240 research outputs found
Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.
Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology
Current nanotechnology advances in diagnostic biosensors
Current diagnostics present challenges that are imposed by increased life expectancy in the worldwide population. These challenges are related, not only to satisfy the need for higher performance of diagnostic tests, but also to the capacity of creating pointĂą ofĂą care, wearable, multiplexing and implantable diagnostic platforms that will allow early detection, continuous monitoring and treatment of health conditions in a personalized manner. These health challenges are translated into technological issues that need to be solved with multidisciplinary knowledge. Nanoscience and technology play a fundamental role in the development of miniaturized sensors that are cheap, accurate, sensitive and consume less power. At nanometre scale, these materials possess higher volumeĂą toĂą surface ratio and display novel properties (composition, charge, reactive sites, physical structure and potential) that are exploited for sensing purposes. These nanomaterials can therefore be integrated into diagnostic sensing platforms allowing the creation of novel technologies that tackle current health challenges. These nanomaterialĂą enhanced sensors are extremely diverse, since they use numerous types of materials, nanostructures and detection modes for a multitude of biomarkers. The purpose of this review is to summarize the current stateĂą ofĂą theĂą art of nanomaterialĂą enhanced sensors, emphasizing and discussing the diagnostic challenges that are addressed by the different engineering and nanotechnology approaches. This review also aims to identify the drawbacks of nanomaterialĂą enhanced sensors, as well as point out future developmental directions.This research was funded by FCT- FUNDAĂĂO PARA A CIĂNCIA E
TECNOLOGIA, grant numbers: PTDC/EMD-EMD/31590/2017 and
PTDC/BTM-ORG/28168/2017
NANOELECTRONIC DEVICES FOR SENSITIVE DETECTION OF BIOMARKERS IN HEALTHCARE MONITORING
In recent years, biosensors have seen an exponential rise of their applications in a number of fields including the field of health care monitoring, particularly in point-of-care diagnostics. With the contemporary rise of nanotechnology, these biosensors have experienced an ever-growing inclusion of nano scale electronic devices or nanoelectronic devices to exploit the plethora of advantages of nanoelectronics. The performances of these nanoelectronic devices, however, largely depend on the nanomaterials used. Especially, carbon-based nanomaterials such as carbon nanotubes (CNTs) and graphene have proven to be superior candidates compared to others because of their multitude of electronic and mechanical properties suitable for biosensing. In particular, graphene-based FET (GFET) that combines the favorable material properties of graphene as well as the device properties of field-effect transistor have demonstrated its potential in biosensing with high sensitivity and signal-to-noise ratio (SNR). Though GFETs have been applied for sensitive detection of a number of analytes, there are still areas for further development in a number of waysâapplication of the platform for sensing new biomarkers, developing an integrated microfluidics platform, etc. in order to improve the sensing performances as well as applicability in real-world setting. Therefore, in this seminar, I will discuss the current states and challenges of the GFET-based sensing and present my work to further advance this platform. Moreover, development of a flexible GFET biosensor compatible with wearable platform will also be discussed. To provide the biosensors with the required selectivity, DNA-based aptamers with specific affinity towards the target analyte are used. However, conventional techniques for functionalization of aptamers suffer from several challenges including low throughput, poor control, and long turnaround time. To address these challenges, I will present my efforts on the development of new strategies to address these challenges both on CNT and graphene-based platforms
Silver nanocluster-based colorimetric/fluorimetric dual-mode sensor for the detection of bromide and sulfite in waters and wastewaters
In this work, the development of a fluorimetric/colorimetric dual-mode nanosensor for the determination of sulfite and fluorimetric determination of bromide involving silver nanoclusters (AgNCs) is reported. SO2 and Br2 were found to significantly modify the optical properties of AgNCs. Particularly, both volatiles weakened the
fluorescence of AgNCs, whereas a color change from nearly colorless to yellowish/brown occurred upon exposure of AgNCs to SO2. Accordingly, three smartphone-based optical assays were devised for sulfite and bromide determination, involving in situ volatile generation and enrichment/trapping of the selectively formed volatiles by AgNCs confined in a droplet and exposed to the headspace above the sample. A hydrophobized cellulose substrate acting as drop holder enabled integrating both the enrichment and the subsequent smartphone-based optical detection in a straightforward manner. Smartphone-based digitization of the enriched AgNCs microdrops and subsequent image processing using a smartphone and its integrated App, respectively, were used for
quantitative purposes. Under optimal conditions, limits of detection (LODs) of 1.1 ÎŒM and 1.5 ÎŒM were achieved for the fluorimetric determination of sulfite and bromide, respectively, whereas sulfite was alternatively determined by colorimetric readout, yielding a LOD of 37.0 ÎŒM. The repeatability, expressed as relative standard
deviation, was found to be in the range of 5.1â5.9 % in all cases (N = 8). The applicability of the method was demonstrated in aqueous samples of increasing complexity, with recoveries in the range 91â109 %. In addition, the responsiveness of AgNCs to SO2 and Br2 rendered them suitable for the monitoring of bromide and sulfite in
increasingly relevant advanced reduction processes such as the UV/sulfite system, as demonstrated in this work.Agencia Estatal de InvestigaciĂłn | Ref. PID2022-136337OB-I00Universidade de Vigo/CISU
Development of nanocatalytic-based assay for the detection of an endocrine disrupting compound in aqueous solution
Endocrine disrupting compound (EDC) pollutants raise a concern among researchers as these pollutants are implicated in the increasing incidence of testicular, breast and thyroid cancers. Some of these chemicals are widely used for plastics production and discharged into the water system as industrial effluents that could harm the ecosystem as well as plant, animal and human life. Thus, rapid detection and quantification of EDCs in water is desired for screening and investigative purposes. For this purpose, nanoparticle-based methods appear to be potentially efficient, quick and cost-effective techniques to rapidly assess this toxic pollutant.
The main focus of this study was to synthesize heterogeneous nanoparticles, iron oxide/gold nanoparticles (IONPs/AuNPs) and to manipulate their synergistic effects for the development of a nanoparticles-based assay, specifically for the EDC compound, 17ÎČ-estradiol. As the first step, IONPs and AuNPs were synthesized separately and heterogeneous nanoparticles were formed by a simple electrostatic- self- assembly technique. The unique physiochemical properties of this hybrid nanoparticle were investigated as a supporting material for biomolecules, as well for its intrinsic peroxidase-like activity using a hydrogen peroxidase dependent system.
The formation of the IONPs/AuNPs was verified using several characterization tools such as UV-Vis spectrophotometry, Dynamic Light Scattering (DLS), Transmission Electron Microscope (TEM), Energy Dispersive X-ray (EDX) and X-ray Photoelectron Spectroscopy (XPS). The diameter calculated from TEM was 16.1 ± 11.1 nm and EDX confirmed the presence of the Fe and Au elements. From a heterostructural analysis using HRTEM and XPS data, an alloy-like morphology (Fe/Au) was suggested for the heterogeneous nanoparticles, rather than a core-shell structure. The Fe/Au nanoparticles showed good potential for the basis of a colorimetric assay for glucose detection using glucose oxidase immobilized on the Fe/Au surface. In addition, the Fe/Au nanoparticles also showed a significant peroxidase-like activity. A nanocatalytic-based assay was developed by modifying the nanoparticles surface with an aptamer in order to specifically âcaptureâ the target molecule, 17ÎČ-estradiol. The formation of a Fe/Au-17ÎČ-estradiol complex significantly hampered the peroxidase-like catalytic activity resulting in the development of a unique nanosensor system based on the extent of loss of peroxidase activity.
Development of the nanocatalytic-based assay suggests the potential application of Fe/Au nanoparticles to capture, separate and detect a selective target as well as a basis for the development of a rapid, simple and reliable detection tool. The heterogeneous Fe/Au nanoparticles show a remarkable synergistic property for application in nanosensor system. Therefore, some of the work presented here can be extended in certain major directions such as heterostructure formation and optimization of nanocatalytic-based assay
Lab-on-a-chip Thermoelectric and Solid-phase Immunodetection of Biochemical Analytes and Extracellular Vesicles: Experimental and Computational Analysis
Microfluidics is the technology of controlling and manipulating fluids at the microscale. Microfluidic platforms provide precise fluidic control coupled with low sample volume and an increase in the speed of biochemical reactions. Lab-on-a-chip platforms are used for detection and quantification of biochemical analytes, capture, and characterization of various proteins, sensitive analysis of cytokines, and isolation and detection of extracellular vesicles (EVs). This study focuses on the development of microfluidic and solid-phase capture pin platforms for the detection of cytokines, extracellular vesicles, and cell co-culture. The fabrication processes of the devices, experimental workflows, numerical analysis to identify optimal design parameters, and reproducibility studies have been discussed. Layer-by-layer assembly of polyelectrolytes has been developed to functionalize glass and stainless-steel substrates with biotin for the immobilization of streptavidinconjugated antibodies for selective capture of cytokines or EVs. Microstructure characterization techniques (SEM, EDX, and fluorescence microscopy) have been implemented to assess the efficiency of substrate functionalization. A detailed overview of current methods for purification and analysis of EVs is discussed as well.
Additionally, the dissertation demonstrates the feasibility of a calorimetric microfluidic immunosensor with an integrated antimony-bismuth (Sb/Bi) thermopile sensor for the detection of cytokines with picomolar sensitivity. The developed platform can be used for the universal detection of both exothermic or endothermic reactions. A three-dimensional numerical model was developed to define the critical design parameters that enhance the sensitivity of the platform. Mathematical analyses identified the optimal combinations of substrate material and dimensions that will maximize the heat transfer to the sensor. Lab-on-a-chip cell co-culture platform with integrated pneumatic valve was designed, numerically characterized, and fabricated. This device enables the reversible separation of two cell culture chambers and serves as a tool for the effective analysis of cell-to-cell communication. Intercellular communication is mediated by extracellular vesicles. A protocol for the functionalization of stainless-steel probe with exosomespecific CD63 antibody was developed. The efficiency of the layer-by-layer deposition of polyelectrolytes and the effectiveness of biotin and streptavidin covalent boding were characterized using fluorescent and scanning electron microscopy
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Development and Application of a Synthetic Near Infrared Fluorescent Probe for Imaging Modulatory Neurotransmitters
Dopamine neurotransmission plays critical roles in brain function in both health anddisease and aberrations in dopamine neurotransmission are implicated in severalpsychiatric and neurological disorders, including schizophrenia, depression, anxiety, andParkinsonâs disease. Until recently, measuring the dynamics of dopamine and otherneurotransmitters of this class could not be achieved at spatiotemporal resolutionsnecessary to study how dopamine regulates the plasticity and function of neurons and neuralcircuits, and how dysfunctions in this regulation lead to disease. Probes that satisfy criticalattributes in spatiotemporal resolution and chemical selectivity are needed to facilitateinvestigations of dopamine neurochemistry.To address this need, this dissertation describes the synthesis and implementation ofan ultrasensitive near-infrared âturn-onâ nanosensor (nIRCat) for the catecholamineneuromodulators dopamine and norepinephrine. To guide probe development, we presentresults from a computational model that offers insight into the spatiotemporal dynamics ofdopamine in the striatum, a subcortical structure that is enriched in dopamine. With thismodel, we elucidated the kinetic requirements for a prototypical optical indicator as well asoptimal imaging frame rates needed for measuring dopamine neurochemical dynamics.Stochastic modeling of dopamine dynamics, driven by kinetic phenomena of vesicularrelease, diffusion and clearance, provide a platform to evaluate dopaminergic volumetransmission arising from a single terminal or ensemble terminal activity. With this work,we illustrate that only probes with kinetic parameters in a particular range are feasible fordopamine imaging at spatiotemporal scales likely to be encountered in brain tissue.In two subsequent chapters, we describe the development and in vitrocharacterization of nIRCats, synthesized from functionalized single wall carbon nanotubes(SWCNT) that fluoresce in the near infrared range of the spectrum. We show that nIRCatsexhibit maximal relative change in fluorescence intensity (ÎF/F0) of up to 35-fold inresponse to catecholamines and have optimal dynamic range that span physiologicalconcentrations of their target brain analytes. Through a combination of experimental andmolecular dynamics approaches, we elucidate the photophysical principles and intermolecularinteractions that govern the molecular recognition and fluorescence modulation of nIRCats by dopamine.Finally, we demonstrate that nIRCat can be used to measure electrically andoptogenetically evoked release of dopamine in striatal brain slices, revealing hotspots ofactivity with a median size of 2 ÎŒm, and exhibiting a log-normal size distribution that extendsup to 10 ÎŒm. Moreover, nIRCats are shown to be compatible with dopamine pharmacologyand permit studies of how receptor-targeting drugs modulate evoked dopamine release. Ourresults suggest nIRCats may uniquely support similar explorations of processes that regulatedopamine neuromodulation at the level of individual synapses, and exploration of the effectsof receptor agonists and antagonists that are commonly used as psychiatric drugs andpsychoactive molecules that modulate the release and clearance profiles of dopamine. Weconclude that nIRCats and other nanosensors of this class can serve as versatile syntheticoptical tools to monitor interneuronal chemical signaling in the brain extracellular space atspatial and temporal scales pertinent to the encoded information
Functionalized nanopipettes: toward label-free, single cell biosensors
Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms
Aptamer-Functionalized Nano-Biosensors
Nanomaterials have become one of the most interesting sensing materials because of their unique size- and shape-dependent optical properties, high surface energy and surface-to-volume ratio, and tunable surface properties. Aptamers are oligonucleotides that can bind their target ligands with high affinity. The use of nanomaterials that are bioconjugated with aptamers for selective and sensitive detection of analytes such as small molecules, metal ions, proteins, and cells has been demonstrated. This review focuses on recent progress in the development of biosensors by integrating functional aptamers with different types of nanomaterials, including quantum dots, magnetic nanoparticles (NPs), metallic NPs, and carbon nanotubes. Colorimetry, fluorescence, electrochemistry, surface plasmon resonance, surface-enhanced Raman scattering, and magnetic resonance imaging are common detection modes for a broad range of analytes with high sensitivity and selectivity when using aptamer bioconjugated nanomaterials (Apt-NMs). We highlight the important roles that the size and concentration of nanomaterials, the secondary structure and density of aptamers, and the multivalent interactions play in determining the specificity and sensitivity of the nanosensors towards analytes. Advantages and disadvantages of the Apt-NMs for bioapplications are focused
Single Molecule Particle Analysis using Nanotechnology
Nanotechnology is the area of science that involves creation of devices/materials or systems in the nanometer scale. The last few decades have seen an increasing demand for rapid, sensitive, and cheaper diagnostic tools in healthcare. Advances in fabrication technologies have led to more miniaturized systems that are satisfying the promise of âmicro total analysisâ or âlab-on-chipâ systems by facilitating the integration of multiple processing steps into a single device or multiple task-specific devices into a fluidic motherboard (i.e., modular microfluidics). The field of nanotechnology has the ability to revolutionize medical diagnostics by facilitating point-of-care testing with greater sensitivity even at the single molecule level. This allows for the screening of diseases at an early stage by identifying biomarkers of the diseases that are in extremely low concentrations in the blood (i.e., liquid biopsy). To this realization, we have used thermoplastics as our choice of material to fabricate microfluidic/nanofluidic hybrid systems that can evaluate how well a patient responds to chemotherapy, identify single nucleotide polymorphisms that cause major life threatening diseases such as stroke and caner, and development of nanofluidic devices to enumerate SARS CoV-2 viral particles that causes the novel coronavirus of 2019. We developed a high-throughput nanofluidic circuit on which single DNA molecules can be stretched to near their full contour length in nanochannels (<100 nm). Patients with cancer undergoing chemotherapy have more oxidative damage in their DNA compared to a healthy individual, which is an indicator of their response to therapy. We tested the device using calf thymus DNA standards labelled with a bis-intercalating dye and the abasic sites were labelled with another dye. Thus, the DNA molecules that were stretched in the nanochannels were parked and visualized using a fluorescent microscope. The abasic sites that were labelled were identified with their position in the DNA and the number of abasic sites per 105 nucleotides identified. This technique can be effectively used on samples having mass limits (picograms range) and where PCR cannot be utilized. Higher the number of abasic sites, better the response of the patient to chemotherapy, such as doxorubicin for breast cancer patients. While this nanofluidic circuit was used only to visualize the abnormalities in DNA, the next device we developed, called the nanosensor, facilitates the integration of multiple processes into a single device. The nanosensor was used to identify point mutations in DNA or mRNA responsible for diseases such as cancer and stroke, respectively. The device featured 8 pixel array populated with 1 ”m pillars, which act as a solid support for Ligase Detection Reactions (spLDR) that can identify a single nucleotide mutation in a DNA from a large majority of wild type DNA. The spLDR can also identify mRNA transcripts from the design of spLDR primers that specifically recognize a unique transcript. The reaction is performed on the pixel arrays and the products are subsequently shuttled into nanometer flight tubes featuring two in-plane nanopores that act as resistive pulse sensors (RPS) to generate a current drop as the products pass through these pores. The time-of-flight (TOF) between the pores in series are used to distinguish between normal and mutated DNA, thus acting as a diagnostic appropriate for the precision medicine initiative. We were able to successfully fabricate the device, run COMSOL simulations to test operation using both hydrodynamic and electrokinetic flows, which were verified via experimentation to establish the functionality of the device to perform the above mentioned processes. The hydrodynamic flow operations used for spLDR was tested using Rhodamine B and the electrokinetic flow to inject the products of the spLDR into the flight tube was tested using oligonucleotides (25mer). Further, plastic-based nanofluidic devices were extended to detect the presence of SARS-CoV-2 viral particles using a nanopore of 350 nm in effective diameter, which has called a nano-coulter Counter (nCC). Briefly, saliva samples containing the viral particles were run through a microfluidic affinity chip containing pillars with surface-immobilized aptamers specific to the SARS-CoV-2 particles. The captured viral particles were released from the microfluidic chip using a blue light and the elute containing only the SARS viral particles were sent to the nCC, which used the RPS technique to count the number of particles. We designed multiple iterations of the nCC and used COMSOL simulations to guide device development. Using the combined principle of hydrodynamic and electrokinetic flow to introduce the viral particles into the nCC, we were able to detect patients with COVID-19 as well as estimate the viral load in SARS CoV-2 standards based on the frequency of the signals generated by correlating the results to a calibration curve. Thus, this combined multi-chip process can diagnose COVID-19 in <20 min thus venturing as an in-home diagnostic kit in the future by automating the operations into a hand-held device
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