Highly sensitive and selective lateral flow aptasensor for anti-coagulant dabigatran etexilate determination in blood.

Dabigatran etexilate (DBG) is a new anticoagulant drug (commercially sold under the names Pradaxa® and Pradax™) that replaces Warfarin, the landmark agent for anticoagulation therapy. Inadequate administration of DBG or in the cases of massive bleeding that occurs after renal impairment, DBG therapy can carry a substantial life-threatening risks. One of the major limitations of DBG treatment is the lack of a simple and quick tool for measuring its level in blood in the case of massive bleedings or emergency operations. In this work, we have incorporated a previously isolated aptamer for DBG to develop a simple competitive lateral flow aptasensor (LFA) for the determination of DBG in buffer and blood samples. A full-length 60-mer aptamer as well as a truncated 38-mer aptamer were conjugated to gold nanoparticles (AuNPs) via thiol-Au coupling chemistry. After appropriate AuNP surface passivation steps, the aptamer's core region was hybridized with 8-mer biotinylated sequences. The conjugated particles could be capture on the test line by the interaction of the biotin molecules with a previously deposited streptavidin. Incubation of the conjugated particles with DBG causes the aptamer to undergo a conformational change that releases the 8-mer biotinylated sequences and result in the disappearance of the test line. Lysozyme protein was used to construct the control line that non-specifically interacts with the conjugated particles whether or not the target compound is present. The developed LFA achieves 20 nM detection level in buffer and blood samples, operates within the nanomolar range, and shows excellent selectivity against potential interfering molecules. The developed sensor could help assessing the levels of DBG in medical conditions that require rapid interventions.

Antibiotic Adsorption by Metal-Organic Framework (UiO-66): A Comprehensive Kinetic, Thermodynamic, and Mechanistic Study.

Bacterial antibiotic resistance has been deemed one of the largest modern threats to human health. One of the root causes of antibiotic resistance is the inability of traditional wastewater management techniques, such as filtration and disinfection, to completely eliminate residual antibiotics from domestic and industrial effluents. In this study, we examine the ability of UiO-66; a metal-organic framework (MOF); in removing the antibiotic Doxycycline from aqueous environments. This study's findings suggest that UiO-66 was able to remove nearly 90% of the initial Doxycycline concentration. To correlate the isothermal data, Langmuir and Freundlich models were used. It was determined that the Langmuir model was best suited. Pseudo-first and -second order models were examined for kinetic data, where the pseudo-second order model was best suited-consistent with the maximum theoretical adsorption capacity found by the Langumir model. Thermodynamic analysis was also examined by studying UiO-66 adsorption under different temperatures. Mechanisms of adsorption were also analyzed through measuring adsorption at varying pH levels, thermogravimetric analysis (TGA), Infrared spectroscopy (IR) and Brunauer-Emmet-Teller (BET). This study also explores the possibility of recycling MOFs through exposure to gamma radiation, heat, and heating under low pressure, in order for UiO-66 to be used in multiple, consecutive cycles of Doxycycline removal.

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.

In vitro selection of DNA aptamers and their integration in a competitive voltammetric biosensor for azlocillin determination in waste water.

The uncontrolled usage of veterinary antibiotics has led to their widespread pollution in waterways and milk products. Potential impact of antibiotic residues on the environment and human health such as increased antibiotic resistance of microorganisms and triggering allergic reactions in humans have been reported. In this work, we developed a highly selective and sensitive voltammetric aptasensor for on-step, sensitive and low cost detection of azlocillin antibiotic, one of the broad spectrum ß-lactam antibiotics. The successful selection of DNA aptamers against azlocillin was accomplished using systemic evolution of ligands by exponential enrichment (SELEX) method. Fluorescence-binding assays showed dissociation constant of 55 nM for one of the selected aptamers (Az9). This aptamer was used to construct a competitive voltammetric aptasensor for azlocillin. A limit of detection of 1.2 pg/mL as well as remarkable selectivity against potential interfering agents, including amoxicillin, were achieved. This signal-off competitive sensor takes 30-50 min to complete the quantification of the target antibiotic. The sensor was challenged by detecting the target directly in complex environments such as tap and waste water where good recovery percentages were achieved.

Decomposition of DNA staining agent ethidium bromide by gamma irradiation: Conditions, kinetics, by-products, biological activity, and removal from wastewater.

Ethidium Bromide (Eth-Br) is an intercalating agent commonly used in medical and biological laboratories as a DNA staining dye. Despite its popular use, aqueous solutions containing Eth-Br showed high toxicity, mutagenic capacity, and deactivate DNA transcription. In this study, the removal of Eth-Br from aqueous solutions by gamma irradiation has been fully investigated. Gamma irradiation was capable of achieving a near complete removal of Eth-Br in neutral and non-buffered aqueous solutions at an absorbed dose of 15 kGy. Various experimental conditions were studied and showed that the removal efficiency is not diminished. The addition of hydrogen peroxide (2 %) to the irradiated solutions reduced the D50 and D90 by 50 %. Modeling Eth-Br decomposition showed that the reaction followed pseudo first-order kinetics and reaches at least 90 % removal under all experimental conditions. TOC and HPLC measurements confirmed that Eth-Br is fully mineralized when the absorbed dose reaches 15 kGy. The biological activity of Eth-Br after irradiation treatment was investigated with synthetic DNA and natural DNA. The biological activity of Eth-Br was deactivated at an absorbed dose as low as 5 kGy. Toxicity measurement with E-coli bacteria also confirmed that the absorbed dose of 5 kGy was sufficient to remove Eth-Br toxicity.

Removal of Antibiotics from Water by Polymer of Intrinsic Microporosity: Isotherms, Kinetics, Thermodynamics, and Adsorption Mechanism.

Traces of antibiotics within domestic and industrial effluents have toxic impact on human health as well as surrounding flora and fauna. Potential increase in antibiotic resistance of microorganisms is likely to rise due to the incomplete removal of antibiotics by traditional wastewater processing, methods such as membrane filtration and biological treatment. In this study, we investigated a novel class of material termed Polymer of Intrinsic Microporosity (PIM) that is based on amorphous microporous organic materials for the application of antibiotic removal form aqueous environments. The adsorption of four commonly used antibiotics (doxycycline, ciprofloxacin, penicillin G, and amoxicillin) was evaluated and found that at least 80% of the initial concentrations was eliminated under the optimized conditions. Langmuir and Freundlich models were then employed to correlate the equilibria data; the Freundlich model fit well the data in all cases. For kinetic data, pseudo-first and second order models were examined. Pseudo-second order model fit well the kinetic data and allowed the calculation of the adsorption rate constants. Thermodynamic parameters were obtained by conducting the adsorption studies at varied reaction temperatures. Surface potential, adsorption at various solution pHs, thermogravimetric analysis (TGA), Infrared spectroscopy (IR), and surface area experiments were conducted to draw possible adsorption mechanisms. The removal of antibiotics from water by PIM-1 is likely to be governed by both surface and pore-filling adsorption and could be facilitated by electrostatic interactions between the aromatic rings and charged functional groups as well as hydrogen bond formation between the adsorbent and adsorbate. Our work shows that the application of such novel microporous material could contribute to the removal of such challenging and persistent contaminants from wastewater with further optimizations of large-scale adsorption processes.

Lateral flow aptasensor for progesterone: Competitive target recognition and displacement of short complementary sequences.

There is a pressing need for simple and accurate analytical tools to assess the level of EDCs in environmental samples. In this work, a simple and highly sensitive competitive lateral flow assay (LFA) was developed for progesterone (P4). Gold nanoparticles (AuNPs) were functionalized with a previously isolated 60-mer aptamer for P4 and further hybridized with 8-mer complementary sequence modified with biotin. In the absence of P4, AuNP-duplexed aptamer conjugates are capture by a test line made with streptavidin. Conformational change within aptamer sequence upon target recognition causes the release of the biotinylated complementary sequence and disappearance of the colored test line. By optimizing the hybridization location of the 8-mer biotinylated sequences, the sensitivity of sensor was improved by 20-folds to achieve 5 nM detection level of P4 in buffer and spiked tap water samples. The simply fabricated sensor demonstrated a dynamic range in the lower nanomolar range and excellent selectivity against potential interfering molecules including the closely similar 17ß-estradiol (E2). The sensor can be used as a fast screening tool to assess the level of P4 in water. Implementing the developed assay avoids applying laborious extraction procedures and lengthily analysis by conventional chromatography based instruments.

Label-Free Fluorescent Aptasensor for Small Targets via Displacement of Groove Bound Curcumin Molecules.

Signal transduction based on fluorescence is one of the most common optical aptasensors for small molecules. Sensors with a number of unique features including high sensitivity, low cost, and simple operation can be constructed easily. However, the label-free fluorescent approach is limited to synthetic dyes that bind strongly to the aptamer sequence and result in a diminished sensor operation with high detection limits. In this study, we report the use of curcumin as a fluorescent probe to signal aptamer/small target binding events. A substantial enhancement in curcumin's fluorescent emission was observed when bound into the grooves of vitamin D3 (VTD3) binding aptamer, as an example. However, the introduction of the target molecule causes the aptamer to undergo a conformational change that favors complexing the target molecule over binding the curcumin dye. The sensor was able to detect VTD3 down to 1 fM concentration in buffer solutions and extracted blood samples, operate at a wide dynamic range, and discriminate against potential biological interfering molecules including VTD2. The operation of the curcumin based fluorescent sensor is at least six orders of magnitude more sensitive than a VTD3 sensor constructed with the synthetic dye SYBR Green I. The generality of the reported label-free approach was applied with a previously isolated 75-mer bisphenol-A (BPA) aptamer, confirming that the reported sensing strategy is not confined on a particular aptamer sequence. Our work not only reports a novel sensor format for the detection of small molecules, but also serves fluorescent sensor's most pressing need being novel fluorophores for multiplex targets detection.

In Vitro Selection of Specific DNA Aptamers Against the Anti-Coagulant Dabigatran Etexilate.

Dabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8-208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.

Colorimetric Aptasensor of Vitamin D3: A Novel Approach to Eliminate Residual Adhesion between Aptamers and Gold Nanoparticles.

Colorimetric aptasensors based on gold nanoparticles (AuNPs) commonly feature ssDNA probes nonspecifically adsorbed to surface gold particles. A major limitation of this versatile method is the incomplete dissociation of the adsorbed nontarget binding segments of the aptamer sequence upon target binding. This results in weak or nonexistent sensor performance by preventing the particles from aggregating when the optimized salt concentration is added. Rather than removing the nonbinding nucleotides flanking the binding region of the aptamer, proposed herein is an alternative strategy, simply introducing a centrifugation and resuspension step after target recognition that eliminates residual binding between the aptamer and the surface of the particles. The performance of two different vitamin D3 (VTD3) aptamers were tested. The method enhanced the performance of the sensor that used the higher detection limit (1 µM) aptamer by fourfold. The superiority of the proposed method became apparent in a nonworking colorimetric sensor became a highly sensitive sensor with a one nanomolar detection level and excellent discrimination against potential interfering molecules including VTD2 when the centrifugation and resuspension process was implemented. The level of VTD3 in human blood was determined colorimetrically after extraction with n-hexane. The results were in agreement with those obtained by HPLC. The proposed method could be applied to aptamers targeting small molecules with no need to reprocess the SELEX-isolated sequence by knowing the binding region and removing the flanking primers.

Removal of antibiotics from water and waste milk by ozonation: kinetics, byproducts, and antimicrobial activity.

The use of antibiotics in the dairy farming for curing and growth promotion results in the production of massive quantities of non-recyclable wastewater by the conventional purification techniques. Additionally, waste milk is produced during the drug withholding periods, which is not suitable for human or animal consumption and cause huge economic loss as well as present serious environmental waste. This study was designed to investigate the decomposition of various antibiotic compounds in un-buffered aqueous solutions and milk samples by ozonation process. Commonly administered broad-spectrum antibiotics such as amoxicillin, doxycycline, ciprofloxacin, and sulphadiazine were selected as model examples in the current investigation. Gradual exposure of these antibiotics to increasing ozone gas concentration induced increasing removal percentages of the antibiotics in spiked water and milk samples. The removal reached 95% across all the tested treated antibiotics with ozone dose as low as 75 mg L-1. It was noted that the removal of antibiotics in milk samples is more efficient with faster rate constants. This was attributed to the self-buffering characteristic of milk that maintains the neutral pH, keeping the amine groups un-protonated and more reactive towards the electrophilic attack by the molecular ozone. 1H NMR as well as HPLC experiments support the near complete removal of antibiotics and indicated the break down to simpler and more soluble fragments of acidic nature. Bacterial growth experiments, conducted with E. coli, and milk ageing experiments provided clear evidences that the resulting decomposition byproducts lack both toxicity effect and antimicrobial activity. This study provides a viable route to remove hazardous materials, which contribute to a growing issue of antibiotic resistance of pathogenic bacteria.

Decomposition byproducts induced by gamma radiation and their toxicity: the case of 2-nitrophenol.

The induced degradation and detoxification of 2-nitrophenol (2-NP) in aqueous media by gamma irradiation were carefully evaluated in this study. Gamma radiation at absorbed doses as low as 20 kGy was able to degrade 2-NP to reach a removal of at least 85% across the investigated range of concentration (50-150 ppm). 2-NP breaks down to aromatic-based compounds with increasing number of byproducts upon increasing the radiation treatment from the absorbed dose of 50% decomposition (D50) to the absorbed dose of 90% decomposition (D90), after which no byproducts could be detected, indicating the formation of undetectable aliphatic hydrocarbons, insoluble, or volatile byproducts. Toxicology studies showed that the degradation of 2-NP under absorbed doses up to D90 resulted in a more toxic byproduct than the parent compound, and a remarkable reduction in the toxicity was observed with the irradiated samples with absorbed doses above D90. Varying the pH of the media to acidic or basic conditions did not significantly alter the degradation behavior of 2-NP. However, a notable improvement of the detoxification was associated with the samples of acidic pH. Adding 0.5% of H2O2 to 2-NP solutions had a positive effect by reducing D90 by a factor of nine and diminishing the toxicity by twofolds.

Lateral Flow Aptasensor for Small Molecule Targets Exploiting Adsorption and Desorption Interactions on Gold Nanoparticles.

A lateral flow assay (LFA) can provide a rapid and cost-effective means to detect targets in situ; however, existing LFA formats (predominantly sandwich assays) are not suitable for small molecule targets. We present a new LFA design that probes the dissociation of aptamers from the surface of gold nanoparticles upon recognition of small targets. The target-induced removal of aptamer molecules from the surface of the colored particles results in the particles being captured on a test line comprised of the protein bovine serum albumin immobilized on nitrocellulose. On the other hand, in the absence of target, aptamer coated particles are protected from capture on the test line and are instead captured at a control line comprised of the protein lysozyme. This protein is strongly positively charged under measurement conditions and therefore captures all gold nanoparticles regardless of the presence of aptamers. The effectiveness and operation mechanism of this simply fabricated sensor was demonstrated by using a previously reported 35-mer aptamer for a small molecule, 17ß-estradiol. The sensor exhibited nanomolar level of detection, excellent selectivity against potential interfering molecules, and robust operation in natural river water samples. The simplicity and performance of the sensor platform renders it applicable to a wide range of other aptamers targeting small molecules, as we demonstrated with a novel bisphenol A aptamer. Additionally, we show that our LFA design is not confined to the specific proteins used as test and control lines, provided that their charge is appropriate to modulate the interaction with aptamer-coated or bare nanoparticles.

Electrostatic gating in carbon nanotube aptasensors.

Synthetic DNA aptamer receptors could boost the prospects of carbon nanotube (CNT)-based electronic biosensors if signal transduction can be understood and engineered. Here, we report CNT aptasensors for potassium ions that clearly demonstrate aptamer-induced electrostatic gating of electronic conduction. The CNT network devices were fabricated on flexible substrates via a facile solution processing route and non-covalently functionalised with potassium binding aptamers. Monotonic increases in CNT conduction were observed in response to increasing potassium ion concentration, with a level of detection as low as 10 picomolar. The signal was shown to arise from a specific aptamer-target interaction that stabilises a G-quadruplex structure, bringing high negative charge density near the CNT channel. Electrostatic gating is established via the specificity and the sign of the current response, and by observing its suppression when higher ionic strength decreases the Debye length at the CNT-water interface. Sensitivity towards potassium and selectivity against other ions is demonstrated in both resistive mode and real time transistor mode measurements. The effective device architecture presented, along with the identification of clear response signatures, should inform the development of new electronic biosensors using the growing library of aptamer receptors.

Label-free electrochemical aptasensor for femtomolar detection of 17ß-estradiol.

We report an electrochemical aptasensor for the rapid, label-free detection of 17ß-estradiol (E2) from femtomolar to micromolar levels. The sensor features an aptamer-functionalised nanoporous conducting polymer electrode whose surface potential is probed via electrochemical impedance spectroscopy. The unprecedented detection limit for E2 is explained via the redistribution of negative charges in the electrode double-layer region when the aptamer adopts a folded conformation around the small neutral target molecule. The sensor responds approximately logarithmically over a wide dynamic range of E2 concentration that spans biological trigger levels, with excellent discrimination against structurally similar molecules including progesterone, and robust operation in human urine. The generality of the approach of using conformationally gated small molecule binding aptamers is highlighted with a further example of adenosine detection via the adenosine binding aptamer.

Ultrasensitive colorimetric detection of 17ß-estradiol: the effect of shortening DNA aptamer sequences.

We report a strategy enabling ultrasensitive colorimetric detection of 17ß-estradiol (E2) in water and urine samples using DNA aptamer-coated gold nanoparticles (AuNPs). Starting from an established sensor format where aggregation is triggered when target-bound aptamers dissociate from AuNP surfaces, we demonstrated that step-change improvements are easily accessible through deletion of excess flanking nucleotides from aptamer sequences. After evaluating the lowest energy two-dimensional configuration of the previously isolated E2 binding 75-mer aptamer (KD ∼25 nM), new 35-mer and 22-mer aptamers were generated with KD's of 14 and 11 nM by simply removing flanking nucleotides on either side of the inner core. The shorter aptamers were found to improve discrimination against other steroidal molecules and to improve colorimetric sensitivity for E2 detection by 25-fold compared with the 75-mer to 200 pM. In comparing the response of all sequences, we find that the excess flanking nucleotides suppress signal transduction by causing target-bound aptamers to remain adhered to AuNPs, which we confirm via surface sensitive electrochemical measurements. However, comparison between the 22-mer and 35-mer systems show that retaining a small number of excess bases is optimal. The performance advances we achieved by specifically considering the signal transduction mechanism ultimately resulted in facile detection of E2 in urine, as well as enabling environmental detection of E2 at levels approaching biological relevance.

Small molecule detection in solution via the size contraction response of aptamer functionalized nanoparticles.

We demonstrate a simple new sensor design that exploits aptamer functionalized nanoparticles (NPs) to transduce the signal of aptamer receptors binding to target small molecules. An aptamer capable of binding to our target 17ß-estradiol (E2) was isolated by SELEX with dissociation constant of 50 nM and tethered to the surface of carboxylated polystyrene NPs. Upon exposing the aptamer functionalized NPs to E2 in buffered water, we use dynamic light scattering (DLS) and resistive pulse sensing (TRPS) to observe a distinct reduction of the conjugated particle size and a less negative zeta potential, which can be correlated to the E2 concentration in the lower nanomolar range. The sensor showed similar affinity towards other hormones of the E2 steroidal family and excellent discrimination against potential non-steroidal interfering agents. The simplicity of the sensing scheme makes it readily applicable to other low molecular weight targets, as we further demonstrate using a known adenosine aptamer. In addition to sensing, our method shows potential to guide the synthetic evolution of aptamers with better binding affinity and specificity.