Paper-Based SERS Platform for One-Step Screening of Tetracycline in Milk

PTDC/CTM-NAN/2912/2014. PTDC/AAG-TEC/5400/2014. POCI-01-0145-FEDER-016637. POCI-01-0145-FEDER-007688. UID/CTM/50025/2019. FCT/MCTES SFRH/BD/115173/2016 and SFRH/BPD/124311/2016 .Throughout the last decade, the expansion of food testing has been gradually moving towards ordinary high throughput screening methods performed on-site. The demand for point-of-care testing, able to distinguish molecular signatures with high accuracy, sensitivity and specificity has been significantly increasing. This new requirement relies on the on-site detection and monitorization of molecular signatures suitable for the surveillance of food production and processing. The widespread use of antibiotics has contributed to disease control of livestock but has also created problems for the dairy industry and consumers. Its therapeutic and subtherapeutic use has increased the risk of contamination in milk in enough concentrations to cause economic losses to the dairy industry and have a health impact in highly sensitive individuals. This study focuses on the development of a simple Surface-Enhanced Raman Spectroscopy (SERS) method for fast high throughput screening of tetracycline (TET) in milk. For this, we integrate a paper-based low-cost, fully recyclable and highly stable SERS platform, with a minimal sample preparation protocol. A two-microliter sample of milk solutions spiked with TET (from 0.01 to 1000 ppm) is dried on a silver nanoparticle coated cardboard substrate and measured via a Raman spectrophotometer. The SERS substrate showed to be extremely stable with a shelf life of several months. A global spectrum principal component analysis approach was used to test all the detected vibrational modes and their correlation with TET concentration. Peak intensity ratios (455 cm−1/1280 cm−1 and 874 cm−1/1397 cm−1) were found to be correlated with TET concentrations in milk, achieving a sensitivity as low as 0.1 ppm. Results indicate that this SERS method combined with portable Raman spectrometer is a potential tool that can be used on-site for the monitoring of TET residues and other antibiotics.publishersversionpublishe

Rapid Raman spectroscopic analysis of stress induced degradation of the pharmaceutical drug tetracycline

Stress factors caused by inadequate storage can induce the unwanted degradation of active compounds in pharmaceutical formulations. Resonance Raman spectroscopy is presented as an analytical tool for rapid monitoring of small concentration changes of tetracycline and the metabolite 4-epianhydrotetracycline. These degradation processes were experimentally induced by changes in temperature, humidity, and irradiation with visible light over a time period of up to 23 days. The excitation wavelength ?exc = 413 nm was proven to provide short acquisition times for the simultaneous Raman spectroscopic detection of the degradation of tetracycline and production of its impurity in small sample volumes. Small concentration changes could be detected (down to 1.4% for tetracycline and 0.3% for 4-epianhydrotetracycline), which shows the potential of resonance Raman spectroscopy for analyzing the decomposition of pharmaceutical products. © 2020 by the authors

Aptamers for pharmaceuticals and their application in environmental analytics

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications

Selection and characterization of DNA aptamers for estradiol and ethynylestradiol for aptasensor development

Small organic contaminants have been widely detected in the surface and ground waters of this nation. A sub-class of these contaminants called endocrine disrupting compounds (EDCs) are known to have adverse effects on aquatic and human health. Among the EDCs, natural hormone 17β-estradiol (E2) and synthetic hormone 17α-ethynylestradiol (EE) possess high estrogenic potency and hence are contaminants of interest. Conventional methods to detect these compounds are expensive, time consuming and need implementation by an expert. By contrast, antibody-based assays are relatively inexpensive and commercially available but suffer from poor selectivity. A promising alternative makes use of DNA aptamers as molecular recognition elements. In order to evaluate the potential of DNA aptamers and aptasensors to detect small organics in natural waters, the following objectives were pursued: (1) critically review DNA aptamers and aptasensors developed for small organic molecules and assess their use for monitoring environmentally relevant organics, (2) select and characterize DNA aptamers that bind to E2 and EE and, (3) study the effect of immobilization on the binding affinity of the selected E2 and EE aptamers. A review of ~80 aptamers and ~200 aptasensors for small organics was conducted to identify factors that affect binding affinity of the aptamer and limits of detection (LODs) of the aptasensor. Based on regression analyses, aptamer binding affinities are found to have a weak relationship with hydrophobicity of the target and length of the aptamer (p-values<0.05). Independent t-tests comparing aptasensor LODs suggest that the electrochemical platform is significantly more sensitive than colorimetric and fluorescence-based platforms. The inherent binding affinity of the aptamer was found to have a significant effect on the LOD of the aptasensor. While some fabricated aptasensors are sufficiently sensitive to detect contaminants at environmentally relevant concentrations, they are often associated with complex fabrication steps, and/or interference from structurally similar analogs. As a result, aptasensor commercialization faces many challenges including reusability, reproducibility and robustness. In vitro selections were conducted with different selection pressures to isolate sensitive and selective DNA aptamers for E2 and EE. An equilibrium-filtration assay was used to determine dissociation constants (Kd) of the aptamer towards its parent target and its analogues. The E2 aptamers, E2Apt1 and E2Apt2 were found to have Kd values of 0.6 µM. They bound to analogue estrone (E1) with a similar affinity but were at least 74-fold more selective over EE. The EE aptamers Kd values are 0.5-1 µM. While one EE aptamer (EEApt1) was 53-fold more selective for EE over E2 and E1, the second EE aptamer (EEApt2) bound to all three EDCs (E1, E2 and EE) with similar affinities. The aptamers maintained their binding affinities in natural waters samples (tap water and lake water). DMS probing of the structure of the DNA aptamer revealed that the binding regions were mostly located in the single-stranded loop regions of the aptamer. Aptasensors typically employ immobilized aptamers though the aptamers are selected and characterized while free or unattached in solution. The Kd values of immobilized selective aptamers were evaluated using magnetic microbeads surface for attachment. E2Apt1 immobilized at either end (5′ or 3′) and E2Apt2 immobilized at the 3′ end retain their binding affinity. The binding affinity is inversely correlated to the average linear distance of the binding pocket from the immobilized end. This result suggests that unwanted interactions between the aptamer and other moieties are more likely when the binding pocket is further away from the surface. Binding curve of E2Apt2 immobilized at the 5′ end indicates potential dimerization at high loadings of aptamer on the beads due to increased proximity between aptamer strands. EEApt1 loses its binding affinity upon immobilization potentially due to disruption in its tertiary structure upon attachment to the surface. Despite no loss in binding affinity upon immobilization, E2Apt1 (5′) shows no significant change in electrochemical current on binding to E2 when incorporated into an electrochemical sensor. This result implies an insufficient conformational change of the aptamer on binding to the target. Overall, this work identifies the first aptamers for EE and selective aptamers for E2, while also highlighting the issues with development of aptamers and their eventual incorporation into aptasensors to detect small organics. Two major concerns are (1) immobilizing aptamers in sensor platforms while selections of aptamers are conducted with free/unattached aptamers, resulting in loss of binding affinity and (2) insufficient conformational change of the aptamer on binding to small molecule targets, resulting in a lack of change in the sensor signal. The findings from this dissertation support additional research directions regarding employing free aptamers in sensors and/or conducting new selections for aptamers using a DNA pool that is attached to a surface

Sample Preparation-Quo Vadis

Sample preparation is and will always be the most important step in chemical analysis. Numerous techniques, methods, methodologies, and approaches are published in the literature offering a wide range of analytical tools to the lab practitioner. Analytical scientists all over the world are trying to develop protocols for a plethora of analytes in various sample matrices. In the last decade, sample pre-treatment advances have followed green chemistry and green analytical chemistry demands, focusing on miniaturization and automation, using the least possible amount of organic solvents. The question is how far we have been till now, and what the future perspectives are. To answer this question, analytical chemists were invited to share their experience in the field and report on the recent advances in sample-preparation approaches. The outcome of our invitation was eleven excellent manuscripts, including four review articles and seven original research articles in the first edition of the Special Issue “Sample Preparation-Quo Vadis: Current Status of Sample Preparation Approaches”.The second edition is a collection of ten significant contributions to the field of sample preparation. It includes two highly interesting and comprehensive review articles and eight innovative research articles

Enhanced Raman spectroscopy as a tool for the improvement of cervical cancer screening: a comparison of global and targeted approaches

Half a million women worldwide are diagnosed with cervical cancer annually, with 87% of mortalities occurring in developing economies. HPV vaccination has reduced disease incidence significantly but must be supplemented with regular cervical screening. In established practice, cells from the cervix are fixed, stained and observed manually using an optical microscope. Inter-observer subjectivity and false diagnoses are a relatively frequent occurrence. The use of molecular pathology allows objective diagnoses to be made based on changes to the molecular content of cells and tissue with disease. This quantitative, objective and automated pathological analysis could be integrated with existing services, as well as creating potential for economically deprived regions. Molecular pathology through spectroscopic methods has shown great promise. For Raman spectroscopy, this is broadly categorised into global comparison or targeted biomarker approaches. In the global paradigm, a direct comparison of wavelength modulated with standard Raman spectroscopy showed marked improvement of sensitivity and specificity in fixed cell classification. Intracellular sampling location studies indicated that the technique is more robust for fixed than live cells. A functional nanoparticle bioconjugate for the established marker p16 was developed for targeted studies following optimisation of covalent, passive and bioconjugation methods. This was applied to fixed smear analogues, both lysed and intact, in lateral flow assay and SERS mapping contexts respectively. Considerations of hybrid SERS-SRS and global-targeted imaging were also explored, combining key vibrational modes and labelling moieties from both arms of the study. After consideration of the clinical context, the SERS-active paper-based dipstick assay was selected for further development. Novel binding peptides for p16 capture were discovered and validated, alternative consensus motifs found through next-generation sequencing and SERS-based quantification demonstrated to be sufficiently sensitive to detect relevant cellular analogues. The investigations described provide both a roadmap for further development of multiple Raman diagnostic tools in a clinical context and the foundational components of a point-of-care, inexpensive test for p16 which could streamline and broaden access to cervical screening

Polysulfone nanocomposites for electrochemical screening of antibiotic residues

Philosophiae Doctor - PhD (Chemistry)Antibiotics are defined as any class of organic molecule that kills or inhibits microbes by specific interactions with bacterial targets. Antibiotics may be classified based on bacterial spectrum (broad versus narrow), route of administration, type of activity (bactericidal versus bacteriostatic), and origin (natural versus synthesized), and can also be classified based on their chemical structure. The intensive use of antibiotics for human (domestic and hospital use), veterinary and agriculture purposes, these compounds are continuously released into the environment from anthropogenic sources, such as wastewater treatment plants which are considered as one of the major source of evolution and spreading of antibiotic resistance into the environment

Fluorescent-based nanosensors for selective detection of a wide range of biological macromolecules: A comprehensive review

Thanks to their unique attributes, such as good sensitivity, selectivity, high surface-to-volume ratio, and versatile optical and electronic properties, fluorescent-based bioprobes have been used to create highly sensitive nano -biosensors to detect various biological and chemical agents. These sensors are superior to other analytical instrumentation techniques like gas chromatography, high-performance liquid chromatography, and capillary electrophoresis for being biodegradable, eco-friendly, and more economical, operational, and cost-effective. Moreover, several reports have also highlighted their application in the early detection of biomarkers associ-ated with drug-induced organ damage such as liver, kidney, or lungs. In the present work, we comprehensively overviewed the electrochemical sensors that employ nanomaterials (nanoparticles/colloids or quantum dots, carbon dots, or nanoscaled metal-organic frameworks, etc.) to detect a variety of biological macromolecules based on fluorescent emission spectra. In addition, the most important mechanisms and methods to sense amino acids, protein, peptides, enzymes, carbohydrates, neurotransmitters, nucleic acids, vitamins, ions, metals, and electrolytes, blood gases, drugs (i.e., anti-inflammatory agents and antibiotics), toxins, alkaloids, antioxidants, cancer biomarkers, urinary metabolites (i.e., urea, uric acid, and creatinine), and pathogenic microorganisms were outlined and compared in terms of their selectivity and sensitivity. Altogether, the small dimensions and capability of these nanosensors for sensitive, label-free, real-time sensing of chemical, biological, and pharma-ceutical agents could be used in array-based screening and in-vitro or in-vivo diagnostics. Although fluorescent nanoprobes are widely applied in determining biological macromolecules, unfortunately, they present many challenges and limitations. Efforts must be made to minimize such limitations in utilizing such nanobiosensors with an emphasis on their commercial developments. We believe that the current review can foster the wider incorporation of nanomedicine and will be of particular interest to researchers working on fluorescence tech-nology, material chemistry, coordination polymers, and related research areas

Sensing at nanostructures for agri-food and enviromental applications

With a predicted population increase of 2.3 billion people, by 2050, agricultural productivity must be vastly improved and made sustainable. Globally, agriculture must deliver a 60% increase in food production to cope with the population demand. Moreover, this needs to be achieved against a changing climate, an exploitation of natural resources, and growing water and land scarcities. New digital technologies can optimise production efficiency and ensure food security and safety while also minimising waste within the production systems and the supply chain. To this end, new sensor technologies are being developed for applications in animal health diagnostics and environmental issues related to the global population, such as food & crop protection, pathogen and toxin detection, and environmental remediation. In this thesis, two new nanosensing diagnostic devices are developed and presented; surface enhanced Raman sensing and electrochemical sensing. Surface-enhanced Raman spectroscopy (SERS) substrates were fabricated by templating a flexible thermoplastic polymer against an aluminium drinks can followed by coating with a silver film, to produce a rough nanostructured metallic surface. SERS is used for both qualitative (molecular fingerprint) and quantitative detection of dye molecules and food toxins. In addition, the SERS technique is also applied in combination with nanoelectrochemical square wave voltammetry to detect nano-concentrations of neonicotinoid pesticides. The enhanced sensitivity and minimum sample preparation requirements provide tremendous opportunities for food safety and security sectors. An impedimetric immunosensor device (with a micro SD style pin-out) was also developed for the serological diagnosis of viruses and antibodies associated with bovine respiratory disease and bovine liver fluke. The silicon chip devices consist of six on-chip nanoband electrodes which can be independently modified with a polymer layer for covalent immobilisation of capture and target biomolecules. This electrochemical biosensor technology provides label-free and cost-efficient sensing capability in a compact size, and demonstrates the potential development of immunoassay-based point-of-use devices for on-farm diagnosis or therapeutic monitoring in animal health applications