Studies on the photophysical characteristics of poly(carboxylic acid)s bound protoporphyrin IX and metal complexes of protoporphyrin IX

The copolymers of methacrylic acid with protoporphyrin IX (PPIX) and the metal complexes, zinc protoporphyrin IX and magnesium protoporphyrin IX were synthesised and characterised. Corresponding acrylic acid copolymers were also synthesised. The steady state absorption and fluorescence spectral properties of the macromolecular bound fluorophores PPIX, Zn-PPIX and Mg-PPIX were investigated. Poly(methacrylic acid) bound protoporphyrin IX, zinc protoporphyrin IX and magnesium protoporphyrin IX show an increase in the fluorescence intensity and lifetime with increase in the pH in the range 2-8 with a marked transition around pH 6.0-7.0. The fluorophore concentration in the dilute solution of the copolymers is micromolar and the fluorophore to the carboxylic acid monomer ratios in the copolymer is around 10-3. The molecular weight of the copolymers is 100 ± 10 kD. The fluorescence decay curves of all the fluorophore bound polymers follow biexponential decay fit independent of pH. Poly(MAA-co-PPIX) and poly(MAA-co-MgPPIX) undergo well marked pH induced structural transitions in the pH range of 6.0-7.0 whereas poly(MAA-co-ZnPPIX) undergoes pH induced structural transitions in the pH range of 4.0. In the case of polyacrylic acid copolymers the changes observed in the steady state and time resolved fluorescence studies are less marked. The distinct hydrophobic and hydrophilic environments experienced by the fluorophore bound to PMMA are attributed to the dynamics of the macromolecules in dilute aqueous solutions manifested by the α-methyl group present in the copolymer. The studies carried out using the fluorophores in the time windows from 2 ns to 12 ns indicate evolving trends in the dynamic coiling and reverse coiling of poly methacrylic acid chain

Exploring the Utility of ssDNA Aptamers Directed against Snake Venom Toxins as New Therapeutics for Snakebite Envenoming.

Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapy for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. In this study, we sought to explore the potential of ssDNA aptamers as toxin-specific inhibitory alternatives to antibodies. As a proof of principle model, we selected snake venom serine protease toxins, which are responsible for contributing to venom-induced coagulopathy following snakebite envenoming, as our target. Using SELEX technology, we selected ssDNA aptamers against recombinantly expressed versions of the fibrinogenolytic SVSPs ancrod from the venom of C. rhodostoma and batroxobin from B. atrox. From the resulting pool of specific ssDNA aptamers directed against each target, we identified candidates that exhibited low nanomolar binding affinities to their targets. Downstream aptamer-linked immobilised sorbent assay, fibrinogenolysis, and coagulation profiling experiments demonstrated that the candidate aptamers were able to recognise native and recombinant SVSP toxins and inhibit the toxin- and venom-induced prolongation of plasma clotting times and the consumption of fibrinogen, with inhibitory potencies highly comparable to commercial polyvalent antivenoms. Our findings demonstrate that rationally selected toxin-specific aptamers can exhibit broad in vitro cross-reactivity against toxin isoforms found in different snake venoms and are capable of inhibiting toxins in pathologically relevant in vitro and ex vivo models of venom activity. These data highlight the potential utility of ssDNA aptamers as novel toxin-inhibiting therapeutics of value for tackling snakebite envenoming

Rapid colorimetric lactoferrin-based sandwich immunoassay on cotton swabs for the detection of foodborne pathogenic bacteria

Cotton swab is the conventional swabbing tool that is usually applied for collecting pathogens from contaminated surfaces, followed by cells lysis and DNA extraction before subjecting to genetic analysis. However, such an approach is time consuming as it involves several steps and requires highly trained personnel to perform the experiment. In this study, we developed a new cotton swab-based detection system that involved integrating bacterial collection, preconcentration and detection on Q-tips. The platform is based on a sandwich assay that can detect different pathogens visually by color changes. Lactoferrin-immobilized cotton is used as a general capturing tool to collect various pathogens from surfaces. The presence of particular bacteria is then detected by immersing the cotton in antibodies attached to different colored nanobeads. The target cell is captured between the lactoferrin and specific antibody-conjugated beads which results in certain color development. The effectiveness of this simply fabricated sensor was demonstrated using Salmonella typhimurium, Salmonella enteritidis, Staphylococcus aureus and Campylobacter jejuni. The intensity of the color on the cotton surfaces increased with increasing the concentration of the pathogenic bacteria. The detection limit was as low as 10 cfu/ml for Salmonella typhimurium and Campylobacter jejuni, 100 cfu/ml for Salmonella enteritidis and 100 cfu/ml for Staphylococcus aureus on chicken meat surface. Moreover, this method showed high selectivity and was further confirmed by loop-mediated isothermal amplification (LAMP). The simplicity and the low cost of this colorimetric sensor renders it applicable to a wide range of other pathogens on different surfaces

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.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

Folding of the lysine riboswitch: importance of peripheral elements for transcriptional regulation

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2–loop L3 and helix P2–loop L4)—all of this for the creation of a specific lysine binding site. We have determined that the interaction P2–L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2–L3 and P2–L4 allows both interactions to fold at lower magnesium ion concentrations. The P2–L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem–loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation

Isolation and Detection of Exosomal Mir210 Using Carbon Nanomaterial-Coated Magnetic Beads

MicroRNAs (miRNAs) are short non-coding RNAs that are found in various cellular compartments and play an important role in regulating gene expression. Extracellular miRNAs, such as those found within extracellular vesicles such as exosomes are involved in cell-to-cell communication. The intercellular transfer of miRNAs has been implicated in various diseases’ pathogenesis including cancer and has been studied extensively as potential cancer biomarkers. However, the extraction of miRNA from exosomes is still a challenging task. The current nucleic acid extraction assays are expensive and labor-intensive. In this study, we demonstrated a microfluidic device for aptamer-based magnetic separation of the exosomes and subsequent detection of the miRNA using a fluorescence switching assay, which was enabled by carbon nanomaterials coated on magnetic beads. In the OFF state, the fluorophore-labelled cDNA is quenched using carbon nanomaterials. However, when the target miRNA210 is introduced, the cDNA detaches from the bead’s surface, which leads to an increase in the fluorescence intensity (ON state). This increment was found to be proportional to miRNA concentration within the dynamic range of 0–100 nM with a detection limit of 5 pM. The assay was validated with spiked miRNA using the standard RT-PCR method. No notable cross-reactivity with other closely related miRNAs was observed. The developed method can be utilized for the minimally invasive detection of cancer biomarkers

On stabilization of a neutral aromatic ligand by π–cation interactions in monoclonal antibodies

International audienceIt has been shown that anti-PAH mAb can bind a particular cross-reactant by adopting two distinct "red" and "blue" conformations of its binding sites [N.M. Grubor et al. PNAS 102, 2005, 7453-7458]. In the case of red conformation of pyrene (Py)/anti-PAH mAb (with a broad fluorescence (0,0)-band with fwhm ~140 cm(-1)), the central role in complex formation was played by π-π interactions. The nature of the blue-shifted conformation with very narrow fluorescence (0,0)-band (fwhm ~75 cm(-1)) was left unclear due to the lack of suitable data for comparison. In this work, we suggest spectroscopic and modeling results obtained for the blue conformation of Py in several mAb (including 4D5 mAb) are consistent with π-cation interactions, underscoring the importance of π-cation interaction in ligand binding and stabilization in agreement with earlier modeling studies [J-L. Pellequer, et al. J. Mol. Biol. 302, 2000, 691-699]. We propose considerable narrowing of the fluorescence origin band of ligand in the protein environment could be regarded as a simple indicator of π-cation interactions. Since 4D5 mAb forms only the blue-shifted conformation, while anti-PAH and 8E11 mAbs form both blue- and red-shifted conformations, we suggest mAb interactions, with Py molecules lacking H-bonding functionality, may induce distinct conformations of mAb binding sites that allow binding by π-π and/or π-cation interactions

Aptamers: Potential Diagnostic and Therapeutic Agents for Blood Diseases

Aptamers are RNA/DNA oligonucleotide molecules that specifically bind to a targeted complementary molecule. As potential recognition elements with promising diagnostic and therapeutic applications, aptamers, such as monoclonal antibodies, could provide many treatment and diagnostic options for blood diseases. Aptamers present several superior features over antibodies, including a simple in vitro selection and production, ease of modification and conjugation, high stability, and low immunogenicity. Emerging as promising alternatives to antibodies, aptamers could overcome the present limitations of monoclonal antibody therapy to provide novel diagnostic, therapeutic, and preventive treatments for blood diseases. Researchers in several biomedical areas, such as biomarker detection, diagnosis, imaging, and targeted therapy, have widely investigated aptamers, and several aptamers have been developed over the past two decades. One of these is the pegaptanib sodium injection, an aptamer-based therapeutic that functions as an anti-angiogenic medicine, and it is the first aptamer approved by the U.S. Food and Drug Administration (FDA) for therapeutic use. Several other aptamers are now in clinical trials. In this review, we highlight the current state of aptamers in the clinical trial program and introduce some promising aptamers currently in pre-clinical development for blood diseases

Label-free bacteria detection using evanescent mode of a suspended core terahertz fiber

We propose for the first time an E. coli bacteria sensor based on the evanescent field of the fundamental mode of a suspended-core terahertz fiber. The sensor is capable of E. coli detection at concentrations in the range of 104-109 cfu/ml. The polyethylene fiber features a 150 {\mu}m core suspended by three deeply sub-wavelength bridges in the center of a 5.1 mm-diameter cladding tube. The fiber core is biofunctionalized with T4 bacteriophages which bind and eventually destroy (lyse) their bacterial target. Using environmental SEM we demonstrate that E. coli is first captured by the phages on the fiber surface. After 25 minutes, most of the bacteria is infected by phages and then destroyed with ~1{\mu}m-size fragments remaining bound to the fiber surface. The bacteria-binding and subsequent lysis unambiguously correlate with a strong increase of the fiber absorption. This signal allows the detection and quantification of bacteria concentration. Presented bacteria detection method is label-free and it does not rely on the presence of any bacterial "fingerprint" features in the THz spectrum

Aptasensors Are Conjectured as Promising ALT and AST Diagnostic Tools for the Early Diagnosis of Acute Liver Injury

Abnormal levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in human serum are the most sensitive indicator of hepatocellular damage. Because liver-related health problems are directly linked to elevated levels of ALT and AST, it is important to develop accurate and rapid methods to detect these enzymes for the early diagnosis of liver disease and prevention of long-term liver damage. Several analytical methods have been developed for the detection of ALT and AST. However, these methods are based on complex mechanisms and require bulky instruments and laboratories, making them unsuitable for point-of-care application or in-house testing. Lateral flow assay (LFA)-based biosensors, on the other hand, provide rapid, accurate, and reliable results, are easy to operate, and are affordable for low-income populations. However, due to the storage, stability, batch-to-batch variations, and error margins, antibody-based LFAs are considered unaffordable for field applications. In this hypothesis, we propose the selection of aptamers with high affinity and specificity for the liver biomarkers ALT and AST to build an efficient LFA device for point-of-care applications. Though the aptamer-based LFA would be semiquantitative for ALT and AST, it would be an inexpensive option for the early detection and diagnosis of liver disease. Aptamer-based LFA is anticipated to minimize the economic burden. It can also be used for routine liver function tests regardless of the economic situation in each country. By developing a low-cost testing platform, millions of patients suffering from liver disease can be saved