivo imaging of ovarian cancer masses in the peritoneum whereas the aptamers coupled to drugs can be used for the treatment of ovarian cancer. Citation Format: Rebecca J. Whelan, Arvinder Kapur, Mildred Felder, Jamie Shallcross, Jeff Nie, Manish S. Patankar. Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1429.

Identification and application of ssDNA aptamers against H₃₇Rv in the detection of Mycobacterium tuberculosis.

Microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used in tuberculosis diagnosis. However, this method suffers from low specificity and is difficult to distinguish Mycobacterium tuberculosis (MTB) from nontuberculosis mycobacterial (NTM), since all mycobacterial species are positive in Ziehl-Neelsen stain. In this study, we utilized whole cell SELEX to obtain species-specific aptamers for increasing the specificity of MTB detection. Whole cell SELEX was performed in MTB reference strain H37Rv by two selection processes based on enzyme-linked plate or Eppendorf tube, respectively. To increase success rate of generating aptamers, the selection processes were systematically monitored to understand the dynamic evolution of aptamers against complex structure of target bacteria. Two preponderant groups and ten high-affinity aptamers were obtained by analyzing the dynamic evolution. Preponderant aptamer MA1 from group I showed relatively high binding affinity with apparent dissociation constant (KD value) of 12.02 nM. Sandwich ELISA assay revealed five aptamer combinations effectively bound MTB strains in preliminary evaluation, especially the combination based on aptamer MA2 (another preponderant aptamer from group II) and MA1. Further evaluated in many other strains, MA2/MA1 combination effectively identified MTB from NTM or other pathogenic bacteria, and displayed the high specificity and sensitivity. Binding analysis of aptamer MA1 or MA2 by fluorescence microscopy observation showed high binding reactivity with H37Rv, low apparent cross-reactivity with M. marinum, and no apparent cross-reactivity with Enterobacter cloacae. Taken together, this study provides attractive candidate species-specific aptamers to effectively capture or discriminate MTB strains.

Label-free capacitance based aptasensor platform for the detection of HER2/ErbB2 cancer biomarker in serum

In this study, a label-free capacitive aptasensor was developed based on capturing of human epidermal growth factor receptor 2 (HER2) protein by anti-HER2 ssDNA aptamers functionalized on interdigitated microelectrodes of capacitor as bio-recognition elements. The aptasensor response was measured by non-Faradaic Impedance Spectroscopy (nFIS) method. Capacitance signal specific to target HER2 protein is based on changes occurred due to charge distribution upon interaction of aptamer–protein molecules against to the applied AC frequency (50–350MHz). HER2 protein in spiked human serum was successfully detected through concentration dependent changes in impedance/capacitance values as a result of the formation of aptamer–HER2 protein complex (0.2–2ngmL−1 of HER2) on capacitor microelectrodes. The label-free capacitive aptasensing is a versatile and promising approach for early detection of cancer biomarkers in dilute human serum and the method potentially be extended to a wide variety of diseases biomarkers.

Intercalation of Proflavine in ssDNA Aptamers: Effect on Binding of the Specific Target Chloramphenicol

AbstractThe structural modification of ssDNA‐based aptamers upon specific binding of its target molecule leads to changes of the charge‐transfer resistance (Rct) of a negatively‐charged free‐diffusing redox probe. The aptamer adopts a structure due to self‐hybridization which is stabilized using profalvine as intercalator. The pre‐organized aptamer structure is used to detect chloramphenicol (CAP) requiring a substantial change of the aptamer structure indicated by a CAP concentration dependent increase in the Rct values. Pre‐incubation of the aptamer‐modified electrode with an intercalator allows for the modulation of the aptamer/target interaction and hence for a modulation of the CAP‐dependent variation of the Rct values.

Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain

Human noroviruses (NoV) are the leading cause of acute viral gastroenteritis worldwide. Significant antigenic diversity of NoV strains has limited the availability of broadly reactive ligands for design of detection assays. The purpose of this work was to produce and characterize single stranded (ss)DNA aptamers with binding specificity to human NoV using an easily produced NoV target—the P domain protein. Aptamer selection was done using SELEX (Systematic Evolution of Ligands by EXponential enrichment) directed against an Escherichia coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of 14 virus-like particles (VLPs) showed these aptamers had broad reactivity and exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs. Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested. Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P domain protein as a functional target for the selection of nucleic acid aptamers that specifically bind and broadly recognize diverse human NoV strains.

Superior fluorescent probe for detection of potassium ion

Here, a simple, and highly sensitive fluorescent assay is designed to monitor K+. The versatile, robust biosensing strategy is based on the specific recognition utility of label-free aptamers with their targets and PicoGreen dye as the signal probe. The aptamers undergo a conformational change to a secondary structure such as G-quadruplex in the presence of targets. In addition to a conformational change with its targets, the remaining single-stranded DNA (ssDNA) aptamer form a duplex structure with its complete complementary sequence. Conformational changes of aptamers as well as fluorescence amplification produce clear signal-off in the presence of targets. Fluorescent assay employing this mechanism for the detection of K+ is highly sensitive, and selective. The detection limit of the K+ assay is determined to be 2.37pM. The sensing strategy is low-cost and simple in its operation without requirement for complex labeling of probe DNA or sophisticated synthesis of the fluorescent compound. Also, the method has less structural requirement of complexes of aptamers with their targets, thus rending its wilder applications for various targets.

Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA–protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36μg/ml and 6.24μg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.

Development of Electrochemical Aptamer-Based Biosensors for the Detection of Hormonal Contaminants in Water

Endocrine disrupters (EDs) are compounds that specifically interfere with the normal functions of the body’s endocrine system (system of glands for hormonal secretion) by causing adverse effects in both human and wildlife. Therefore, routine, sensitive, reliable and cost effective detection methods for EDs are highly demanded. In this work we present the selection and characterization of ssDNA aptamers that exhibit high affinity, specificity and sensitivity to progesterone (P4), 17b-estradiol (E2) and, norethisterone (NET). The aptamer selection was conducted by in vitro selections by incubating a highly diverse ssDNA library of about random 60mer sequences with the target analyte. After 15 cycles of selections, the enriched aptamers pool was cloned and sequenced. The dissociation constants (KD) of the selected aptamers determined by fluorometry and by electrochemical impedance spectroscopy (EIS) methods are in the subnanomolar order. Cross-reactivity tests for each aptamer demonstrated the high specificity to their target analyte. In addition, circular dichroism (CD) spectroscopy measurements of the selected aptamers for progesterone showed a marked conformational change upon binding the target analyte. This change in the conformation was exploited to design a biosensing platform to generate a measurable signal after binding the specific analyte by following the redox process of a couple composed for [Fe(CN)6]3-/4-. For first time, an impedimetric aptasensor for progesterone detection was developed with limit of detection of 0.90 ng/mL. We believe, based in our finding, that the continuous selection of high affinity aptamers for other EDs and their integration in a biosensing platform will facilitate the routine monitoring of EDs in environment as well as clinical and medical diagnosis purposes. Figure 1

Isolation of single-stranded DNA aptamers that distinguish influenza virus hemagglutinin subtype H1 from H5.

Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus.

Hi-Fi SELEX: A High-Fidelity Digital-PCR Based Therapeutic Aptamer Discovery Platform.

Current technologies for aptamer discovery typically leverage the systematic evolution of ligands by exponential enrichment (SELEX) concept by recursively panning semi-combinatorial ssDNA or RNA libraries against a molecular target. The expectation is that this iterative selection process will be sufficiently stringent to identify a candidate pool of specific high-affinity aptamers. However, failure of this process to yield promising aptamers is common, due in part to (i) limitations in library designs, (ii) retention of non-specific aptamers during screening rounds, (iii) excessive accumulation of amplification artifacts, and (iv) the use of screening criteria (binding affinity) that does not reflect therapeutic activity. We report a new selection platform, High-Fidelity (Hi-Fi) SELEX, that introduces fixed-region blocking elements to safeguard the functional diversity of the library. The chemistry of the target-display surface and the composition of the equilibration solvent are engineered to strongly inhibit non-specific retention of aptamers. Partition efficiencies approaching 10(6) are thereby realized. Retained members are amplified in Hi-Fi SELEX by digital PCR in a manner that ensures both elimination of amplification artifacts and stoichiometric conversion of amplicons into the single-stranded library required for the next selection round. Improvements to aptamer selections are first demonstrated using human α-thrombin as the target. Three clinical targets (human factors IXa, X, and D) are then subjected to Hi-Fi SELEX. For each, rapid enrichment of ssDNA aptamers offering an order-nM mean equilibrium dissociation constant (Kd) is achieved within three selection rounds, as quantified by a new label-free qPCR assay reported here. Therapeutic candidates against factor D are identified.

Label-free sensing of adenosine based on force variations induced by molecular recognition.

We demonstrate a simple force-based label-free strategy for the highly sensitive sensing of adenosine. An adenosine ssDNA aptamer was bound onto an atomic force microscopy (AFM) probe by covalent modification, and the molecular-interface adsorption force between the aptamer and a flat graphite surface was measured by single-molecule force spectroscopy (SMFS). In the presence of adenosine, the molecular recognition between adenosine and the aptamer resulted in the formation of a folded, hairpin-like DNA structure and hence caused a variation of the adsorption force at the graphite/water interface. The sensitive force response to molecular recognition provided an adenosine detection limit in the range of 0.1 to 1 nM. The addition of guanosine, cytidine, and uridine had no significant interference with the sensing of adenosine, indicating a strong selectivity of this sensor architecture. In addition, operational parameters that may affect the sensor, such as loading rate and solution ionic strength, were investigated.

Ras ssDNA aptamer inhibits vascular smooth muscle cell proliferation and migration through MAPK and PI3K pathways.

Proliferation and migration of vascular smooth muscle cells (VSMCs) mediated by Ras proteins are crucial in restenosis following percutaneous coronary intervention (PCI) and coronary artery bypass graft (CABG). In this study, a novel, single-stranded DNA (ssDNA) aptamer designated as Ras-a1 with high affinity and specificity to human Ras protein was isolated using systematic evolution of ligands by exponential enrichment. Ras-a1 was delivered into VSMCs by electroporation using one square waveform of 200 V for 20 msec. Proliferation of VSMCs was determined using a cell counting kit‑8 assay, which revealed the maximal inhibitory rate (40%) was obtained at 24 h after Ras-a1 transfection. The migration of VSMCs, determined using a Transwell assay, was significantly inhibited in Rasa1 cells in a time-dependent manner. To investigate the potential mechanisms of transfected Ras-a1 on the migration and proliferation of VSMCs, the phosphorylation of MEK1/2, ERK1/2, and Akt was determined using western blot analysis, which showed that a marked downregulation was observed in the phosphorylation of MEK1/2, ERK1/2, and Akt following the delivery of Ras-a1. This result demonstrated that Ras-a1 inhibits the proliferation and migration of VSMCs by inhibiting the phosphorylation of Ras and interrupting signal transduction in the Ras‑MEK1/2‑ERK1/2 and phosphoinositide-3 kinase/Akt pathways. The novel Ras protein-targeted ssDNA aptamer selected may be applicable for the prevention of restenosis after PCI and CABG.

Indirect purification method provides high yield and quality ssDNA sublibrary for potential aptamer selection

The quality and yield of single-stranded DNA (ssDNA) play key roles in ssDNA aptamer selection. However, current methods for generating and purifying ssDNA provides either low yield due to ssDNA loss during the gel purification process or low specificity due to tertiary structural damage of ssDNA by alkaline or exonuclease treatment in removing dsDNA and by-products. This study developed an indirect purification method that provides a high yield and quality ssDNA sublibrary. Symmetric PCR was applied to generate a sufficient template, while asymmetric PCR using an excessive nonbiotinylated forward primer and an insufficient biotinylated reverse primer combined with a biotin–strepavidin system was applied to eliminate dsDNA, hence, leading to ssDNA purification. However, no alkaline or exonuclease were involved in treating dsDNA, so as to warrant the tertiary structure of ssDNA for potential aptamer SELEX selection. Agarose gel imaging indicated that no dsDNA or by-product contamination was detected in the ssDNA sublibrary generated by the indirect purification method. Purified ssDNA concentration reached 1020±210nM, which was much greater than previous methods. In conclusion, this novel method provided a simple and fast tool for generating and purifying a high yield and quality ssDNA sublibrary.

A novel colorimetric aptasensor using cysteamine-stabilized gold nanoparticles as probe for rapid and specific detection of tetracycline in raw milk

In this paper, a sensitive and specific colorimetric aptasensor for rapid detection of tetracycline (TET) in raw milk was developed by using cysteamine-stabilized gold nanoparticles (CS-AuNPs) as the probe. The highly specific single-stranded DNA (ssDNA) aptamers which bind to TET with high affinity were employed to discriminate other antibiotics, such as oxytetracycline, doxycycline, ciprofloxacin, kanamycin, lomefloxacin, chloramphenicol, ofloxacin, enoxacin. The sensing approach is based on the strongly specific interactions between the TET-binding aptamers and TET over the electrostatic interactions between the negatively-charged TET-binding aptamers and the positively-charged CS-AuNPs. Thus a simple colorimetric aptasensor for TET determination is developed using CS-AuNPs as the colorimetric probe. Under the optimum conditions, the absorbance change (ΔA527) of CS-AuNPs was linearly proportional with TET concentration in the range of 0.20–2.0 μg/mL with the detection limit of 0.039 μg/mL (3σ), which is lower than the permissible level in the European Union (EU) (225 nM) and China (100 μg/kg). The application of this method for samples of TET-spiked raw milk suggested satisfactory recoveries between 91.28% and 100.87%. Owing to the advantages of simple operation procedure, reduced detection time, and excellent selectivity, practical application of the proposed colorimetric assay could be expected to realize in on-site and real-time detection of TET in raw milk.

Aptamer-based label-free impedimetric biosensor for detection of progesterone.

Rising progesterone (P4) levels in humans due to its overconsumption through hormonal therapy, food products, or drinking water can lead to many negative health effects. Thus, the simple and accurate assessment of P4 in both environmental and clinical samples is highly important to protect public health. In this work, we present the selection, identification, and characterization of ssDNA aptamers with high binding affinity to P4. The aptamers were selected in vitro from a single-stranded DNA library of 1.8 × 10(15) oligonucleotides showing dissociation constants (KD) in the low nanomolar range. The dissociation constant of the best aptamer, designated as P4G13, was estimated to be 17 nM by electrochemical impedance spectroscopy (EIS) as well as fluorometric assay. Moreover, the aptamer P4G13 did not show cross-reactivity to analogues similar to progesterone such as 17β-estradiol (E2) and norethisterone (NET). An impedimetric aptasensor for progesterone was then fabricated based on the conformational change of P4G13 aptamer, immobilized on the gold electrode by self-assembly, upon binding to P4, which results in an increase in electron transfer resistance. Aptamer-complementary DNA (cDNA) oligonucleotides were tested to maximize the signal gain of the aptasensor after binding with progesterone. Significant signal enhancement was observed when the aptamer hybridized with a short complementary sequence at specific site was used instead of pure aptamer. This signal gain is likely due to the more significant conformational change of the aptamer-cDNA than the pure aptamer upon binding with P4, as confirmed by circular dichroism (CD) spectroscopy. The developed aptasensor exhibited a linear range for concentrations of P4 from 10 to 60 ng/mL with a detection limit of 0.90 ng/mL. Moreover, the aptasensor was applied in spiked tap water samples and showed good recovery percentages. The new selected progesterone aptamers can be exploited in further biosensing applications for environmental, clinical, and medical diagnostic purposes.