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In JoVE (1)
Other Publications (28)
- Analytical Chemistry
- Journal of Biomechanics
- Journal of the American Chemical Society
- Proceedings of the National Academy of Sciences of the United States of America
- Carcinogenesis
- The Analyst
- The Analyst
- Environmental Health Perspectives
- Pharmacological Research : the Official Journal of the Italian Pharmacological Society
- Annual Review of Biomedical Engineering
- Nanotechnology
- Free Radical Biology & Medicine
- Biomedical Microdevices
- JALA (Charlottesville, Va.)
- The Analyst
- Biosensors & Bioelectronics
- JALA (Charlottesville, Va.)
- Analytical Chemistry
- Bioanalysis
- PloS One
- Biomaterials
- Trends in Pharmacological Sciences
- Lab on a Chip
- Journal of Biological Engineering
- Analytical Chemistry
- PloS One
- Journal of Cell Science
- The Journal of Urology
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Articles by Pak Kin Wong in JoVE
JoVE Bioengineering
Padronização de plasma de superfície litografia para a Criação de Redes de celular
1Aerospace and Mechanical Engineering, University of Arizona, 2Biomedical Engineering IDP and BIO5 Institute, University of Arizona
A técnica de litografia versátil plasma foi desenvolvido para gerar padrões de superfície estável para orientar adesão celular. Esta técnica pode ser aplicada para criar redes de células, incluindo as que imitam tecidos naturais e tem sido usada para estudar vários tipos de células distintas.
Other articles by Pak Kin Wong on PubMed
Electrokinetic Bioprocessor for Concentrating Cells and Molecules
Bioprocessors for concentrating bioparticles, such as cells and molecules, are commonly needed in bioanalysis systems. In this microfluidic processor, a global flow field generated by ac electroosmosis transports the embedded particles to the regions near the electrode surface. The processor then utilizes electrophoretic and dielectrophoretic forces, which are effective in short range, to trap the target cells and molecules on the electrode surface. By optimizing the operating parameters, we have concentrated various biological objects in a large range of sizes, including Escherichia coli bacteria, lambda phage DNA, and single-stranded DNA fragments as small as 20 bases that have a radius of gyration of only 3 nm.
Cell Relaxation After Electrodeformation: Effect of Latrunculin A on Cytoskeletal Actin
Precise measurement of the mechanical properties of a cell provides useful information about its structural organization and physiological state. It is interesting to understand the effect of individual components on the mechanical properties of the entire cell. In this study, we investigate the influence of the cytoskeletal actin on the viscoelastic properties of a cell. Actin-specific agents, including latrunculin A and jasplakinolide, are used to alter the organization of the cytoskeletal actin. Brassica oleracea protoplasts are treated with the drugs and deformed under an external electric potential. The relaxation processes of single protoplasts after electrodeformation are measured. The data are analyzed by a model-independent spectrum recovery algorithm. Two distinct characteristic time constants are obtained from the relaxation spectra. Treatment with latrunculin A increases both of the relaxation time constants. The longest relaxation times for control, latrunculin A treated, and jasplakinolide treated cells are determined to be 0.28, 1.0, and 0.21 s, respectively.
Single-molecule Tracing on a Fluidic Microchip for Quantitative Detection of Low-abundance Nucleic Acids
Here, we report a method capable of quantitative detection of low-abundance DNA/RNA molecules by incorporating confocal fluorescence spectroscopy, molecular beacons, and a molecular-confinement microfluidic reactor. By using a combination of ac and dc fields via a trio of 3-D electrodes in the microreactor, we are able to precisely direct the transport of individual molecules to a minuscule laser-focused detection volume ( approximately 1 fL). A burst of fluorescence photons is detected whenever a molecular beacon-target hybrid flows through the detection region, and the amount of targets can be directly quantified according to the number of recorded single-molecule flow-through events. This assay consumes only attomoles of molecular probes and is able to quantitatively detect subpicomolar DNA targets. A measurement time of less than 2 min is sufficient to complete the detection.
Closed-loop Control of Cellular Functions Using Combinatory Drugs Guided by a Stochastic Search Algorithm
A mixture of drugs is often more effective than using a single effector. However, it is extremely challenging to identify potent drug combinations by trial and error because of the large number of possible combinations and the inherent complexity of the underlying biological network. With a closed-loop optimization modality, we experimentally demonstrate effective searching for potent drug combinations for controlling cellular functions through a large parametric space. Only tens of iterations out of one hundred thousand possible trials were needed to determine a potent combination of drugs for inhibiting vesicular stomatitis virus infection of NIH 3T3 fibroblasts. In addition, the drug combination reduced the required dosage by approximately 10-fold compared with individual drugs. In another example, a potent mixture was identified in thirty iterations out of a possible million combinations of six cytokines that regulate the activity of nuclear factor kappa B in 293T cells. The closed-loop optimization approach possesses the potential of being an effective approach for manipulating a wide class of biological systems.
Nrf2 Enhances Resistance of Cancer Cells to Chemotherapeutic Drugs, the Dark Side of Nrf2
Drug resistance during chemotherapy is the major obstacle to the successful treatment of many cancers. Here, we report that inhibition of NF-E2-related factor 2 (Nrf2) may be a promising strategy to combat chemoresistance. Nrf2 is a critical transcription factor regulating a cellular protective response that defends cells against toxic insults from a broad spectrum of chemicals. Under normal conditions, the low constitutive amount of Nrf2 protein is maintained by the Kelch-like ECH-associated protein1 (Keap1)-mediated ubiquitination and proteasomal degradation system. Upon activation, this Keap1-dependent Nrf2 degradation mechanism is quickly inactivated, resulting in accumulation and activation of the antioxidant response element (ARE)-dependent cytoprotective genes. Since its discovery, Nrf2 has been viewed as a 'good' transcription factor that protects us from many diseases. In this study, we demonstrate the dark side of Nrf2: stable overexpression of Nrf2 resulted in enhanced resistance of cancer cells to chemotherapeutic agents including cisplatin, doxorubicin and etoposide. Inversely, downregulation of the Nrf2-dependent response by overexpression of Keap1 or transient transfection of Nrf2-small interfering RNA (siRNA) rendered cancer cells more susceptible to these drugs. Upregulation of Nrf2 by the small chemical tert-butylhydroquinone (tBHQ) also enhanced the resistance of cancer cells, indicating the feasibility of using small chemical inhibitors of Nrf2 as adjuvants to chemotherapy to increase the efficacy of chemotherapeutic agents. Furthermore, we provide evidence that the strategy of using Nrf2 inhibitors to increase efficacy of chemotherapeutic agents is not limited to certain cancer types or anticancer drugs and thus can be applied during the course of chemotherapy to treat many cancer types.
Separation-free Detection of Nuclear Factor Kappa B with Double-stranded Molecular Probes
This paper reports a simple, rapid molecular binding scheme for the detection and quantification of the transcription factor nuclear factor kappa B and other DNA binding proteins without any separation or immobilization step.
A Double-stranded Molecular Probe for Homogeneous Nucleic Acid Analysis
This paper reports the design and optimization of a double-stranded molecular probe for homogeneous detection of specific nucleotide sequences. The probes are labeled with either a fluorophore or a quencher such that the probe hybridization brings the two labels into close proximity, and this diminishes the fluorescence signal in the absence of a target. In the presence of a target, the fluorophore probe is thermodynamically driven to unzip from its hybridized form and bind with the target. An equilibrium analysis, which successfully describes all the major features of the assay without any fitting parameter, is performed to generalize the design of the probe. Several key parameters affecting the performance of the assay are examined. We show that the dynamic range and the signal-to-noise ratio of the assay can be optimized by the probe concentration, the quencher-to-fluorophore ratio, and the probe strand sequence. By proper design of the sequence, the probe discriminates single nucleotide mismatches in a single step without any separation step or measurement of melting profile.
Oridonin Confers Protection Against Arsenic-induced Toxicity Through Activation of the Nrf2-mediated Defensive Response
Groundwater contaminated with arsenic imposes a big challenge to human health worldwide. Using natural compounds to subvert the detrimental effects of arsenic represents an attractive strategy. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of the cellular antioxidant response and xenobiotic metabolism. Recently, activation of the Nrf2 signaling pathway has been reported to confer protection against arsenic-induced toxicity in a cell culture model.
Dual Roles of Nrf2 in Cancer
In response to oxidative stress, the transcription factor NF-E2-related factor 2 (Nrf2) controls the fate of cells through transcriptional upregulation of antioxidant response element (ARE)-bearing genes, including those encoding endogenous antioxidants, phase II detoxifying enzymes, and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis. As a result, activation of the Nrf2 pathway, by naturally-occurring compounds or synthetic chemicals at sub-toxic doses, confers protection against subsequent toxic/carcinogenic exposure. Thus, the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention. Interestingly, recent emerging data has revealed the "dark" side of Nrf2. Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, it may be necessary to inhibit the Nrf2 pathway during chemotherapy. This review is primarily focused on the role of Nrf2 in cancer, with emphasis on the recent findings indicating the cancer promoting function of Nrf2 and its role in acquired chemoresistance.
Microengineered Platforms for Cell Mechanobiology
Mechanical forces play important roles in the regulation of various biological processes at the molecular and cellular level, such as gene expression, adhesion, migration, and cell fate, which are essential to the maintenance of tissue homeostasis. In this review, we discuss emerging bioengineered tools enabled by microscale technologies for studying the roles of mechanical forces in cell biology. In addition to traditional mechanobiology experimental techniques, we review recent advances of microelectromechanical systems (MEMS)-based approaches for cell mechanobiology and discuss how microengineered platforms can be used to generate in vivo-like micromechanical environment in in vitro settings for investigating cellular processes in normal and pathophysiological contexts. These capabilities also have significant implications for mechanical control of cell and tissue development and cell-based regenerative therapies.
Hybrid Electrokinetics for Separation, Mixing, and Concentration of Colloidal Particles
The advent of nanotechnology has facilitated the preparation of colloidal particles with adjustable sizes and the control of their size-dependent properties. Physical manipulation, such as separation, mixing, and concentration, of these colloidal particles represents an essential step for fully utilizing their potential in a wide spectrum of nanotechnology applications. In this study, we investigate hybrid electrokinetics, the combination of dielectrophoresis and electrohydrodynamics, for active manipulation of colloidal particles ranging from nanometers to micrometers in size. A concentric electrode configuration, which is optimized for generating electrohydrodynamic flow, has been designed to elucidate the effectiveness of hybrid electrokinetics and define the operating regimes for different microfluidic operations. The results indicate that the relative importance of electrohydrodynamics increases with decreasing particle size as predicted by a scaling analysis and that electrohydrodynamics is pivotal for manipulating nanoscale particles. Using the concentric electrodes, we demonstrate separation, mixing, and concentration of colloidal particles by adjusting the relative strengths of different electrokinetic phenomena. The effectiveness of hybrid electrokinetics indicates its potential to serve as a generic technique for active manipulation of colloidal particles in various nanotechnology applications.
Nrf2 Promotes Neuronal Cell Differentiation
The transcription factor Nrf2 has emerged as a master regulator of the endogenous antioxidant response, which is critical in defending cells against environmental insults and in maintaining intracellular redox balance. However, whether Nrf2 has any role in neuronal cell differentiation is largely unknown. In this report, we have examined the effects of Nrf2 on cell differentiation using a neuroblastoma cell line, SH-SY5Y. Retinoic acid (RA) and 12-O-tetradecanoylphorbol 13-acetate, two well-studied inducers of neuronal differentiation, are able to induce Nrf2 and its target gene NAD(P)H quinone oxidoreductase 1 in a dose- and time-dependent manner. RA-induced Nrf2 up-regulation is accompanied by neurite outgrowth and an induction of two neuronal differentiation markers, neurofilament-M and microtubule-associated protein 2. Overexpression of Nrf2 in SH-SY5Y cells promotes neuronal differentiation, whereas inhibition of endogenous Nrf2 expression inhibited neuronal differentiation. More remarkably, the positive role of Nrf2 in neuronal differentiation was verified ex vivo in primary neuron culture. Primary neurons isolated from Nrf2-null mice showed a retarded progress in differentiation, compared to those from wild-type mice. Collectively, our data demonstrate a novel role for Nrf2 in promoting neuronal cell differentiation, which will open new perspectives for therapeutic uses of Nrf2 activators in patients with neurodegenerative diseases.
Computational Simulation of a Magnetic Microactuator for Tissue Engineering Applications
The next generation of tissue engineered constructs (TECs) requires the incorporation of a controllable and optimized microstructure if they are to chemically, mechanically, and biologically mimic tissue function. In order to obtain TECs with optimized microstructures, a combination of spatiotemporally regulated mechanical and biochemical stimuli is necessary during the formation of the construct. While numerous efforts have been made to create functional tissue constructs, there are few techniques available to stimulate TECs in a localized manner. We herein describe the design of a microdevice which can stimulate TECs in a localized, inhomogeneous, and predefined anisotropic fashion using ferromagnetically doped polydimethylsiloxane microflaps (MFs). Specifically, a sequential magneto-structural finite element model of the proposed microdevice is constructed and utilized to understand how changes in magnetic and geometrical properties of the device affect MF deflection. Our study indicates that a relatively small density of ferromagnetic material is required to result in adequate force and MF defection (175 microm approximately 7% TEC strain). We also demonstrate that MF to magnet distance is more important than inherent MF magnetic permeability in determining resulting MF deflection. An experimental validation test setup was used to validate the computational solutions. The comparison shows reasonable agreement indicating a 5.9% difference between experimentally measured and computationally predicted MF displacement. Correspondingly, an apparatus with two MFs and two magnets has been made and is currently undergoing construct testing. The current study presents the design of a novel magnetic microactuator for tissue engineering applications. The computational results reported here will form the foundation in the design and optimization of a functional microdevice with multiple MFs and magnets capable of stimulating TECs in nonhomogenous and preferred directions with relevant spatial resolution.
A Microfluidic Cartridge System for Multiplexed Clinical Analysis
Cartridge-based microfluidics is a promising technology for clinical diagnostics. By miniaturizing the fluid-handling processes required for genomic and proteomic analyses, reagent and specimen volume is minimized along with the size of the system. We demonstrate an automated microfluidic system capable of performing six multiplexed genomic and proteomic analyses simultaneously, by means of an integrated electrochemical sensor and embedded controls.
Hybridization Kinetics of Double-stranded DNA Probes for Rapid Molecular Analysis
This study reports the hybridization kinetics of double-stranded DNA probes for rapid molecular analysis. Molecular binding schemes based on double-stranded DNA probes have been developed for quantitative detection of various biomolecules, such as nucleic acids and DNA binding proteins recently. The thermodynamic competition between the target and the competitor in binding to the probe provides a highly specific mechanism for molecular detection. The kinetics of the double-stranded DNA probe, on the other hand, represent another key aspect toward its general applicability for a wide set of biomedical applications. Herein we report a systematic investigation of the kinetics of double-stranded DNA probes. The signal-to-background ratio and assay time of the double-stranded DNA probes are optimized at a high ionic strength (over 100 mM NaCl). Both the donor probe and the quencher probe sequences are shown to be important in the hybridization kinetics. A long sticky end of the probe is able to dramatically accelerate the kinetics of the assay. To provide a quantitative description of the kinetics, a two-stage binding model is developed to describe the major features of the kinetics of the assay. The sensitivity of the kinetic model and the dominant affinity constants are studied. The study provides a general guideline for the design of the probes for reducing the total assay time. With an appropriate design of the probes, the assay can be finished within minutes at room temperature.
Electrochemical Immunosensor Detection of Urinary Lactoferrin in Clinical Samples for Urinary Tract Infection Diagnosis
Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but also more informative than the current standard.
Electrothermal Fluid Manipulation of High-Conductivity Samples for Laboratory Automation Applications
Electrothermal flow is a promising technique in microfluidic manipulation toward laboratory automation applications, such as clinical diagnostics and high throughput drug screening. Despite the potential of electrothermal flow in biomedical applications, relative little is known about electrothermal manipulation of highly conductive samples, such as physiological fluids and buffer solutions. In this study, the characteristics and challenges of electrothermal manipulation of fluid samples with different conductivities were investigated systematically. Electrothermal flow was shown to create fluid motion for samples with a wide range of conductivity when the driving frequency was above 100 kHz. For samples with low conductivities (below 1 S/m), the characteristics of the electrothermal fluid motions were in quantitative agreement with the theory. For samples with high conductivities (above 1 S/m), the fluid motion appeared to deviate from the model as a result of potential electrochemical reactions and other electrothermal effects. These effects should be taken into consideration for electrothermal manipulation of biological samples with high conductivities. This study will provide insights in designing microfluidic devices for electrokinetic manipulation of biological samples toward laboratory automation applications in the future.
Antimicrobial Susceptibility Testing Using High Surface-to-volume Ratio Microchannels
This study reports the use of microfluidics, which intrinsically has a large surface-to-volume ratio, toward rapid antimicrobial susceptibility testing at the point of care. By observing the growth of uropathogenic Escherichia coli in gas permeable polymeric microchannels with different dimensions, we demonstrate that the large surface-to-volume ratio of microfluidic systems facilitates rapid growth of bacteria. For microchannels with 250 microm or less in depth, the effective oxygenation can sustain the growth of E. coli to over 10(9) cfu/mL without external agitation or oxygenation, which eliminates the requirement of bulky instrumentation and facilitates rapid bacterial growth for antimicrobial susceptibility testing at the point of care. The applicability of microfluidic rapid antimicrobial susceptibility testing is demonstrated in culture media and in urine with clinical bacterial isolates that have different antimicrobial resistance profiles. The antimicrobial resistance pattern can be determined as rapidly as 2 h compared to days in standard clinical procedures facilitating diagnostics at the point of care.
Transfection of Molecular Beacons in Microchannels for Single-cell Gene-expression Analysis
Efficient transfection of molecular beacons has to be performed in the microscale in order to fully utilize the potential of molecular beacons and microfluidics for studying the real-time gene-expression dynamics in living cells. Nevertheless, there has been relatively little study on transfection of molecular beacons in microfluidic channels.
Statistical Metamodeling for Revealing Synergistic Antimicrobial Interactions
Many bacterial pathogens are becoming drug resistant faster than we can develop new antimicrobials. To address this threat in public health, a metamodel antimicrobial cocktail optimization (MACO) scheme is demonstrated for rapid screening of potent antibiotic cocktails using uropathogenic clinical isolates as model systems. With the MACO scheme, only 18 parallel trials were required to determine a potent antimicrobial cocktail out of hundreds of possible combinations. In particular, trimethoprim and gentamicin were identified to work synergistically for inhibiting the bacterial growth. Sensitivity analysis indicated gentamicin functions as a synergist for trimethoprim, and reduces its minimum inhibitory concentration for 40-fold. Validation study also confirmed that the trimethoprim-gentamicin synergistic cocktail effectively inhibited the growths of multiple strains of uropathogenic clinical isolates. With its effectiveness and simplicity, the MACO scheme possesses the potential to serve as a generic platform for identifying synergistic antimicrobial cocktails toward management of bacterial infection in the future.
Probing Cell Migration in Confined Environments by Plasma Lithography
Cellular processes are regulated by various mechanical and physical factors in their local microenvironment such as geometric confinements, cell-substrate interactions, and cell-cell contact. Systematic elucidation of these regulatory mechanisms is crucial for fundamental understanding of cell biology and for rational design of biomedical devices and regenerative medicine. Here, we report a generally applicable plasma lithography technique, which performs selective surface functionalization on large substrate areas, for achieving long-term, stable confinements with length scales from 100 nm to 1 cm toward the investigation of cell-microenvironment interactions. In particular, we applied plasma lithography for cellular confinement of neuroblastomas, myoblasts, endothelial cells, and mammary gland epithelial cells, and examined the motion of mouse embryonic fibroblasts in directionality-confined environments for studying the effect of confinements on migratory behavior. In conjunction with live cell imaging, the distance traveled, velocity, and angular motion of individual cells and collective cell migration behaviors were measured in confined environments with dimensions comparable to a cell. A critical length scale that a cell could conceivably occupy and migrate to was also identified by investigating the behaviors of cells using confined environments with subcellular length scales.
Biosensor Diagnosis of Urinary Tract Infections: a Path to Better Treatment?
Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. The standard culture-based diagnosis of UTI has a typical delay of two to three days. In the absence of definitive microbiological diagnosis at the point of care, physicians frequently initiate empirical broad-spectrum antibiotic treatment, and this has contributed to the emergence of resistant pathogens. Biosensors are emerging as a powerful diagnostic platform for infectious diseases. Paralleling how blood glucose sensors revolutionized the management of diabetes, and how pregnancy tests are now conducted in the home, biosensors are poised to improve UTI diagnosis significantly. Biosensors are amenable to integration with microfluidic technology for point-of-care (POC) applications. This review focuses on promising biosensor technology for UTI diagnosis, including pathogen identification and antimicrobial susceptibility testing, and hurdles to be surpassed in the translation of biosensor technology from bench to bedside.
Hybrid Electrokinetic Manipulation in High-conductivity Media
This study reports a hybrid electrokinetic technique for label-free manipulation of pathogenic bacteria in biological samples toward medical diagnostic applications. While most electrokinetic techniques only function in low-conductivity buffers, hybrid electrokinetics enables effective operation in high-conductivity samples, such as physiological fluids (∼1 S m(-1)). The hybrid electrokinetic technique combines short-range electrophoresis and dielectrophoresis, and long-range AC electrothermal flow to improve its effectiveness. The major technical hurdle of electrode instability for manipulating high conductivity samples is tackled by using a Ti-Au-Ti sandwich electrode and a 3-parallel-electrode configuration is designed for continuous isolation of bacteria. The device operates directly with biological samples including urine and buffy coats. We show that pathogenic bacteria and biowarfare agents can be concentrated for over 3 orders of magnitude using hybrid electrokinetics.
System Integration - A Major Step Toward Lab on a Chip
Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.
Molecular Detection of Bacterial Pathogens Using Microparticle Enhanced Double-stranded DNA Probes
Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.
Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis
Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.
Cellular Self-organization by Autocatalytic Alignment Feedback
Myoblasts aggregate, differentiate and fuse to form skeletal muscle during both embryogenesis and tissue regeneration. For proper muscle function, long-range self-organization of myoblasts is required to create organized muscle architecture globally aligned to neighboring tissue. However, how the cells process geometric information over distances considerably longer than individual cells to self-organize into well-ordered, aligned and multinucleated myofibers remains a central question in developmental biology and regenerative medicine. Using plasma lithography micropatterning to create spatial cues for cell guidance, we show a physical mechanism by which orientation information can propagate for a long distance from a geometric boundary to guide development of muscle tissue. This long-range alignment occurs only in differentiating myoblasts, but not in non-fusing myoblasts perturbed by microfluidic disturbances or other non-fusing cell types. Computational cellular automata analysis of the spatiotemporal evolution of the self-organization process reveals that myogenic fusion in conjunction with rotational inertia functions in a self-reinforcing manner to enhance long-range propagation of alignment information. With this autocatalytic alignment feedback, well-ordered alignment of muscle could reinforce existing orientations and help promote proper arrangement with neighboring tissue and overall organization. Such physical self-enhancement might represent a fundamental mechanism for long-range pattern formation during tissue morphogenesis.
A Biosensor Platform for Rapid Antimicrobial Susceptibility Testing Directly from Clinical Samples
A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test.
