RESUMO
A reduced channel height in microfluidic Lab-on-a-Chip (LOC) devices enables a reduction in the required volume of sample and reagents. LOC devices are most often manufactured by microstructuring a planar substrate and subsequently sealing it with a cover film. However, shallow chip designs, made from polymers, are sensitive to channel deformation during the sealing of the microfluidic device. Inappropriate bonding conditions often result in the loss of the microfluidic functionality. A systematic and practical approach for the identification of suitable bonding process parameters is missing. In this article, a straightforward approach for the optimization of channel integrity in the sealing of shallow microfluidic devices made from Cyclic Olefin Polymer (COP) is presented. Two COP materials were tested: COP Zeonex 690R (Glass transition temperature Tg = 135 °C) both as a cover film and substrate material, and COP ZF14 (Tg = 135 °C) as a film material. A mechanical analysis using microstructured Zeonex 690R substrates was performed to generate a matrix of low-distortion bonding parameters, including temperature, pressure and time. The well-established method of solvent-assisted bonding was used to enhance the characteristically low bond strengths of the native COP material. In addition, plasma-assisted bonding was tested and compared. The optimization approach was validated by the manufacture of a microfluidic test device, the demonstration of its microfluidic functionality, and the quantitative evaluation of the achieved channel integrity.
Assuntos
Cicloparafinas/química , Dispositivos Lab-On-A-Chip , Polímeros/química , Desenho de Equipamento , Gases em Plasma/química , Propriedades de SuperfícieRESUMO
One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstract Photograph of the RT-PCR thermoplastic chip.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Vírus de RNA/isolamento & purificação , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Ebolavirus/isolamento & purificação , Desenho de Equipamento , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Humanos , Dispositivos Lab-On-A-Chip , Limite de Detecção , Infecções por Vírus de RNA/diagnóstico , Infecções por Vírus de RNA/virologiaRESUMO
Traditional methods for identifying pathogens in bacteremic patients are slow (24-48+ h). This can lead to physicians making treatment decisions based on an incomplete diagnosis and potentially increasing the patient's mortality risk. To decrease time to diagnosis, we have developed a novel technology that can recover viable bacteria directly from whole blood and identify them in less than 7 h. Our technology combines a sample preparation process with surface-enhanced Raman spectroscopy (SERS). The sample preparation process enriches viable microorganisms from 10 mL of whole blood into a 200 µL aliquot. After a short incubation period, SERS is used to identify the microorganisms. We further demonstrated that SERS can be used as a broad detection method, as it identified a model set of 17 clinical blood culture isolates and microbial reference strains with 100% identification agreement. By applying the integrated technology of sample preparation and SERS to spiked whole blood samples, we were able to correctly identify both Staphylococcus aureus and Escherichia coli 97% of the time with 97% specificity and 88% sensitivity.
Assuntos
Escherichia coli/isolamento & purificação , Staphylococcus aureus/isolamento & purificação , Humanos , Análise Espectral Raman/instrumentação , Propriedades de SuperfícieRESUMO
In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm × 30.9 mm × 0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device.
Assuntos
Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , Custos e Análise de Custo , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Desenho de Equipamento , Procedimentos Analíticos em Microchip , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
A strong natural selection for microbial antibiotic resistance has resulted from the extensive use and misuse of antibiotics. Though multiple factors are responsible for this crisis, the most significant factor - widespread prescription of broad-spectrum antibiotics - is largely driven by the fact that the standard process for determining antibiotic susceptibility includes a 1-2-day culture period, resulting in 48-72 h from patient sample to final determination. Clearly, disruptive approaches, rather than small incremental gains, are needed to address this issue. The field of microfluidics promises several advantages over existing macro-scale methods, including: faster assays, increased multiplexing, smaller volumes, increased portability for potential point-of-care use, higher sensitivity, and rapid detection methods. This Perspective will cover the advances made in the field of microfluidic, phenotypic antibiotic susceptibility testing (AST) over the past two years. Sections are organized based on the functionality of the chip - from simple microscopy platforms, to gradient generators, to antibody-based capture devices. Microfluidic AST methods that monitor growth as well as those that are not based on growth are presented. Finally, we will give our perspective on the major hurdles still facing the field, including the need for rapid sample preparation and affordable detection technologies.
Assuntos
Antibacterianos/farmacologia , Dispositivos Lab-On-A-Chip , Testes de Sensibilidade Microbiana/instrumentação , Fenótipo , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Farmacorresistência Bacteriana , Fatores de TempoRESUMO
BACKGROUND: Compared with traditional open surgery, minimally invasive surgery may improve recovery and patient satisfaction while maintaining surgical principles. Laparoscopic, single incision, natural orifice, and robotic approaches hold their own appeal. However, they lack the ability to manipulate organs as easily as the human hand. Advances in minimally invasive surgical techniques require new tools with increased functionality of the end effectors. Multifunctional tools with greater dexterity than those currently available are highly desired. METHODS: To address this need, we designed, fabricated, and tested the first prototype of a laparoscopic tool that provides the dexterity of a hand. The "hand" has two jointed fingers and a jointed thumb attached to a laparoscopic sheath that can be collapsed to fit through a 12-mm trocar or small orifice. The handle provides control for three independent degrees of freedom: finger motion (bending/spreading), fingertip bending, and thumb bending. The tool can be used for pinching, grasping, and spreading motions. Furthermore, the thumb is "double jointed" so that the tool can be converted to a rake configuration to allow lifting motions. The initial prototype has been tested in a cadaver lab to demonstrate its utility. RESULTS: Our "lap-hand" was used to complete standard surgical tasks in a simulation device in a time comparable to open and laparoscopic approaches, including "bowel" manipulation and peg movement. Cadaver testing confirmed the ability to grasp, elevate, and move liver, stomach, colon, and small bowel in a fashion expected by the hand. No adverse events were noted, and no bowel injury or perforation resulted from over-grasping. CONCLUSIONS: We have designed, built, and tested a first prototype of an artificial hand for minimally invasive surgery. Use of such tools could both reduce the number of hand-incisions required and potentially transition more patients to undergo their abdominal procedures laparoscopically.
Assuntos
Laparoscopia/instrumentação , Procedimentos Cirúrgicos Minimamente Invasivos/instrumentação , Robótica/instrumentação , Cadáver , Mãos , Força da Mão , Humanos , Laparoscopia/métodos , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Instrumentos CirúrgicosRESUMO
The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications.
Assuntos
Polímeros/química , RNA/isolamento & purificação , Extração em Fase Sólida/métodos , Ar , Animais , Automação , Soluções Tampão , Linhagem Celular , Cães , Porosidade , Pressão , Reprodutibilidade dos Testes , Extração em Fase Sólida/economia , Extração em Fase Sólida/instrumentaçãoRESUMO
In this paper, we present a fully integrated lab-on-a-chip and associated instrument for the detection of bacteria from liquid samples. The system conducts bacterial lysis, nucleic acid isolation and concentration, polymerase chain reaction (PCR), and end-point fluorescent detection. To enable truly low-cost manufacture of the single-use disposable chip, we designed the plastic chip in a planar format without any active components to be amenable to injection molding and utilized a novel porous polymer monolith (PPM) embedded with silica that has been shown to lyse bacteria and isolate the nucleic acids from clinical samples (M. D. Kulinski, M. Mahalanabis, S. Gillers, J. Y. Zhang, S. Singh and C. M. Klapperich, Biomed. Microdevices, 2009, 11, 671-678).(1) The chip is made of Zeonex(R), a thermoplastic with a high melting temperature to allow PCR, good UV transmissibility for UV-curing of the PPM, and low auto-fluorescence for fluorescence detection of the amplicon. We have built a prototype instrument to automate control of the fluids, temperature cycling, and optical detection with the capability of accommodating various chip designs. To enable fluid control without including valves or pumps on the chip, we utilized a remote valve switching technique. To allow fluid flow rate changes on the valveless chip, we incorporated speed changing fluid reservoirs. The PCR thermal cycling was achieved with a ceramic heater and air cooling, while end-point fluorescence detection was accomplished with an optical spectrometer; all integrated in the instrument. The chip seamlessly and automatically is mated to the instrument through an interface block that presses against the chip. The interface block aligns and ensures good contact of the chip to the temperature controlled region and the optics. The integrated functionality of the chip was demonstrated using Bacillus subtilis as a model bacterial target. A Taqman assay was employed on-chip to detect the isolated bacterial DNA.
Assuntos
Bacillus subtilis/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Bacillus subtilis/genética , Bacteriólise , Custos e Análise de Custo , Técnicas Analíticas Microfluídicas/economia , Reação em Cadeia da Polimerase , Polímeros/química , PorosidadeRESUMO
Bacteremia is a life-threatening condition for which antibiotics must be prescribed within hours of clinical diagnosis. Since the current gold standard for bacteremia diagnosis is based on conventional methods developed in the mid-1800s-growth on agar or in broth-identification and susceptibility profiling for both Gram-positive and Gram-negative bacterial species requires at least 48-72 h. Recent advancements in accelerated phenotypic antibiotic susceptibility testing have centered on the microscopic growth analysis of small bacterial populations. These approaches are still inherently limited by the bacterial growth rate. Our approach is fundamentally different. By applying environmental stress to bacteria in a microfluidic platform, we can correctly assign antibiotic susceptibility profiles of clinically relevant Gram-negative bacteria within two hours of antibiotic introduction rather than 8-24 h. The substantial expansion to include a number of clinical isolates of important Gram-negative species-Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa-reported here underscores the broad utility of our approach, complementing the method's proven utility for Gram-positive bacteria. We also demonstrate that the platform is compatible with antibiotics that have varying mechanisms of action-meropenem, gentamicin, and ceftazidime-highlighting the versatility of this platform.
Assuntos
Técnicas Bacteriológicas/métodos , Farmacorresistência Bacteriana , Enterobacteriaceae/efeitos dos fármacos , Microfluídica/métodos , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacologia , Técnicas Bacteriológicas/instrumentação , Enterobacteriaceae/classificação , Microfluídica/instrumentação , Pseudomonas aeruginosa/classificação , Estresse FisiológicoRESUMO
Due to its relatively low level of antigenicity and high durability, titanium has successfully been used as the major material for biological implants. However, because the typical interface between titanium and tissue precludes adequate transmission of load into the surrounding bone, over time, load-bearing implants tend to loosen and revision surgeries are required. Osseointegration of titanium implants requires presentation of both biological and mechanical cues that promote attachment of and trigger mineral deposition by osteoblasts. While many factors contribute to differentiation, the relative importance of the various cues is unclear. To substantially improve osseointegration of titanium implants, we generated a gelatin methacryloyl (GelMA) scaffold, using an extrusion-based 3D bioprinter, which can be directly printed on and grafted to the titanium implant surface. We demonstrate that this scaffold is able to trigger mineral deposition of both MG63 osteoblasts and primary normal human osteoblasts in the absence of any exogenous osteogenic factors. Films of the same formulation failed to promote mineral deposition suggesting that the three dimensional scaffold was able to tip the balance in favor of differentiation despite other potentially unfavorable differentiation cues of the material. We further show that these GelMA lattices can be directly grafted to titanium alloy and are secure in vitro over a period of seven weeks. When grafted within a groove system, the GelMA hydrogel is protected from shearing forces in a marrow implantation model. This prepares the way for osteogenic coatings to be directly manufactured on the implant surface and packaged for surgery.
Assuntos
Bioimpressão/métodos , Minerais/metabolismo , Alicerces Teciduais/química , Actinas/metabolismo , Animais , Bovinos , Movimento Celular , Células Cultivadas , Gelatina/química , Humanos , Hidrogéis/química , Minerais/química , Modelos Animais , Osseointegração , Osteoblastos/citologia , Osteoblastos/metabolismo , Impressão Tridimensional , Próteses e Implantes , Resistência ao Cisalhamento , Propriedades de Superfície , Suínos , Titânio/químicaRESUMO
We present a lab-on-a-chip and associated instrument for heterogeneous enzyme-linked immunosorbent assay (ELISA)-based detection of proteins from liquid samples. The system performs all necessary ELISA steps (starting from antigen incubation) in a quarter of the time required for corresponding plate-based protocols. We have previously described the instrument, which automates fluidic control via remote valve switching and detects fluorescence from reacted substrate, for use in a molecular diagnostics application. The ELISA chip reported here utilizes a high surface area bead bed to enhance capture efficiency and increase the dynamic range of the assay as compared to a standard plate-based ELISA. Its functionality is demonstrated using human IL-10 as a model antigen, but theoretically any sandwich ELISA could be ported onto this "open source platform." We show that our automated on-chip assays have greater sensitivities than the corresponding standard manual plate-based ELISAs, and that single samples can be assayed in a fraction of the time.
RESUMO
Appropriate care for bacteremic patients is dictated by the amount of time needed for an accurate diagnosis. However, the concentration of microbes in the blood is extremely low in these patients (1-100 CFU/mL), traditionally requiring growth (blood culture) or amplification (e.g., PCR) for detection. Current culture-based methods can take a minimum of two days, while faster methods like PCR require a sample free of inhibitors (i.e., blood components). Though commercial kits exist for the removal of blood from these samples, they typically capture only DNA, thereby necessitating the use of blood culture for antimicrobial testing. Here, we report a novel, scaled-up sample preparation protocol carried out in a new microbial concentration device. The process can efficiently lyse 10 mL of bacteremic blood while maintaining the microorganisms' viability, giving a 30-µL final output volume. A suite of six microorganisms (Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, and Candida albicans) at a range of clinically relevant concentrations was tested. All of the microorganisms had recoveries greater than 55% at the highest tested concentration of 100 CFU/mL, with three of them having over 70% recovery. At the lowest tested concentration of 3 CFU/mL, two microorganisms had recoveries of ca. 40-50% while the other four gave recoveries greater than 70%. Using a Taqman assay for methicillin-sensitive S. aureus (MSSA)to prove the feasibility of downstream analysis, we show that our microbial pellets are clean enough for PCR amplification. PCR testing of 56 spiked-positive and negative samples gave a specificity of 0.97 and a sensitivity of 0.96, showing that our sample preparation protocol holds great promise for the rapid diagnosis of bacteremia directly from a primary sample.
Assuntos
Bacteriemia/diagnóstico , Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/métodos , Bacteriemia/microbiologia , HumanosRESUMO
We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step - bacterial growth - is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane (PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.
Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Técnicas Analíticas Microfluídicas/métodos , Dimetilpolisiloxanos/química , Farmacorresistência Bacteriana , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Estresse Fisiológico/fisiologiaRESUMO
To accurately diagnose microbial infections in blood, it is essential to recover as many microorganisms from a sample as possible. Unfortunately, recovering such microorganisms depends significantly on their adhesion to the surfaces of diagnostic devices. Consequently, we sought to minimize the adhesion of methicillin-sensitive Staphylococcus aureus (MSSA) to the surface of polypropylene- and acrylic-based bacteria concentration devices. These devices were treated with 11 different coatings having various charges and hydrophobicities. Some coatings promoted bacterial adhesion under centrifugation, whereas others were more likely to prevent it. Experiments were run using a simple buffer system and lysed blood, both inoculated with MSSA. Under both conditions, Hydromer's 7-TS-13 and Aqua65JL were most effective at reducing bacterial adhesion.
RESUMO
Many new and exciting portable HIV viral load testing technologies are emerging for use in global medicine. While the potential to provide fast, isothermal, and quantitative molecular diagnostic information to clinicians in the field will soon be a reality, many of these technologies lack a robust front end for sample clean up and nucleic acid preparation. Such a technology would enable many different downstream molecular assays. Here, we present a portable system for centrifuge-free room temperature nucleic acid extraction from small volumes of whole blood (70 µL), using only thermally stable reagents compatible with storage and transport in low resource settings. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of simulated samples demonstrate a lower limit of detection of 1000 copies/ml, with the ability to detect differences in viral load across four orders of magnitude. The system can also be used to store extracted RNA on detachable cartridges for up to one week at ambient temperature, and can be operated using only hand generated air pressure.
RESUMO
Sample preparation for DNA and RNA assays is a prime candidate for laboratory automation. A novel, parallel processing device that performs the three separate liquid-handling functions necessary for such sample preparation-dispensing, pipetting, and pressurizing-is presented. The device comprises an array of fine nozzles connected by fluidic channels to automatically and precisely distribute flow between one source and an array of points. The design principles, as well as the experimental and computational methods used to develop the device, are described. Test results, including accuracy, uniformity, volume range, and timing, are presented. The functionality of the device is demonstrated by performing a solid-phase extraction of DNA with two types of microcolumns.
Assuntos
Automação Laboratorial/métodos , Técnicas de Laboratório Clínico/métodos , Ácidos Nucleicos/isolamento & purificação , Manejo de Espécimes/métodos , HumanosRESUMO
A fully automated "factory" was developed that uses tobacco plants to produce large quantities of vaccines and other therapeutic biologics within weeks. This first-of-a-kind factory takes advantage of a plant viral vector technology to produce specific proteins within the leaves of rapidly growing plant biomass. The factory's custom-designed robotic machines plant seeds, nurture the growing plants, introduce a viral vector that directs the plant to produce a target protein, and harvest the biomass once the target protein has accumulated in the plants-all in compliance with Food and Drug Administration (FDA) guidelines (e.g., current Good Manufacturing Practices). The factory was designed to be time, cost, and space efficient. The plants are grown in custom multiplant trays. Robots ride up and down a track, servicing the plants and delivering the trays from the lighted, irrigated growth modules to each processing station as needed. Using preprogrammed robots and processing equipment eliminates the need for human contact, preventing potential contamination of the process and economizing the operation. To quickly produce large quantities of protein-based medicines, we transformed a laboratory-based biological process and scaled it into an industrial process. This enables quick, safe, and cost-effective vaccine production that would be required in case of a pandemic.
Assuntos
Automação Laboratorial/métodos , Produtos Biológicos/isolamento & purificação , Biotecnologia/métodos , Plantas Geneticamente Modificadas , Tecnologia Farmacêutica/métodos , Vacinas/isolamento & purificação , Produtos Biológicos/metabolismo , Vetores Genéticos , Fidelidade a Diretrizes , Humanos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Vírus de Plantas/genética , Nicotiana/genética , Nicotiana/metabolismo , Estados Unidos , United States Food and Drug Administration , Vacinas/metabolismoRESUMO
The emergence and spread of bacterial resistance to ever increasing classes of antibiotics intensifies the need for fast phenotype-based clinical tests for determining antibiotic susceptibility. Standard susceptibility testing relies on the passive observation of bacterial growth inhibition in the presence of antibiotics. In this paper, we present a novel microfluidic platform for antibiotic susceptibility testing based on stress-activation of biosynthetic pathways that are the primary targets of antibiotics. We chose Staphylococcus aureus (S. aureus) as a model system due to its clinical importance, and we selected bacterial cell wall biosynthesis as the primary target of both stress and antibiotic. Enzymatic and mechanical stresses were used to damage the bacterial cell wall, and a ß-lactam antibiotic interfered with the repair process, resulting in rapid cell death of strains that harbor no resistance mechanism. In contrast, resistant bacteria remained viable under the assay conditions. Bacteria, covalently-bound to the bottom of the microfluidic channel, were subjected to mechanical shear stress created by flowing culture media through the microfluidic channel and to enzymatic stress with sub-inhibitory concentrations of the bactericidal agent lysostaphin. Bacterial cell death was monitored via fluorescence using the Sytox Green dead cell stain, and rates of killing were measured for the bacterial samples in the presence and absence of oxacillin. Using model susceptible (Sanger 476) and resistant (MW2) S. aureus strains, a metric was established to separate susceptible and resistant staphylococci based on normalized fluorescence values after 60 min of exposure to stress and antibiotic. Because this ground-breaking approach is not based on standard methodology, it circumvents the need for minimum inhibitory concentration (MIC) measurements and long wait times. We demonstrate the successful development of a rapid microfluidic-based and stress-activated antibiotic susceptibility test by correctly designating the phenotypes of 16 additional clinically relevant S. aureus strains in a blinded study. In addition to future clinical utility, this method has great potential for studying the effects of various stresses on bacteria and their antibiotic susceptibility.
Assuntos
Antibacterianos/farmacologia , Técnicas Analíticas Microfluídicas/métodos , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Testes de Sensibilidade Microbiana , Técnicas Analíticas Microfluídicas/instrumentação , Estresse MecânicoRESUMO
Blast exposure is associated with traumatic brain injury (TBI), neuropsychiatric symptoms, and long-term cognitive disability. We examined a case series of postmortem brains from U.S. military veterans exposed to blast and/or concussive injury. We found evidence of chronic traumatic encephalopathy (CTE), a tau protein-linked neurodegenerative disease, that was similar to the CTE neuropathology observed in young amateur American football players and a professional wrestler with histories of concussive injuries. We developed a blast neurotrauma mouse model that recapitulated CTE-linked neuropathology in wild-type C57BL/6 mice 2 weeks after exposure to a single blast. Blast-exposed mice demonstrated phosphorylated tauopathy, myelinated axonopathy, microvasculopathy, chronic neuroinflammation, and neurodegeneration in the absence of macroscopic tissue damage or hemorrhage. Blast exposure induced persistent hippocampal-dependent learning and memory deficits that persisted for at least 1 month and correlated with impaired axonal conduction and defective activity-dependent long-term potentiation of synaptic transmission. Intracerebral pressure recordings demonstrated that shock waves traversed the mouse brain with minimal change and without thoracic contributions. Kinematic analysis revealed blast-induced head oscillation at accelerations sufficient to cause brain injury. Head immobilization during blast exposure prevented blast-induced learning and memory deficits. The contribution of blast wind to injurious head acceleration may be a primary injury mechanism leading to blast-related TBI and CTE. These results identify common pathogenic determinants leading to CTE in blast-exposed military veterans and head-injured athletes and additionally provide mechanistic evidence linking blast exposure to persistent impairments in neurophysiological function, learning, and memory.