RESUMO
The unprecedented global pandemic known as SARS-CoV-2 has exercised to its limits nearly all aspects of modern viral diagnostics. In doing so, it has illuminated both the advantages and limitations of current technologies. Tremendous effort has been put forth to expand our capacity to diagnose this deadly virus. In this work, we put forth key observations in the functionality of current methods for SARS-CoV-2 diagnostic testing. These methods include nucleic acid amplification-, CRISPR-, sequencing-, antigen-, and antibody-based detection methods. Additionally, we include analysis of equally critical aspects of COVID-19 diagnostics, including sample collection and preparation, testing models, and commercial response. We emphasize the integrated nature of assays, wherein issues in sample collection and preparation could impact the overall performance in a clinical setting.
Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pandemias , SARS-CoV-2/isolamento & purificação , COVID-19/virologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , RNA Viral/análise , SARS-CoV-2/genética , Manejo de Espécimes/métodosRESUMO
The availability of clean drinking water is a significant problem worldwide. Many technologies exist for purifying drinking water, however, many of these methods require chemicals or use simple methods, such as boiling and filtering, which may or may not be effective in removing waterborne pathogens. Present methods for detecting pathogens in point-of-use (POU) sterilized water are typically time prohibitive or have limited ability differentiating between active and inactive cells. This work describes a rapid electrochemical sensor to differentially detect the presence of active Escherichia coli (E. coli) O157:H7 in samples that have been partially or completely sterilized using a new POU electrocatalytic water purification technology based on superradicals generated by defect laden titania (TiO2) nanotubes. The sensor was also used to detect pathogens sterilized by UV-C radiation for a comparison of different modes of cell death. The sensor utilizes immunomagnetic bead separation to isolate active bacteria by forming a sandwich assay comprised of antibody functionalized secondary magnetic beads, E. coli O157:H7, and polyguanine (polyG) oligonucleotide functionalized secondary polystyrene beads as an electrochemical tag. The assay is formed by the attachment of antibodies to active receptors on the membrane of E. coli, allowing the sensor to differentially detect viable cells. Ultravioloet (UV)-C radiation and an electrocatalytic reactor (ER) with integrated defect-laden titania nanotubes were used to examine the sensors’ performance in detecting sterilized cells under different modes of cell death. Plate counts and flow cytometry were used to quantify disinfection efficacy and cell damage. It was found that the ER treatments shredded the bacteria into multiple fragments, while UV-C treatments inactivated the bacteria but left the cell membrane mostly intact.
RESUMO
In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic) beads for capture and polyguanine (polyG) oligonucleotide functionalized secondary (polystyrene) beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/E. coli secondary bead). While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 108 guanine tags per secondary bead (7.5 x 106 biotin-FITC per secondary bead, 20 guanines per oligonucleotide) bound to the target (E. coli). A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV) was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)3(2+)) as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3. We also demonstrate the use of the protocol for detection of E. coli O157:H7 seeded in waste water effluent samples.
Assuntos
Técnicas Bacteriológicas/métodos , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Escherichia coli O157/isolamento & purificação , Guanina/química , Separação Imunomagnética/métodos , Escherichia coli O157/citologiaRESUMO
Tuberculosis (TB) represents a major public health threat, with millions of new cases reported worldwide each year. A major hurdle to curtailing the spread of this disease is the need for low-cost, point-of-care (PoC) diagnostics. Mannose-capped lipoarabinomannan, a significant component of the Mycobacterium tuberculosis bacillus, has been heavily studied as a biomarker for TB, but with little success due to its complexation with endogenous components of body fluids in a manner that sterically interferes with its detection by ELISA and other immunoassays. Recent work by our group and others has shown that complexation can be disrupted with protein-denaturing protocols. By way of followup, we recently described an enzymatic digestion (Proteinase K) sample pretreatment that enables quantitative recovery of ManLAM spiked into healthy human control serum. Herein, we report on the transfer of our benchtop sample pretreatment methodology to an automated microfluidic platform. We show that this platform can be configured to: (1) carry out the pretreatment process with very little user interaction and, (2) yield recoveries for ManLAm spiked into control serum which are statistically indistinguishable from those achieved by the benchtop process. Plans to integrate this device with a portable sample reader as a possible basis for a PoC TB diagnostic system and analyze patient samples are briefly discussed.
Assuntos
Biomarcadores , Lipopolissacarídeos , Manose , Tuberculose , Humanos , Lipopolissacarídeos/sangue , Biomarcadores/sangue , Tuberculose/sangue , Tuberculose/diagnóstico , Manose/química , Manose/sangue , Mycobacterium tuberculosis/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodosRESUMO
Characterization of polymerized liposomes (PolyPIPosomes) was carried out using a combination of normal dc electrical field-flow fractionation and cyclical electrical field-flow fractionation (CyElFFF) as an analytical technique. The constant nature of the carrier fluid and channel configuration for this technique eliminates many variables associated with multidimensional analysis. CyElFFF uses an oscillating field to induce separation and is performed in the same channel as standard dc electrical field-flow fractionation separation. Theory and experimental methods to characterize nanoparticles in terms of their sizes and electrophoretic mobilities are discussed in this paper. Polystyrene nanoparticles are used for system calibration and characterization of the separation performance, whereas polymerized liposomes are used to demonstrate the applicability of the system to biomedical samples. This paper is also the first to report separation and a higher effective field when CyElFFF is operated at very low applied voltages. The technique is shown to have the ability to quantify both particle size and electrophoretic mobility distributions for colloidal polystyrene nanoparticles and PolyPIPosomes.
Assuntos
Campos Eletromagnéticos , Fracionamento por Campo e Fluxo , Lipossomos/análise , Lipossomos/síntese química , Tamanho da Partícula , PolimerizaçãoRESUMO
The electric field that drives separation and retention in electrical field flow fractionation (ElFFF) and cyclical electrical field-flow fractionation (CyElFFF) is a complex function of many parameters such as carrier ionic strength and pH, voltage, channel dimensions, flowrate, and electrode material. Currently there is no accurate or in situ method to measure the field during system operation. This paper introduces a technique to measure the effective electric field during ElFFF and CyElFFF operation using transient electrical spikes. With this technique we can determine the relationship between changes in carrier conductivity and flowrate during a run and their combined effect on effective field and retention in ElFFF. This technique can also be used to measure the voltage drop due to double layer capacitance in CyElFFF and the variation in effective field with frequency of the applied field. The measured effective fields for the CyElFFF and DC ElFFF techniques are also tested with a high ionic-strength buffer solution as carrier. For a high ionic-strength buffer, DC ElFFF generates a near-zero effective field (0.2% in 100 s), whereas CyElFFF can sustain much higher effective fields (~8%) even at relatively high voltages. The ability to measure the effective field allows for experiments to provide better data and for tuning and optimization of the separation run.
Assuntos
Campos Eletromagnéticos , Fracionamento por Campo e Fluxo/métodos , Concentração de Íons de Hidrogênio , Nanopartículas/química , Concentração OsmolarRESUMO
A previously reported microvascular coupler was shown to effectively create vascular anastomoses, but was too large for practical clinical use. To safely reduce coupler size, certain failure modes needed to be better understood. The coupler functions, in part, by compressing the vessel wall between two concentric rings, creating a friction fit that anchors the device to the vessel. This work investigates the relationship between vessel wall compression and resulting friction fit strength to ensure reducing coupler size will not unduly increase the risk that this friction fit might fail. Vascular walls were compressed to a specified strain and the tensile force required to overcome the resulting friction was measured. Experiments were conducted with various vessel types (Porcine common carotid artery, splenic artery, and jugular vein), across a range of compressive strains (55-95%), and by using either PEEK or HDPE to compress the vessel. Tensile force was increased at a rate of 5 g/min or held constant for 24 h. For experiments with incrementally increasing force, the force at failure varied with compressive strain via a power function. At 70% compression, PEEK produced 4.6 times stronger friction fits than HDPE, and common carotid arteries and splenic arteries produced 1.8 and 1.3 times stronger fits than jugular veins respectively. For experiments where tensile force was applied for 24 h, much lower forces were required to overcome friction. These results were compared to friction fit failure in a coupler prototype and it was found that the prototypes failed at just 30% of the force required to cause vessel slip under the other test conditions. These results were used to develop a model that predicts the probability of device failure via vessel slipping (one design, smaller than previously reported, was estimated to fail at maximum in vivo axial stress once in 500 anastomoses, a potentially safe level of risk).
Assuntos
Veias Jugulares , Fenômenos Mecânicos , Anastomose Cirúrgica , Animais , Fricção , Pressão , SuínosRESUMO
Cyclical electrical field flow fractionation (CyElFFF) is a variation on electrical field flow fractionation (ElFFF) where cyclical electrical fields are used instead of steady DC fields to increase the effective field experienced by particles in the flow channel. Even though the effective field increases more than 20-fold compared to normal ElFFF, the retention and resolution in CyElFFF has not been shown to be better than in ElFFF. In this paper we report how one can optimize operational parameters in CyElFFF to obtain good retention and resolution in CyElFFF. The effects of offset voltage, frequency, flowrate, concentration of particles and sample size on retention, resolution and retained peak/void peak ratio have been observed. The results obtained from these experiments were analyzed and suggestions have been made to improve both retention and resolution. A 4-fold improvement in retention without a significant increase in band broadening is reported.
Assuntos
Eletroforese/métodos , Algoritmos , Nanopartículas/químicaRESUMO
Foodborne illnesses are a major contributor to misery and health challenges in both rich and poor nations. Illnesses from pathogens such as Escherichia coli and Cryptosporidium parvum oocysts account for most of the cases of diarrhea in the world. Many standard methods exist for detecting these pathogens in water. However, these standard methods do not readily translate to the detection of the same pathogens in food. Detection techniques for pathogens in food are often inadequate, due to their inability to completely separate pathogens from food matrices. In this paper, we present a technique to separate and detect both Escherichia coli cells and Cryptosporidium parvum oocysts that have been embedded in ground meat. We achieve this objective by combining enzymatic digestion of the meat, hydrodynamic cavitation to disassemble pathogens from the meat, immunomagnetic separation to purify meat samples and indirect electrochemical detection of the target pathogens. Our use of hydrodynamic cavitation to separate pathogens is compared against an industry standard separation technique. Results indicate that the use of hydrodynamic cavitation amplifies the detection capabilities of our sensing technique and is overall comparable to or better than conventional stomacher sample preparation.
Assuntos
Cryptosporidium parvum/isolamento & purificação , Escherichia coli O157/isolamento & purificação , Análise de Alimentos/métodos , Carne Vermelha/microbiologia , Animais , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/métodos , Bovinos , Criptosporidiose/diagnóstico , Criptosporidiose/microbiologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Análise de Alimentos/economia , Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/diagnóstico , Doenças Transmitidas por Alimentos/microbiologia , Hidrodinâmica , Separação Imunomagnética/economia , Separação Imunomagnética/métodos , Fatores de TempoRESUMO
This paper examines geometric scaling models for field flow fractionation systems to understand how channel dimensions affect resolution and retention. Specifically, the changing contribution of the instrumental plate height during miniaturization of field flow fractionation (FFF) systems is reported. The work is directed towards determining the optimal geometrical parameters for miniaturization of field flow fractionation systems. The experimental relationship between channel height in FFF systems and instrumental plate heights is reported. FFF scaling models are modified to: (i) better clarify the dependence of plate height and resolution on channel height in FFF and (ii) include a more complete geometrical scaling analysis and model comparison in the low retention regime. Electrical field flow fractionation has been shown to benefit from miniaturization, so this paper focuses on that subtype, but surprisingly, the results also indicate the possibility of improvement in performance with miniaturization of other field flow fractionation systems including general FFF subtypes in which the applied field does not vary with channel height. This paper also discusses the potential role of more powerful microscale field flow fractionation systems as a new class of sample preparation units for micro-total-analysis systems (mu-TAS).
Assuntos
Fracionamento por Campo e Fluxo , MicrofluídicaRESUMO
Successful development of siRNA therapies has significant potential for the treatment of skin conditions (alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, pachyonychia congenital) caused by aberrant gene expression. Although hypodermic needles can be used to effectively deliver siRNA through the stratum corneum, the major challenge is that this approach is painful and the effects are restricted to the injection site. Microneedle arrays may represent a better way to deliver siRNAs across the stratum corneum. In this study, we evaluated for the first time the ability of the solid silicon microneedle array for punching holes to deliver cholesterol-modified housekeeping gene (Gapdh) siRNA to the mouse ear skin. Treating the ear with microneedles showed permeation of siRNA in the skin and could reduce Gapdh gene expression up to 66% in the skin without accumulation in the major organs. The results showed that microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively.
Assuntos
Terapia Genética/instrumentação , Terapia Genética/métodos , RNA Interferente Pequeno/farmacologia , Pele , Animais , Feminino , Camundongos , AgulhasRESUMO
Delivery of drugs and biomolecules into skin has significant advantages. To achieve this, herein, a nanomaterial-strengthened dissolving microneedle patch for transdermal delivery is reported. The patch comprises thousands of microneedles, which are composed of dissolving polymers, nanomaterials, and drug/biomolecules in their interior. With the addition of nanomaterials, the mechanical property of generally weak dissolving polymers can be dramatically improved without sacrificing dissolution rate within skin. In this experiments, layered double hydroxides (LDH) nanoparticles are incorporated into sodium carboxymethylcellulose (CMC) to form a nanocomposite. The results show that, by adding 5 wt% of LDH nanoparticles into CMC, the mechanical strength significantly increased. Small and densely packed CMC-LDH microneedles penetrate human and pig skin more reliably than pure CMC ones and attractively the nanocomposite-strengthened microneedles dissolve in skin and release payload within only 1 min. Finally, the application of using the nanocomposite-strengthened microneedle arrays is tested for in vivo vaccine delivery and the results show that significantly stronger antibody response could be induced when compared with subcutaneous injection. These data suggest that nanomaterials could be useful for fabricating densely packed and small polymer microneedles that have robust mechanical properties and rapid dissolution rates and therefore potential use in clinical applications.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Microinjeções/instrumentação , Nanocompostos/química , Agulhas , Administração Cutânea , Animais , Carboximetilcelulose Sódica/química , Humanos , Hidróxidos/química , Nanocompostos/ultraestrutura , Ovalbumina/administração & dosagem , Ovalbumina/química , Ovalbumina/imunologia , Ovalbumina/farmacocinética , SuínosRESUMO
Dissolving polymer microneedles have attracted much attention for their biocompatibility, fast dissolution, and high drug loading. Among them, polyvinylpyrrolidone (PVP) is widely used, but its high water absorption and poor mechanical properties constrain its broad applications. Herein we show that adding cyclodextrin (CD) to form PVP-CD inclusion complexes can alleviate these problems. The water absorption of PVP was reduced by 36-40% at different RHs as the PVP-CD inclusion complexes formed. Attractively, the water absorption at 10 and 20 days remained almost the same for the complexes while it could dramatically increase for the pure PVP samples, particularly in high humidity environments, indicating a possibly longer storage time for the complexes. It was also found that the Young's modulus and hardness of the PVP-CD could be greatly improved, especially for low molecular weight PVP. Furthermore, the glass transition temperature (Tg) of the PVP-CD increased by up to 39 °C. With the improved properties, the fabricated PVP-CD microneedles possessed much sharper needle tips and the patch had less cracks than those made from pure PVP. Pig skin application results suggested that the PVP-CD microneedle arrays were able to reliably pierce the stratum corneum of the skin while it was not achievable for the PVP microneedles with the same geometry. We anticipate that these PVP-CD complex microneedles are more suitable for vaccine and drug delivery because of their superior properties.
RESUMO
Diagnostic assays implemented in microfluidic devices have developed rapidly over the past decade and are expected to become commonplace in the next few years. Hundreds of microfluidics-based approaches towards clinical diagnostics and pathogen detection have been reported with a general theme of rapid and customizable assays that are potentially cost-effective. This chapter reviews microfluidics in molecular diagnostics based on application areas with a concise review of microfluidics in general. Basic principles of microfabrication are briefly reviewed and the transition to polymer fabricated devices is discussed. Most current microfluidic diagnostic devices are designed to target a single disease, such as a given cancer or a variety of pathogens, and there will likely be a large market for these focused devices; however, the future of molecular diagnostics lies in highly multiplexed microfluidic devices that can screen for potentially hundreds of diseases simultaneously.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Animais , Bactérias/isolamento & purificação , Biomarcadores Tumorais/análise , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Diagnóstico Molecular/instrumentação , Saúde Pública/estatística & dados numéricos , Vírus/isolamento & purificaçãoRESUMO
A microscale thermal-electrical field-flow fractionation (ThElFFF) channel is reported for the first time and preliminary characterization results show high retention at certain operating conditions including relatively high flow rates when compared to standard microscale electrical or thermal field-flow fractionation instruments. A new design is presented that simplifies manufacturing and assembly of the prototype and that can provide both an electrical field and a high temperature gradient (â¼106 °C/m). Monodisperse particle retention is carried out with polystyrene nanoparticle samples to characterize the device. Retention ratios as low as 0.045 are observed with the ThElFFF instrument. Size selectivity of 1.77 was achieved for ThElFFF. The comparison with theory shows a marked deviation from the existing theory. Separation of a mixture of polystyrene particles is demonstrated for the first time using a ThElFFF system by separating 130 nm carboxylated polystyrene and 209 nm polystyrene particles.
Assuntos
Fracionamento por Campo e Fluxo/instrumentação , Fracionamento por Campo e Fluxo/métodos , Desenho de Equipamento , Microfluídica , Nanopartículas/química , Tamanho da Partícula , Poliestirenos/química , TemperaturaRESUMO
A diffusion Split-Flow Thin Cell (SPLITT) system was used to partially remove small peptides such as ß2 microglobulin (ß2M) and parathyroid hormone (PTH) in a continuous manner from an input flow stream while preserving most (over 97%) of the larger protein in the sample, such as albumin. To help determine the operating conditions for this work, a two-dimensional numerical model based on the Navier-Stokes equation and convection-diffusion equations was developed for diffusional SPLITT using COMSOL multiphysics software (COMSOL Inc., MA). These simulations were used to obtain the relationship between important operational parameters and the purification efficiency for proteins of interest. The diffusion-based SPLITT system was fabricated using xurography and was used to demonstrate protein purification based on the differences in size or diffusion coefficient of the sample. The results obtained from the experiments are compared with the mathematical model and show good agreement, while the variations between these results are discussed. The results show that significant portions of small peptides (>25%) can be removed while preserving larger proteins (up to 95%) in the carrier stream. A potential application of this technique is to be used as an additional step in kidney dialysis to remove toxins that are not effectively removed by current dialysis protocols.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Proteínas/isolamento & purificação , Simulação por Computador , Diálise/instrumentação , Diálise/métodos , Difusão , Modelos Teóricos , Peptídeos/isolamento & purificaçãoRESUMO
Contemporary bridging techniques for repairing nerve gaps caused by trauma require autologous nerve grafts, which are difficult to harvest and handle and result in significant donor site deficit. Several nerve conduits with axon growth-enhancing potential have been proposed, developed and tested over the past fifteen years. In this work, prototypes of a nerve conduit designed to bridge large nerve gaps (≥10mm) end-to-end were incorporated with concentric drug reservoirs for constant and controlled drug delivery to enhance axon growth. These devices were designed, fabricated and tested in vitro in amber glass vials with bovine serum albumin in order to determine the drug release kinetics for future application. Our devices have shown the capability to deliver the drug of interest over a 6-day period.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Fatores de Crescimento Neural/administração & dosagem , Regeneração Nervosa/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Traumatismos dos Nervos Periféricos/cirurgia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Bovinos , Desenho de Equipamento , Humanos , Fatores de Crescimento Neural/farmacocinética , Regeneração Nervosa/fisiologia , Soroalbumina Bovina/administração & dosagem , Soroalbumina Bovina/farmacocinéticaRESUMO
A channel configuration for the elimination of end effects in field-flow fractionation (FFF) channels is simulated and demonstrated for a microfabricated FFF system. In field-flow fractionation, the carrier liquid and sample particles are transferred from a point injection to the full breadth of the rectangular channel using a triangular end piece at the inlet. The nonuniformity in streamline length generated by this end piece results in an increased instrument-related plate height. An additional contribution from the end piece at the outlet of the channel further increases the total band broadening. This paper presents a novel approach to minimize end-effect contributions to plate height by fabricating microstructures in the channel end sections to redistribute the flow streams and force streamline lengths to be more uniform. Numerical analysis of the flow profile and sample dispersion (including spreading of particles due to diffusion and advection) is carried out to investigate the optimized microstructure column size, shape, and placement in the end pieces. The configuration obtained from the numerical simulation results is used to design a prototype device. Experimental measurement of the plate heights for this prototype with an on-chip impedance-based detector shows marked improvement in performance due to the presence of the microstructures in comparison to conventional FFF channel geometry with an average 50% reduction in plate height. The redesigned inlet triangle results in a uniform transition of the point-injected sample into a thin and straight band across the width of the channel at the start of the rectangular section of the fractionation channel.