Cristian Ionescu-Zanetti

A General Model for Amyloid Fibril Assembly Based on Morphological Studies Using Atomic Force Microscopy

Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.

New Device for High-Throughput Viability Screening of Flow Biofilms

ABSTRACT Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.

On-chip cell lysis by local hydroxide generation

We present a novel method for on-chip cell lysis based on local hydroxide electro-generation. Hydroxide ions porate the cell membrane, leading to cell lysis. After lysis occurs, hydrogen ions, also generated on chip, react with excess hydroxide, creating a neutral pH lysate and eliminating the need for a wash step. Three different cell types are shown to be effectively lysed by this method: red blood cells, HeLa (human tumor line) and Chinese Hamster Ovary (CHO) cell lines. The release of cytoplasmic molecules from HeLa and CHO cells is demonstrated by monitoring the escape of a membrane impermeant dye from the cytoplasm. In the vicinity of the cathode, the hydroxide concentration is predicted by finite element simulations and shown to fit the lysis rates at different distances from the generating cathode. For flow-through experiments, a second device integrating a mechanical filter with hydroxide generation is fabricated and tested. The purpose of the filter is to trap whole cells and only allow lysate to pass through. The flow rate dependence of hydroxide concentration at the lysis filter is modeled and lysis efficiency is experimentally determined to be proportional to the hydroxide concentration for flow rates from 15 to 30 microl min(-1).

Single-cell electroporation arrays with real-time monitoring and feedback control

Rapid well-controlled intracellular delivery of drug compounds, RNA, or DNA into a cell--without permanent damage to the cell--is a pervasive challenge in basic cell biology research, drug discovery, and gene delivery. To address this challenge, we have developed a bench-top system comprised of a control interface, that mates to disposable 96-well-formatted microfluidic devices, enabling the individual manipulation, electroporation and real-time monitoring of each cell in suspension. This is the first demonstrated real-time feedback-controlled electroporation of an array of single-cells. Our computer program automatically detects electroporation events and subsequently releases the electric field, precluding continued field-induced damage of the cell, to allow for membrane resealing. Using this novel set-up, we demonstrate the reliable electroporation of an array (n = 15) of individual cells in suspension, using low applied electric fields (<1 V) and the rapid and localized intracellular delivery of otherwise impermeable compounds (Calcein and Orange Green Dextran). Such multiplexed electrical and optical measurements as a function of time are not attainable with typical electroporation setups. This system, which mounts on an inverted microscope, obviates many issues typically associated with prototypical microfluidic chip setups and, more importantly, offers well-controlled and reproducible parallel pressure and electrical application to individual cells for repeatability.

Circulating Tumor Cells as Potential Biomarkers in Bladder Cancer.

PURPOSE We explored the diagnostic use of circulating tumor cells in patients with neoadjuvant bladder cancer using enumeration and next generation sequencing. MATERIALS AND METHODS A total of 20 patients with bladder cancer who were eligible for cisplatin based neoadjuvant chemotherapy were enrolled in an institutional review board approved study. Subjects underwent blood draws at baseline and after 1 cycle of chemotherapy. A total of 11 patients with metastatic bladder cancer and 13 healthy donors were analyzed for comparison. Samples were enriched for circulating tumor cells using the novel IsoFlux™ System microfluidic collection device. Circulating tumor cell counts were analyzed for repeatability and compared with Food and Drug Administration cleared circulating tumor cells. Circulating tumor cells were also analyzed for mutational status using next generation sequencing. RESULTS Median circulating tumor cell counts were 13 at baseline and 5 at followup in the neoadjuvant group, 29 in the metastatic group and 2 in the healthy group. The concordance of circulating tumor cell levels, defined as low-fewer than 10, medium-11 to 30 and high-greater than 30, across replicate tubes was 100% in 15 preparations. In matched samples the IsoFlux test showed 10 or more circulating tumor cells in 4 of 9 samples (44%) while CellSearch® showed 0 of 9 (0%). At cystectomy 4 months after baseline all 3 patients (100%) with medium/high circulating tumor cell levels at baseline and followup had unfavorable pathological stage disease (T1-T4 or N+). Next generation sequencing analysis showed somatic variant detection in 4 of 8 patients using a targeted cancer panel. All 8 cases (100%) had a medium/high circulating tumor cell level with a circulating tumor cell fraction of greater than 5% purity. CONCLUSIONS This study demonstrates a potential role for circulating tumor cell assays in the management of bladder cancer. The IsoFlux method of circulating tumor cell detection shows increased sensitivity compared with CellSearch. A next generation sequencing assay is presented with sufficient sensitivity to detect genomic alterations in circulating tumor cells.

Well plate microfluidic system for investigation of dynamic platelet behavior under variable shear loads

The study of platelet behavior in real‐time under controlled shear stress offers insights into the underlying mechanisms of many vascular diseases and enables evaluation of platelet‐focused therapeutics. The two most common methods used to study platelet behavior at the vessel wall under uniform shear flow are parallel plate flow chambers and cone‐plate viscometers. Typically, these methods are difficult to use, lack experimental flexibility, provide low data content, are low in throughput, and require large reagent volumes. Here, we report a well plate microfluidic (WPM)‐based system that offers high throughput, low reagent consumption, and high experimental flexibility in an easy to use well plate format. The system consists of well plates with an integrated array of microfluidic channels, a pneumatic interface, an automated microscope, and software. This WPM system was used to investigate dynamic platelet behavior under shear stress. Multiple channel designs are presented and tested for shear loads with whole blood to determine their applicability to study thrombus formation. Normal physiological shear (0.1–20 dyn/cm2) and pathological shear (20–200 dyn/cm2) devices were used to study platelet behavior in vitro under various shear, matrix coating, and monolayer conditions. The high physiological relevance, low blood consumption, and increased throughput create a valuable technique available to vascular biology researchers. The approach also has extensibility to other research areas including inflammation, cancer biology, and developmental/stem cell research. Biotechnol. Bioeng. 2011;108: 2978–2987.

Nanogap capacitors: Sensitivity to sample permittivity changes

In this article, detailed models for a number of nanogap capacitor geometries are presented, and their agreement with impedance spectroscopy data is evaluated. The detection limit is investigated using a technique for the modification of inter-electrode permittivity. Precise changes in permittivity of the sample region are introduced by timed etching of the capacitor spacer and measured in order to determine the system’s sensitivity to changes in sample permittivity. We have focused on a frequency range far below the relaxation frequencies of the dielectric materials used and we were concerned solely with the real part of the complex permittivity of the materials used . The models developed here can be used to determine the sensitivity of nanogap capacitors to dielectric changes of biomolecular materials present in the sample region. Additionally, it is demonstrated that such devices could function as metrology tools for monitoring the rate of removal/deposition of material in nanocavities, thus aiding in fabrication accuracy. The sensitivity of the measured parameters Z impedance magnitude and phase shift to permittivity changes is measured and compared to model predictions. In conjunction with standard deviation of Z and data over a number of devices 3‐6, sensitivity values can be used to determine detection limits for such sensors. These validated models will be useful to researchers using nanogap-based sensors, and will enable the optimization of such devices as they are developed into genomic or proteomic sensor arrays.

Soft-state biomicrofluidic pulse generator for single cell analysis

We present the design, fabrication, and characterization of a soft-state biomicrofluidic pulse generator for single cell analysis. Hydrodynamic cell trapping via lateral microfluidic junctions allows the trapping of single cells from a bulk suspension. Microfluidic injection sites adjacent to the cell-trapping channels enable the pulsed delivery of nanoliter volumes of biochemical reagent. We demonstrated the application and removal of reagent at a frequency of 10Hz with a rise time of less than 33ms and a reagent consumption rate of 0.2nL∕s. It is shown that this system operates as a low-pass filter with a cutoff frequency of 7Hz.

IonFlux: A Microfluidic Patch Clamp System Evaluated with Human Ether-a`-go-go Related Gene Channel Physiology and Pharmacology

Ion channel assays are essential in drug discovery, not only for identifying promising new clinical compounds, but also for minimizing the likelihood of potential side effects. Both applications demand optimized throughput, cost, and predictive accuracy of measured membrane current changes evoked or modulated by drug candidates. Several competing electrophysiological technologies are available to address this demand, but important gaps remain. We describe the industrial application of a novel microfluidic-based technology that combines compounds, cells, and buffers on a single, standard well plate. Cell trapping, whole cell, and compound perfusion are accomplished in interconnecting microfluidic channels that are coupled to pneumatic valves, which emancipate the system from robotics, fluidic tubing, and associated maintenance. IonFlux™ is a state-of-the-art, compact system with temperature control and continuous voltage clamp for potential application in screening for voltage- and ligand-gated ion channel modulators. Here, ensemble recordings of the IonFlux system were validated with the human Ether-à-go-go related gene (hERG) channel (stably expressed in a Chinese hamster ovary cell line), which has established biophysical and pharmacological characteristics in other automated planar patch systems. We characterized the temperature dependence of channel activation and its reversal potential. Concentration response characteristics of known hERG blockers and control compounds obtained with the IonFlux system correlated with literature and internal data obtained on this cell line with the QPatch HT system. Based on the biophysical and pharmacological data, we conclude that the IonFlux system offers a novel, versatile, automated profiling, and screening system for ion channel targets with the benefit of temperature control.

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.