RESUMO
With the rapid development of digital precision medicine, the digital polymerase chain reaction (dPCR) deoxyribonucleic acid (DNA) gene chip integrates more channels with smaller size and larger area, which leads to a higher technical requirement for commercial optical fluorescence microscopy. The multitime image splicing method is widely used for DNA detection. However, it consumes time and has visible seamless image results. This work has demonstrated the design and fabrication of a three channel reversed and reduced fluorescence microscopic imaging system with high-resolution and large field of view for one-time imaging. We introduced the super ultra-thin dichroic mirror into the space between the objective lens and the gene chip to achieve a uniform illumination and a strong signal for the large area gene chip. The fabricated new fluorescence microscopy can take a one-time imaging for the 28×18mm dPCR gene chip with more than 20,000 multi micro-droplets within FAM, HEX, and ROX fluorescence channels. The optical system was designed with a numerical aperture (NA) of 0.106. Modulation transfer function (MTF) is higher than 0.675 at 70 lp/mm, and the function resolution capability is 10 µm with the whole magnification of -0.65times. The fly's eye lens-based illumination system was tested with a uniform output of over 90% in the whole Ï34.7mm chip area. The design was tested, and the experimental results showed that this new system provides a fast, efficient, and professional optical imaging method for detection of the new emerged digital PCR gene chip, which has larger area and more channels.
Assuntos
Dispositivos Lab-On-A-Chip , Microscopia de Fluorescência/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , DNA/genética , Desenho de Equipamento , Microquímica/instrumentação , Imagem ÓpticaRESUMO
O/W nanoemulsions are isotropic colloidal systems constituted of oil droplets dispersed in continuous aqueous media and stabilised by surfactant molecules. Nanoemulsions hold applications in more widespread technological domains, more crucially in the pharmaceutical industry. Innovative nanoemulsion-based drug delivery system has been suggested as a powerful alternative strategy through the useful means of encapsulating, protecting, and delivering the poorly water-soluble bioactive components. Consequently, there is a need to generate an emulsion with small and consistent droplets. Diverse studies acknowledged that ultrasonic cavitation is a feasible and energy-efficient method in making pharmaceutical-grade nanoemulsions. This method offers more notable improvements in terms of stability with a lower Ostwald ripening rate. Meanwhile, a microstructured reactor, for instance, microchannel, has further been realised as an innovative technology that facilitates combinatorial approaches with the acceleration of reaction, analysis, and measurement. The recent breakthrough that has been achieved is the controlled generation of fine and monodispersed multiple emulsions through microstructured reactors. The small inner dimensions of microchannel display properties such as short diffusion paths and high specific interfacial areas, which increase the mass and heat transfer rates. Hence, the combination of ultrasonic cavitation with microstructures (microchannel) provides process intensification of creating a smaller monodispersed nanoemulsion system. This investigation is vital as it will then facilitate the creation of new nanoemulsion based drug delivery system continuously. Following this, the fabrication of microchannel and setup of its combination with ultrasound was conducted in the generation of O/W nanoemulsion, as well as optimisation to analyse the effect of varied operating parameters on the mean droplet diameter and dispersity of the nanoemulsion generated, besides monitoring the stability of the nanoemulsion. Scanning transmission electron microscopy (STEM) images were also carried out for the droplet size measurements. In short, the outcomes of this study are encouraging, which necessitates further investigations to be carried out to advance a better understanding of coupling microchannel with ultrasound to produce pharmaceutical-grade nanoemulsions.
Assuntos
Emulsões/química , Microquímica/instrumentação , Nanoestruturas/química , Óleo de Palmeira/química , Ultrassom/métodos , Água/química , Hexoses/química , Microquímica/métodos , Sonicação/métodos , TensoativosRESUMO
We demonstrate the use of accelerated reactions with desorption electrospray ionization mass spectrometry (DESI-MS) as a tool for predicting the outcome of microfluidic reactions. DESI-MS was employed as a high throughput experimentation tool to provide qualitative predictions of reaction outcomes, so that vast regions of chemical reactivity space may be more rapidly explored and areas of optimal efficiency identified. This work is part of a larger effort to accelerate reaction optimization to enable the rapid development of continuous-flow syntheses of small molecules in high yield. In order to build confidence in this approach, however, it is necessary to establish a robust predictive connection between reactions performed under analogous DESI-MS, batch, and microfluidic reaction conditions. In the present work, we explore the potential of high throughput DESI-MS experiments to identify trends in reactivity based on chemical structure, solvent, temperature, and stoichiometry that are consistent across these platforms. N-alkylation reactions were used as the test case due to their ease of reactant and product detection by electrospray ionization mass spectrometry (ESI-MS) and their great importance in API synthesis. While DESI-MS narrowed the scope of possibilities for reaction selection among some parameters such as solvent, others like stoichiometry and temperature still required further optimization under continuous synthesis conditions. DESI-MS high throughput experimentation (HTE) reaction evaluation significantly reduced the search space for flow chemistry optimization, thus representing a significant savings in time and materials to achieve a desired transformation with high efficiency.
Assuntos
Técnicas de Química Sintética/métodos , Microquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Alquilação , Compostos de Anilina/síntese química , Compostos de Anilina/química , Técnicas de Química Sintética/instrumentação , Desenho de Equipamento , Dispositivos Lab-On-A-Chip , Microquímica/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Espectrometria de Massas por Ionização por Electrospray/instrumentaçãoRESUMO
Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.
Assuntos
Encéfalo/metabolismo , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Microquímica/métodos , Mitocôndrias/metabolismo , Oximetria/métodos , Consumo de Oxigênio , Difosfato de Adenosina/farmacologia , Animais , Encéfalo/ultraestrutura , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Fluorometria/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microquímica/instrumentação , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/análise , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Oligomicinas/farmacologia , Fosforilação Oxidativa , Oximetria/instrumentação , Oxigênio/análise , Consumo de Oxigênio/efeitos dos fármacos , PrótonsRESUMO
Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Microquímica/métodos , Análise de Sequência de RNA/métodos , Automação Laboratorial , Redução de Custos , Estudos de Viabilidade , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Metagenômica/economia , Metagenômica/instrumentação , Microquímica/economia , Microquímica/instrumentação , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/instrumentaçãoRESUMO
This review describes briefly the high rate of counterfeiting of antimicrobial drugs with focus upon its immediate health consequences. The major part of this review encompasses accounts of the improvements achieved in the domain of miniaturization of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D). The application of this principle into the development of portable devices as well as its application to counter the health-system-crippling phenomenon of counterfeit antibiotic formulations, are discussed in the context of developing countries.
Assuntos
Antibacterianos/análise , Medicamentos Falsificados/análise , Fraude/prevenção & controle , Química Verde/métodos , Microquímica/métodos , Países em Desenvolvimento/economia , Condutividade Elétrica , Eletroforese Capilar/economia , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Eletroforese Capilar/tendências , Fraude/economia , Química Verde/economia , Química Verde/instrumentação , Química Verde/tendências , Microquímica/economia , Microquímica/instrumentação , Microquímica/tendências , Sensibilidade e EspecificidadeAssuntos
Biomarcadores/análise , Lágrimas/química , Pesquisa Translacional Biomédica , Humanos , Inflamação/diagnóstico , Inflamação/metabolismo , Lipídeos/análise , Metaloproteinase 9 da Matriz/análise , Microquímica/instrumentação , Microquímica/métodos , Concentração Osmolar , Osmometria/instrumentação , Osmometria/métodos , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
This is a historical account on the development of capillary LC from its beginning to the present day. The first investigations into the viability of capillary LC date back to the late 1970s, a decade after the pioneering efforts in HPLC. The drastically reduced column dimensions were required to counter the slow solute diffusion in liquids. There were numerous instrumental difficulties with sample introduction and detection in the picoliter or even femtoliter volumes. High-efficiency separations were needed in the analysis of complex biological mixtures. Miniaturization brought distinct advantages in spectroscopic and electrochemical detection. Since the 1980s, column technologies underwent significant changes: (a) from glass-drawn microcapillaries to slurry-packed, small-diameter fused silica columns; and (b) in microcapillaries packed alternatively with sub-2-µm particles or monoliths. The viability of LC-MS combination has dramatically promoted the use of small-diameter capillaries. Through "omics technologies", capillary LC/tandem MS accounts for most applications in proteomics, glycomics and metabolomics.
Assuntos
Cromatografia Líquida/história , Microquímica/instrumentação , História do Século XX , História do Século XXI , Espectrometria de Massas , Metabolômica , Miniaturização , Proteômica , Dióxido de Silício/químicaRESUMO
Glucometers present an important self-monitoring tool for diabetes patients and, therefore, must exhibit high accuracy as well as good usability features. Based on an invasive photometric measurement principle that drastically reduces the volume of the blood sample needed from the patient, we present a framework that is capable of dealing with small blood samples, while maintaining the required accuracy. The framework consists of two major parts: 1) image segmentation; and 2) convergence detection. Step 1 is based on iterative mode-seeking methods to estimate the intensity value of the region of interest. We present several variations of these methods and give theoretical proofs of their convergence. Our approach is able to deal with changes in the number and position of clusters without any prior knowledge. Furthermore, we propose a method based on sparse approximation to decrease the computational load, while maintaining accuracy. Step 2 is achieved by employing temporal tracking and prediction, herewith decreasing the measurement time, and, thus, improving usability. Our framework is tested on several real datasets with different characteristics. We show that we are able to estimate the underlying glucose concentration from much smaller blood samples than is currently state of the art with sufficient accuracy according to the most recent ISO standards and reduce measurement time significantly compared to state-of-the-art methods.
Assuntos
Algoritmos , Automonitorização da Glicemia/métodos , Glicemia/análise , Coleta de Amostras Sanguíneas/métodos , Microquímica/métodos , Fotometria/métodos , Automonitorização da Glicemia/instrumentação , Humanos , Microquímica/instrumentação , Fotometria/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Reprodutibilidade dos Testes , Tamanho da Amostra , Sensibilidade e EspecificidadeRESUMO
Bipolar disorder is characterized as a manic-depressive psychiatric syndrome with life-threatening risks to the patient. Diagnosed individuals undergo long-term lithium therapy which has proven to be effective for mood stabilization. Maintaining blood lithium concentration levels within a narrow therapeutic window between 0.6 and 1.5 mM is vital for the patient as slightly elevated concentrations of the order of 0.1 mM can be toxic. This paper aims to evaluate the merits of tetrapolar electrical impedance spectroscopy as an alternative method in monitoring blood lithium levels. Measurements were performed using a custom-made tetrapolar probe in human blood plasma with lithium concentrations covering the therapeutic range. The results indicate a limit of detection less than 0.1 mM and a response time of less than 5 s. Prediction of lithium concentration levels using impedance values is in good agreement with conventional standard techniques to approximately 0.05 mM. This technique provides a basis for further development of instrumentation for point of care healthcare technologies.
Assuntos
Análise Química do Sangue/instrumentação , Espectroscopia Dielétrica/instrumentação , Monitoramento de Medicamentos/instrumentação , Eletrodos , Compostos de Lítio/sangue , Microquímica/instrumentação , Análise Química do Sangue/métodos , Desenho Assistido por Computador , Espectroscopia Dielétrica/métodos , Monitoramento de Medicamentos/métodos , Condutividade Elétrica , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Compostos de Lítio/uso terapêutico , Microquímica/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
This review shows the recent advances and state of the art in paper-based analytical devices (PADs) through the analysis of their integration with microfluidics and LOC micro- and nanotechnologies, electrochemical/optical detection and electronic devices as the convergence of various knowledge areas. The important role of the paper design/architecture in the improvement of the performance of sensor devices is discussed. The discussion is fundamentally based on µPADs as the new generation of paper-based (bio)sensors. Data about the scientific publication ranking of PADs, illustrating their increase as an experimental research topic in the past years, are supplied. In addition, an analysis of the simultaneous evolution of PADs in academic lab research and industrial commercialization highlighting the parallelism of the technological transfer from academia to industry is displayed. A general overview of the market behaviour, the leading industries in the sector and their commercialized devices is given. Finally, personal opinions of the authors about future perspectives and tendencies in the design and fabrication technology of PADs are disclosed.
Assuntos
Pesquisa Biomédica/métodos , Desenho de Equipamento , Pesquisa Interdisciplinar , Dispositivos Lab-On-A-Chip/tendências , Papel , Animais , Pesquisa Biomédica/instrumentação , Pesquisa Biomédica/tendências , Celulose/química , Colódio/química , Eletroquímica/instrumentação , Eletroquímica/métodos , Eletroquímica/tendências , Desenho de Equipamento/tendências , Humanos , Proteínas Imobilizadas/metabolismo , Pesquisa Interdisciplinar/tendências , Microquímica/instrumentação , Microquímica/métodos , Microquímica/tendências , Microfluídica/instrumentação , Microfluídica/métodos , Microfluídica/tendências , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Nanotecnologia/tendências , Óptica e Fotônica/instrumentação , Óptica e Fotônica/métodos , Óptica e Fotônica/tendências , Testes Imediatos/tendênciasRESUMO
Here, we introduce a simple and fast method for bonding a poly(dimethylsiloxane) (PDMS) silicone elastomer to different plastics. In this technique, surface modification and subsequent bonding processes are performed at room temperature. Furthermore, only one chemical is needed, and no surface oxidation step is necessary prior to bonding. This bonding method is particularly suitable for encapsulating biomolecules that are sensitive to external stimuli, such as heat or plasma treatment, and for embedding fracturable materials prior to the bonding step. Microchannel-fabricated PDMS was first oxidized by plasma treatment and reacted with aminosilane by forming strong siloxane bonds (Si-O-Si) at room temperature. Without the surface oxidation of the amine-terminated PDMS and plastic, the two heterogeneous substrates were brought into intimate physical contact and left at room temperature. Subsequently, aminolysis occurred, leading to the generation of a permanent seal via the formation of robust urethane bonds after only 5 min of assembling. Using this method, large-area (10 × 10 cm) bonding was successfully realized. The surface was characterized by contact angle measurements and X-ray photoelectron spectroscopy (XPS) analyses, and the bonding strength was analyzed by performing peel, delamination, leak, and burst tests. The bond strength of the PDMS-polycarbonate (PC) assembly was approximately 409 ± 6.6 kPa, and the assembly withstood the injection of a tremendous amount of liquid with the per-minute injection volume exceeding 2000 times its total internal volume. The thermal stability of the bonded microdevice was confirmed by performing a chamber-type multiplex polymerase chain reaction (PCR) of two major foodborne pathogens - Escherichia coli O157:H7 and Salmonella typhimurium - and assessing the possibility for on-site direct detection of PCR amplicons. This bonding method demonstrated high potential for the stable construction of closed microfluidic systems socketed with biomolecule-immobilized surfaces such as DNA, antibody, enzyme, peptide, and protein microarrays.
Assuntos
Técnicas de Tipagem Bacteriana/instrumentação , Materiais Biomiméticos/química , Dimetilpolisiloxanos/química , Inspeção de Alimentos/instrumentação , Microquímica/instrumentação , Tipagem Molecular/instrumentação , Plásticos/química , Escherichia coli O157/classificação , Escherichia coli O157/isolamento & purificação , Estudos de Viabilidade , Humanos , Teste de Materiais , Oxirredução , Cimento de Policarboxilato/química , Resinas Sintéticas/química , Salmonella typhimurium/classificação , Salmonella typhimurium/isolamento & purificação , Propriedades de Superfície , TemperaturaRESUMO
Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.
Assuntos
DNA Viral/análise , Magnetometria/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Análise de Sequência de DNA/instrumentação , Zika virus/genética , Zika virus/isolamento & purificação , DNA Viral/genética , Desenho de Equipamento , Análise de Falha de Equipamento , Nanopartículas Metálicas/química , Microquímica/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We report an approach for the simultaneous estimation of vitamin K1 (VK1) and heparin via cascaded channel multianalyte sensing probe employing fiber optic surface plasmon resonance technique. Cladding from two well separated portions of the fiber is removed and are respectively coated with thin films of silver (channel-1) and copper (channel-2). The nanohybrid of multiwalled carbon nanotube in chitosan is fabricated over silver layer for the sensing of VK1 whereas core shell nanostructure of polybrene@ZnO is coated over copper layer for the sensing of heparin. Spectral interrogation method is used for the characterization of the sensor. Analyte selectivity of both the channels is performed by carrying out experiments using independent solutions of VK1 and heparin. Experiments performed on the solution of the mixture of VK1 and heparin show red shifts in both the channels on changing the concentration of both the analytes in the mixture. The operating range of both VK1 and heparin is from 0 to 10(-3)g/l. The limit of detection of the sensor is 2.66×10(-4)µg/l and 2.88×10(-4)µg/l for VK1 and heparin respectively which are lower than the reported ones. The additional advantages of the present sensor are low cost, possibility of online monitoring and remote sensing.
Assuntos
Testes de Coagulação Sanguínea/instrumentação , Tecnologia de Fibra Óptica/instrumentação , Heparina/sangue , Ressonância de Plasmônio de Superfície/instrumentação , Vitamina K 1/sangue , Análise Química do Sangue/instrumentação , Misturas Complexas/sangue , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/instrumentação , Humanos , Microquímica/instrumentação , Nanotubos de Carbono/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Óxido de Zinco/químicaRESUMO
UNLABELLED: Small-animal nuclear imaging modalities have become essential tools in the development process of new drugs, diagnostic procedures, and therapies. Quantification of metabolic or physiologic parameters is based on pharmacokinetic modeling of radiotracer biodistribution, which requires the blood input function in addition to tissue images. Such measurements are challenging in small animals because of their small blood volume. In this work, we propose a microfluidic counting system to monitor rodent blood radioactivity in real time, with high efficiency and small detection volume (â¼1 µL). METHODS: A microfluidic channel is built directly above unpackaged p-i-n photodiodes to detect ß-particles with maximum efficiency. The device is embedded in a compact system comprising dedicated electronics, shielding, and pumping unit controlled by custom firmware to enable measurements next to small-animal scanners. Data corrections required to use the input function in pharmacokinetic models were established using calibrated solutions of the most common PET and SPECT radiotracers. Sensitivity, dead time, propagation delay, dispersion, background sensitivity, and the effect of sample temperature were characterized. The system was tested for pharmacokinetic studies in mice by quantifying myocardial perfusion and oxygen consumption with (11)C-acetate (PET) and by measuring the arterial input function using (99m)TcO4 (-) (SPECT). RESULTS: Sensitivity for PET isotopes reached 20%-47%, a 2- to 10-fold improvement relative to conventional catheter-based geometries. Furthermore, the system detected (99m)Tc-based SPECT tracers with an efficiency of 4%, an outcome not possible through a catheter. Correction for dead time was found to be unnecessary for small-animal experiments, whereas propagation delay and dispersion within the microfluidic channel were accurately corrected. Background activity and sample temperature were shown to have no influence on measurements. Finally, the system was successfully used in animal studies. CONCLUSION: A fully operational microfluidic blood-counting system for preclinical pharmacokinetic studies was developed. Microfluidics enabled reliable and high-efficiency measurement of the blood concentration of most common PET and SPECT radiotracers with high temporal resolution in small blood volume.
Assuntos
Análise Química do Sangue/instrumentação , Dispositivos Lab-On-A-Chip , Tomografia por Emissão de Pósitrons/instrumentação , Radiometria/instrumentação , Compostos Radiofarmacêuticos/sangue , Tomografia Computadorizada de Emissão de Fóton Único/instrumentação , Animais , Sistemas Computacionais , Avaliação Pré-Clínica de Medicamentos/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Camundongos Endogâmicos BALB C , Microquímica/instrumentação , Farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Droplet-based microfluidics enabling exquisite liquid-handling has been developed for diagnosis, drug discovery and quantitative biology. Compartmentalization of samples into a large number of tiny droplets is a great approach to perform multiplex assays and to improve reliability and accuracy using a limited volume of samples. Despite significant advances in microfluidic technology, individual droplet handling in pico-volume resolution is still a challenge in obtaining more efficient and varying multiplex assays. We present a highly addressable static droplet array (SDA) enabling individual digital manipulation of a single droplet using a microvalve system. In a conventional single-layer microvalve system, the number of microvalves required is dictated by the number of operation objects; thus, individual trap-and-release on a large-scale 2D array format is highly challenging. By integrating double-layer microvalves, we achieve a "balloon" valve that preserves the pressure-on state under released pressure; this valve can allow the selective releasing and trapping of 7200 multiplexed pico-droplets using only 1 µL of sample without volume loss. This selectivity and addressability completely arranged only single-cell encapsulated droplets from a mixture of droplet compositions via repetitive selective trapping and releasing. Thus, it will be useful for efficient handling of miniscule volumes of rare or clinical samples in multiplex or combinatory assays, and the selective collection of samples.
Assuntos
Escherichia coli/isolamento & purificação , Dispositivos Lab-On-A-Chip , Análise em Microsséries/instrumentação , Microquímica/instrumentação , Modelos Químicos , Análise de Célula Única/instrumentação , Dimetilpolisiloxanos/química , Módulo de Elasticidade , Emulsões , Desenho de Equipamento , Escherichia coli/química , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ensaios de Triagem em Larga Escala/instrumentação , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Mutação , Estudo de Prova de Conceito , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Estereolitografia , Imagem com Lapso de TempoRESUMO
A highly sensitive and ultrasensitive electrochemical aptasensor for platelet-derived growth factor BB (PDGF-BB) detection is fabricated based on layered molybdenum selenide-graphene (MoSe2-Gr) composites and Exonuclease III (Exo III)-aided signal amplification. MoSe2-Gr is prepared by a simple hydrothermal method and used as a promising sensing platform. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity is limited on the duplex DNAs with more than 4 mismatched terminal bases at 3' ends. Herein, aptamer and complementary DNA (cDNA) sequences are designed with four thymine bases on 3' ends. In the presence of target protein, the aptamer associates with it and facilitates the formation of duplex DNA between cDNA and signal DNA. The duplex DNA then is digested by Exo III and releases cDNA, which hybridizes with signal DNA to perform a new cleavage process. Nevertheless, in the absence of target protein, the aptamer hybridizes with cDNA will inhibit the Exo III-assisted nucleotides cleavage. The signal DNA then hybridizes with capture DNA on the electrode. Subsequently, horse radish peroxidase is fixed on electrode by avidin-biotin reaction and then catalyzes hydrogen peroxide and hydroquinone to produce electrochemical response. Therefore, a bridge can be established between the concentration of target protein and the degree of the attenuation of the obtained signal, providing a quantitative measure of target protein with a broad detection range of 0.0001-1 nM and a detection limit of 20 fM.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Condutometria/instrumentação , Exodesoxirribonucleases/química , Microquímica/instrumentação , Nanocompostos/química , Desenho de Equipamento , Análise de Falha de Equipamento , Grafite/química , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Molibdênio/química , Nanocompostos/ultraestrutura , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Fator de Crescimento Derivado de Plaquetas/química , Selênio/químicaRESUMO
We demonstrate the detection of low concentrations of allergen-specific Immunoglobulin E (IgE) in human sera using a Photonic Crystal Enhanced Fluorescence (PCEF) microarray platform. The Photonic Crystal (PC) surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cy5, was used to amplify the fluorescence signal intensity measured from a multiplexed allergen microarray. Surface-based sandwich immunoassays were used to detect and quantify specific IgE antibodies against a highly purified cat allergen (Fel d1). A comparison of the lowest detectable concentration of IgE measured by the PC microarray system and a commercially available clinical analyzer demonstrated that the PCEF microarray system provides higher sensitivity. The PCEF system was able to detect low concentrations of specific IgE (~0.02 kU/L), which is 5-17-fold more sensitive than the commercially available FDA-approved analyzers. In preliminary experiments using multi-allergen arrays, we demonstrate selective simultaneous detection of IgE antibodies to multiple allergens.
Assuntos
Autoanticorpos/sangue , Glicoproteínas/sangue , Imunoensaio/instrumentação , Imunoglobulina E/sangue , Microquímica/instrumentação , Espectrometria de Fluorescência/instrumentação , Animais , Autoanticorpos/imunologia , Técnicas Biossensoriais/instrumentação , Gatos , Desenho de Equipamento , Análise de Falha de Equipamento , Glicoproteínas/imunologia , Humanos , Imunoglobulina E/imunologia , FótonsRESUMO
An ultra-sensitive electrochemical sensor for the detection of insulin was fabricated, using low-cost and environmentally friendly nickel nanoparticles (NiNPs) by ion implantation. The morphology and structure of the NiNPs are characterized by scanning electron microscopy (SEM), revealing diameters ranging from 4 to 8 nm. The insulin assay performances were evaluated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (I-t). The NiNPs-modified indium tin oxide electrode (NiNPs/ITO) showed excellent analytical features, including ultra-high sensitivity (2140 µAµM(-1)) for detecting low concentrations of insulin, an incredibly low detection limit (10 pM) and a wide dynamic range (100 pM to 2400 pM and 1 nM to 125 nM). In addition, the NiNPs/ITO electrode was also used to analyze the insulin concentration in bovine insulin injections. The NiNPs/ITO electrode is expected to be used as a potential biosensor for insulin.