In situ formed magnetic ionic liquids: DNA extraction performance and fluorescence-compatibility in bioanalytical applications

Extracting genomic DNA from complex biological sample matrices is often the first step in numerous molecular biology procedures such as polymerase chain reaction (PCR), cloning, and gene therapy. Obtaining high yields and pure DNA presents a significant sample preparation challenge in nucleic acid analysis. Current methodologies such as the phenol-chloroform extraction use toxic organic solvents and commercially available kits are often very expensive and have limited reusability. Magnetic ionic liquids (MILs) have gained popularity as inexpensive, environmentally benign and tunable extraction solvents. MILs are a subclass of ionic liquids containing a paramagnetic component in the cation or anion, allowing them to be manipulated using an external magnetic field. This thesis describes the use of a new class of MILs featuring metal-containing cations for DNA extraction and their compatibility with fluorescence-based detection methods. Two studies were conducted to address this goal. The first study focused on the DNA extraction efficiency of a new class of MILs using in situ dispersive liquid-liquid microextraction (DLLME) versus conventional DLLME to assess the extraction of DNA sequences of varied sizes. Extraction efficiencies were obtained using indirect detection using anion-exchange high performance liquid chromatography with diode array detection and fluorescence spectroscopy. However, to minimize steps in the sample preparation process, it is useful to directly analyze the DNA within the enriched MIL microdroplet therefore, in a second study the fluorescence quenching effects of the MIL were evaluated. These studies provide an insight into how the paramagnetic metal (Ni, Co, Mn) and ligand used in the design of the MIL can be tailored in order to achieve highly efficient DNA extraction and the subsequent influence of the MIL on the fluorescence signal in downstream analysis

A portable device for time-resolved fluorescence based on an array of CMOS SPADs with integrated microfluidics

[eng] Traditionally, molecular analysis is performed in laboratories equipped with desktop instruments operated by specialized technicians. This paradigm has been changing in recent decades, as biosensor technology has become as accurate as desktop instruments, providing results in much shorter periods and miniaturizing the instrumentation, moving the diagnostic tests gradually out of the central laboratory. However, despite the inherent advantages of time-resolved fluorescence spectroscopy applied to molecular diagnosis, it is only in the last decade that POC (Point Of Care) devices have begun to be developed based on the detection of fluorescence, due to the challenge of developing high-performance, portable and low-cost spectroscopic sensors. This thesis presents the development of a compact, robust and low-cost system for molecular diagnosis based on time-resolved fluorescence spectroscopy, which serves as a general-purpose platform for the optical detection of a variety of biomarkers, bridging the gap between the laboratory and the POC of the fluorescence lifetime based bioassays. In particular, two systems with different levels of integration have been developed that combine a one-dimensional array of SPAD (Single-Photon Avalanch Diode) pixels capable of detecting a single photon, with an interchangeable microfluidic cartridge used to insert the sample and a laser diode Pulsed low-cost UV as a source of excitation. The contact-oriented design of the binomial formed by the sensor and the microfluidic, together with the timed operation of the sensors, makes it possible to dispense with the use of lenses and filters. In turn, custom packaging of the sensor chip allows the microfluidic cartridge to be positioned directly on the sensor array without any alignment procedure. Both systems have been validated, determining the decomposition time of quantum dots in 20 nl of solution for different concentrations, emulating a molecular test in a POC device.[cat] Tradicionalment, l'anàlisi molecular es realitza en laboratoris equipats amb instruments de sobretaula operats per tècnics especialitzats. Aquest paradigma ha anat canviant en les últimes dècades, a mesura que la tecnologia de biosensor s'ha tornat tan precisa com els instruments de sobretaula, proporcionant resultats en períodes molt més curts de temps i miniaturitzant la instrumentació, permetent així, traslladar gradualment les proves de diagnòstic fora de laboratori central. No obstant això i malgrat els avantatges inherents de l'espectroscòpia de fluorescència resolta en el temps aplicada a la diagnosi molecular, no ha estat fins a l'última dècada que s'han començat a desenvolupar dispositius POC (Point Of Care) basats en la detecció de la fluorescència, degut al desafiament que suposa el desenvolupament de sensors espectroscòpics d'alt rendiment, portàtils i de baix cost. Aquesta tesi presenta el desenvolupament d'un sistema compacte, robust i de baix cost per al diagnòstic molecular basat en l'espectroscòpia de fluorescència resolta en el temps, que serveixi com a plataforma d'ús general per a la detecció òptica d'una varietat de biomarcadors, tancant la bretxa entre el laboratori i el POC dels bioassaigs basats en l'anàlisi de la pèrdua de la fluorescència. En particular, s'han desenvolupat dos sistemes amb diferents nivells d'integració que combinen una matriu unidimensional de píxels SPAD (Single-Photon Avalanch Diode) capaços de detectar un sol fotó, amb un cartutx microfluídic intercanviable emprat per inserir la mostra, així com un díode làser UV premut de baix cost com a font d'excitació. El disseny orientat a la detecció per contacte de l'binomi format pel sensor i la microfluídica, juntament amb l'operació temporitzada dels sensors, permet prescindir de l'ús de lents i filtres. Al seu torn, l'empaquetat a mida de l'xip sensor permet posicionar el cartutx microfluídic directament sobre la matriu de sensors sense cap procediment d'alineament. Tots dos sistemes han estat validats determinant el temps de descomposició de "quantum dots" en 20 nl de solució per a diferents concentracions, emulant així un assaig molecular en un dispositiu POC

Application of SERS for Nanomaterials

This Special Issue of “Applications of SERS” for Nanomaterials is a collection of articles which is representative of much of the current research being undertaken in the field of Surface-Enhanced Raman Scattering (SERS) spectroscopy. SERS is a fascinating, multidisciplinary field of scientific study which combines elements from chemistry, physics, material science, and engineering. Essentially, SERS is a molecular spectroscopy technique by which a measurable Raman signal for molecules on metal and semiconductor surfaces is generated through the interaction of laser light, absorbed molecules, and structured nanomaterial surfaces. This Special Issue contains an article regarding the fabrication of metal nanostructured Ag-Cu chips for SERS chemical analysis and ] the electromagnetic properties of Ag, Au, and Al nano-tips for use in the SERS imaging technique for tip-enhanced Raman Scattering (TERS). In another article, SERS spectra were simulated using density Functional Theory (DFT/TD-DFT) for the N3 dye molecule on a TiO2 nanocluster, which can be compared to experimental SERS spectra found in studies of Dye-Sensitized Solar Cells (DSSCs). In two other articles, the SERS photoinduced charge-transfer mechanism was studied experimentally in wide-bandgap semiconductors regarding molecules on ZrO2 and a composite system with molecules that linked Au nanorods and a CuO2 shell. An example of the use of SERS in solid-state physics is shown in an article which examined the effect of oxygen vacancy defects in MO3 on the SERS mechanism. Finally, this Special Issue contains two noteworthy examples of SERS applications for biochemical and chemical analysis. One paper addresses the detection of the COVID-19 coronavirus SARS-CoV-2 using SERS, and the other examines a SERS assay of the notorious herbicide glyphosate. In these papers, nanomaterials all served as the enhancing substrate, while some acted as the physical–chemical system which was being investigated

Genotoxic effects in human peripheral lymphocytes from healthy individuals and head and neck cancer patients after treatment with hydrogen peroxide and pembrolizumab liposome

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. It has commonly been associated with exposure to tobacco-derived carcinogens and alcohol consumption. Pembrolizumab has shown to be effective in the treatment of many types of cancers such as melanoma, non-small cell lung cancer, due to its antiproliferative, immunoregulatory properties. The aim of this study was to investigate the effects of naked Pembrolizumab and Pembrolizumab liposome on the level of DNA damage, gene, and protein expressions in peripheral lymphocytes from HNC patients and compared to the healthy individuals by using the Comet and micronucleus assays. Western blotting and real-time polymerase chain reaction were performed to assess the potential of improving the repair mechanisms after treatment with naked Pembrolizumab and Pembrolizumab liposome. According to the results, Comet assay and micronucleus assay showed a significantly decreased DNA damage in the lymphocytes from HNC patients after being treated with naked Pembrolizumab and pembrolizumab liposome. Furthermore, the results have shown that naked Pembrolizumab and pembrolizumab liposomes (10 μg/ml) greatly decreased the oxidative stress produced by H2O2. Both forms of pembrolizumab have also demonstrated improving the repair mechanisms in lymphocytes from HNC patients by modulating the expression of P53, P21, and Bcl-2 at mRNA and protein levels. This study suggested that Pembrolizumab naked and liposome could have an antioxidant role alongside other actions in the treatment of HNSCC. However, further studies on Cancer cell lines and in vivo observation are required to validate the anticancer potential of pembrolizumab naked with liposome in HNC.Ministry of Higher Education and Scientific Research, Liby

Synthesis, characterisation and biological activity of FITC-insulin for the development of an artificial pancreas

In advanced drug development and delivery, fluorescence studies have clarified and improved aspectssuch as biodistribution, stability and metabolism with respect to the complexities imposed by thebiological systems. The ultimate aim of this study was to assess the delivery kinetics of a synthesisedfluorescently labelled insulin in an implantable artificial pancreas (INsmart device) developed by Tayloret al, 2016, which is currently being tested in-vivo. The first objective of this thesis was to produce a fluorescein isothiocyanate (FITC)-insulin conjugatewhich shows equivalent biological activity to native insulin under novel reaction conditions without theneed for using protecting groups and multi-step synthetic conditions. Secondly, the stability andsolubility profiles of the synthesised FITC-insulin conjugate in solution will be investigated for long-term storage and future applications. Thirdly, physiologically relevant glucose concentrations will beused to assess the performance of FITC-insulin delivery from an INsmart device. Lastly, other dyessuch as eosin isothiocyanate (EITC) and rhodamine B isothiocyanate (RBITC) will be assessed aslabelling candidates to produce other derivatised insulin conjugates. Mono-labelled FITC-insulin conjugate was successfully synthesised using a molar ratio of 2:1 (FITC:insulin) with short reaction times (up to 18h) at pH7 after studies were conducted to examine theeffects of reaction time, molar ratio and pH. The labelling position of this mono-labelled species wasidentified by MS-Orbitrap Fusion at the B1 residue (MonoB1). However, during synthesis,MonoB1conjugate always contains some trace amounts of unlabelled insulin that required furtherpurification by RP-HPLC using a gradient method. This HPLC method could identify four FITC-insulinconjugates including two mono-labelled species (labelled at the A1 or B1 position), di-labelled species(labelled at the A1 and B1) and tri-labelled species (labelled at the A1, B1 and B29). Further analysiswas performed using MALDI-MS to confirm the molecular weight of each conjugate produced. The biological activity of four FITC-insulin conjugates was assessed in human umbilical veinendothelial cells (HUVEC) and skeletal muscle cells (C2C12) via the insulin signalling pathway byexamining the levels of AKT phosphorylation (pAKT) and cell surface GLUT4. There was no significantdifference in pAKT and the GLUT4 cell surface levels observed for synthesised MonoB1 compared tonative insulin, highlighting that this conjugate was as biologically active as native insulin. The enhanced stability and solubility of FITC-insulin conjugate using diluting fluid containing m-cresol,glycerol and zinc oxide, which is typically contained in most commercial insulin formulations arebeneficial for setting up the in-vitro delivery study of the INsmart device. Because it allows aconcentrated depot of FITC-insulin in the device for release over an extended time. Improvement inthe smart gel formulation with diluting fluid and the use of correct membrane pore size showedpromising and reproducible results in the extended experiments where the INsmart device has beenset up and triggered multiple times with glucose and other dietary saccharides which act as controls toshow the specificity of the gel to glucose challenges. The results indicated that the device is capable ofdelivering basal insulin dose which can be boosted in response to multiple (11x) mealtime glucosesurges over a 5-day period. EITC and RBITC were used as fluorescent candidates to assess whether the same syntheticmethodology of fluorescently labelled insulin was applicable to different dye sizes. Mono-labelledinsulin conjugate with EITC was achieved using the same reaction conditions as in FITC-insulinsynthesis. Further development is still needed to remove unreacted RBITC during the synthesis ofRBITC-insulin conjugate

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials