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Articles by Shramik Sengupta in JoVE
JoVE Bioengineering
Detecção e isolamento de células de melanoma circulantes usando Fluxometria Fotoacústica
Nós desenvolvemos um citômetro de fluxo, utilizando ultra-som para detectar a laser induzida circulando células de melanoma como um indicador precoce de doença metastática.
Other articles by Shramik Sengupta on PubMed
Multistage Electrophoresis II: Treatment of a Kinetic Separation As a Pseudoequilibrium Process
An electrophoresis device is described which separates cells, particles, proteins and other separands by collecting samples having decreasing electrophoretic mobility in a train of inverted cavities while an electric field is applied between the inverted cavities and a sample cuvette containing a mixture of cells, particles, proteins or other separands. A circular plate is provided for the inverted cavities, and this circular plate is rotated to collect fractions. The system utilizes an innovative purification method that combines free electrophoresis and multistage extraction in an instrument capable of separating living cells, particles, and proteins in useful quantities at high concentrations. Most multistage processes are based on equilibrium separations, but electrophoresis is a kinetic separation; therefore, a pseudoequilibrium paradigm was developed for use in optimizing separation parameters including number of stages and electrophoresis time per stage. This paradigm allows the application of McCabe-Thiele type analysis, and it was calculated, for example, that two separands differing by 20% in electrophoretic mobility can be purified to 95% purity with acceptable yield in about seven stages. Laboratory experiments demonstrated a 95% purification in four stages of a separand originally present at 4% when electrophoretic mobilities differed by 80%.
Sliding-cavity Fluid Contactors in Low-gravity Fluids, Materials, and Biotechnology Research
The well-known method of sliding-cavity fluid contactors used by Gosting for diffusion measurements and by Tiselius in electrophoresis has found considerable use in low-gravity research. To date, sliding-cavity contactors have been used in liquid diffusion experiments, interfacial transport experiments, biomolecular crystal growth, biphasic extraction, multistage extraction, microencapsulation, seed germination, invertebrate development, and thin-film casting. Sliding-cavity technology has several advantages for spaceflight: it is simple, it accommodates small samples, samples can be fully enclosed, phases can be combined, multiple samples can be processed at high sample density, real-time observations can be made, and mixed and diffused samples can be compared. An analysis of the transport phenomena that govern the sliding-cavity method is offered. During sliding of one liquid over another flow rates between 0.001 and 0.1m/sec are developed, giving Reynolds numbers in the range 0.1-100. Assuming no slip at liquid-solid boundaries shear rates are of the order 1sec(-1). The measured consequence is the transfer of 2-5% of the content of a cavity to the opposite cavity. In the absence of gravity, buoyancy-driven transport is assumed absent. Transport processes are limited to (1) molecular diffusion, in which reactants diffuse toward one another at rates that depend on their diffusion coefficient and concentration gradient (Fick's second law), (2) solutocapillary (Marangoni) flow driven by surface-tension gradients, (3) capillary flow (drop spreading) at liquid-solid three-phase lines leading to immiscible phase demixing, and (4) vapor-phase diffusive mass transfer in evaporative processes. Quantitative treatment of these phenomena has been accomplished over the past few years in low-gravity research in space and on aircraft.
Application of the Lag-after-pulsed-separation (LAPS) Flow Meter to Different Protein Solutions
A lag after pulsed separation (LAPS) meter was previously developed to measure flow rates of protein solutions. The LAPS meter operates on the time-of-flight principle. An upstream event (electrophoretic concentration of the particles in one section of the device) is detected downstream (by change in ac resistance). The time lag between the event and its detection is inversely proportional to the fluid flow rate. We demonstrate the ability of the LAPS meter to measure the flow rate of solutions containing one or more charged biomacromolecules or particles. A prototype of the LAPS meter was used to measure flow rates of solutions of model proteins [bovine serum albumin (BSA), lysozyme and hemoglobin] and mixtures of BSA and lysozyme. Flow rates of 10-50 microl min(-1)(average velocities of 0.24-1.2 mm s(-1)) were measured. When a single ac measurement was used, the results were solution-dependent, which we attribute to the interface between the protein solution and the ac electrodes. A differential mode, in which the signal from a positive and a negative dc pulse were subtracted from each other, eliminated interfacial effects and led to a single universal (solution-independent) calibration curve. The LAPS meter can be used as a non-invasive, no-moving-parts flow sensor in any microfluidic system (such as drug delivery devices or micro-reactor arrays) where one needs to measure the flow rate of a solution or a suspension containing charged species such as proteins or cells.
A Micro-scale Multi-frequency Reactance Measurement Technique to Detect Bacterial Growth at Low Bio-particle Concentrations
The technique described enables the user to detect the presence and proliferation of bacteria through an increase in the bulk capacitance (C) of the suspension, which is proportional to the bacteria count, at practical frequencies less than 1 MHz. The geometry of the micro-capillary design employed increases the bulk resistance (R) of the medium, thus increasing its RC time. This makes the measured reactance sensitive to changes in the bulk capacitance, which is usually masked by the much larger surface capacitance. The sensitivity is further enhanced by the existence of a minimum in the value of the reactance at a frequency proportional to the inverse medium RC time. The value of this reactance minimum and the frequency at which the minimum is recorded are dependent on the bacteria count and permit the detection of an initial concentration of approximately 100 CFU ml(-1) of E. coli within 3 hours of incubation, in comparison with the previous reported values of about 8 hours, with an initial load of 1000 CFU ml(-1).
Bovine Red Blood Cell Starvation Age Discrimination Through a Glutaraldehyde-amplified Dielectrophoretic Approach with Buffer Selection and Membrane Cross-linking
We report a novel buffer electric and dielectric relaxation time tuning technique, coupled with a glutaraldehyde (Glt.) cross-linking cell fixation reaction that allows for sensitive dielectrophoretic analysis and discrimination of bovine red blood cells of different starvation age. Guided by a single-shell oblate spheroid model, a zwitterion buffer composition is selected to ensure that two measurable crossover frequencies (cof's) near 500 kHz exist for dielectrophoresis (DEP) within a small range of each other. It is shown that the low cof is sensitive to changes in the cell membrane dielectric constant, in which cross-linking by Glt. reduces the dielectric constant of the cell membrane from 10.5 to 3.8, while the high cof is sensitive to cell cytoplasm conductivity changes. We speculate that this enhanced particle polarizability that results from the cross-linking reaction is because younger (reduced starvation time) cells possess more amino groups that the reaction can release to enhance the cell interior ionic strength. Such sensitive discrimination of cells with different age (surface protein density) by DEP is not possible without the zwitterion buffer and cleavage by Glt. treatment. It is then expected that rapid identification and sorting of healthy from diseased cells can be similarly sensitized.
Concentration Control for Protein Crystallization Via a Continuously-fed Crystallization Chamber
A continuously-fed crystallization chamber that allows for kinetic path control through the crystallization phase diagram (from labile/nucleation to metastable/growth) was fabricated and used to crystallize lysozyme. A lumped kinetic model was developed, and parameters for heterogeneous nucleation kinetics were determined. Heterogeneous nucleation was found to have faster nucleation kinetics and slower growth kinetics than homogeneous nucleation, as expected. The major contributions of the new device are (1) to allow better control of the chemical environment for studies of crystal nucleation and growth, and (2) to allow lumped-model analysis of those studies to extract kinetic parameters.
Rapid On-chip Genetic Detection Microfluidic Platform for Real World Applications
The development of genetic detection protocols for field applications is an important aspect of modern medical diagnostic technology and environmental monitoring. In this paper, we report a rapid, portable, and inexpensive DNA hybridization technique using a bead-based microfluidic platform that functions by passing fluorescently labeled target DNA through a chamber packed with functionalized beads within a microfluidic channel. DNA hybridization is then assessed using a digital camera attached to a Clare Chemical DR-45M dark reader non-UV transilluminator that uses visible light as an excitation source and a blue and amber filter to reveal fluorescence. This microfluidic approach significantly enhances hybridization by reducing the diffusion time between target DNA and the silica surface. The use of probe-functionalized beads as solid support also enhances the sensitivity and limit of detection due to a larger surface area per unit volume. This platform could be adapted for use in medical applications and environmental monitoring, including the detection of harmful organisms in the ballast water of ships.
Novel Electrical Method for Early Detection of Viable Bacteria in Blood Cultures
We present a novel electrical method for detecting viable bacteria in blood cultures that is 4 to 10 times faster than continuous monitoring blood culture systems (CMBCS) like the Bactec system. Proliferating bacteria are detected via an increase in the bulk capacitance of suspensions, and the threshold concentration for detection is ∼ 10(4) CFU/ml (compared to ∼ 10(8) CFU/ml for the Bactec system).
Development and in Vitro Studies of a Polyethylene Terephthalate-gold Nanoparticle Scaffold for Improved Biocompatibility
Polyethylene terephthalate (PET) mesh is one of the most commonly used synthetic biomaterials for tension-free hernia repair. In an effort to improve the biocompatibility of PET mesh, gold nanoparticles (AuNP) in various concentrations were conjugated to the PET surface to develop PET-AuNP scaffolds. These novel scaffolds were characterized with Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) to assess the addition of functional groups, presence of AuNPs, and thermal stability of the modified PET mesh, respectively. The biocompatibility of the PET-AuNP scaffolds was evaluated through in vitro cell culture assays. The cellularity of cells exposed to the PET-AuNP scaffolds, as well as the scaffolds' ability to reduce reactive oxygen species, was assessed using L929 murine fibroblasts. Antimicrobial properties of AuNPs conjugated to PET mesh were tested against the bacteria Pseudomonas aeruginosa. Results from the FT-IR showed presence of COOH groups while SEM displayed bonding of AuNPs to the PET surface. DSC results indicated that the PET more than likely did not undergo any detrimental degradation due to the surface modification. Results from the in vitro studies showed that AuNPs, in optimal concentrations (1× concentrations), enhanced cellularity, reduced ROS, and reduced bacteria adhesion to PET. These studies demonstrated enhanced biocompatibility of the AuNP conjugated PET mesh over pristine PET mesh.
Sputter-deposition of Silver Nanoparticles into Ionic Liquid As a Sacrificial Reservoir in Antimicrobial Organosilicate Nanocomposite Coatings
We present a new approach for fabricating robust, regenerable antimicrobial coatings containing an ionic liquid (IL) phase incorporating silver nanoparticles (AgNPs) as a reservoir for Ag(0)/Ag(+) species within sol-gel-derived nanocomposite films integrating organosilicate nanoparticles. The IL serves as an ultralow volatility (vacuum-compatible) liquid target, allowing for the direct deposition and dispersion of a high-density AgNP "ionosol" following conventional sputtering techniques. Two like-anion ILs were investigated in this work: methyltrioctylammonium bis(trifluoromethylsulfonyl)imide, [N(8881)][Tf(2)N], and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [emim][Tf(2)N]. Silver ionosols derived from these two ILs were incorporated into silica-based sol-gel films and the resultant antimicrobial activity evaluated against Pseudomonas aeruginosa bacteria. Imaging of the surface morphologies of the as-prepared films established a link between an open macroporous film architecture and the observation of high activity. Nanocomposites based on [N(8881)][Tf(2)N] displayed excellent antimicrobial activity against P. aeruginosa over multiple cycles, reducing cell viability by 6 log units within 4 h of contact. Surprisingly, similar films prepared from [emim][Tf(2)N] presented negligible antimicrobial activity, an observation we attribute to the differing abilities of these IL cations to infiltrate the cell wall, regulating the influx of silver ions to the bacterium's interior.
