Gianni Medoro

A CMOS chip for individual cell manipulation and detection

Investigation of individual biological cell interactions on a large scale creates applications in drug screening, cell separation and cell analysis. The IC is designed to detect and manipulate more than 10k cells in parallel and includes a 320 /spl times/ 320 sensing and actuation array. The chip is implemented in 0.35/spl mu/m 2P 3M CMOS.

A lab-on-a-chip for cell detection and manipulation

This paper presents a lab-on-a-chip for electronic manipulation and detection of microorganisms based on the use of closed dielectrophoretic (DEP) cages combined with impedance sensing. A prototype has been realized using a standard printed circuit board (PCB) technology by which polystyrene microbeads have been trapped, concentrated and quantitated, in agreement with CAD simulations, without the need for external optical components. The experiment was successfully repeated with S. cerevisiae. The approach is suited to be implemented in integrated circuit technology, which would allow to manipulate and detect single cells and reduce the system dimension.

Molecular profiling of single circulating tumor cells with diagnostic intention

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non‐random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC‐positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high‐quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre‐existing cells resistant to ERBB2‐targeted therapies suggesting ongoing microevolution at late‐stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Applications to cancer research of "lab-on-a-chip" devices based on dielectrophoresis (DEP).

The recent development of advanced analytical and bioseparation methodologies based on microarrays and biosensors is one of the strategic objectives of the so-called post-genomic. In this field, the development of microfabricated devices could bring new opportunities in several application fields, such as predictive oncology, diagnostics and anti-tumor drug research. The so called “Laboratory-on-a-chip technology”, involving miniaturisation of analytical procedures, is expected to enable highly complex laboratory testing to move from the central laboratory into non-laboratory settings. The main advantages of Lab-on-a-chip devices are integration of multiple steps of different analytical procedures, large variety of applications, sub-microliter consumption of reagents and samples, and portability. One of the requirement for new generation Lab-on-a-chip devices is the possibility to be independent from additional preparative/analytical instruments. Ideally, Lab-on-a-chip devices should be able to perform with high efficiency and reproducibility both actuating and sensing procedures. In this review, we discuss applications of dielectrophoretic(DEP)-based Lab-on-a-chip devices to cancer research. The theory of dielectrophoresis as well as the description of several devices, based on spiral-shaped, parallel and arrayed electrodes are here presented. In addition, in this review we describe manipulation of cancer cells using advanced DEP-based Lab-on-a-chip devices in the absence of fluid flow and with the integration of both actuating and sensing procedures.

Selective sample recovery of DEP-separated cells and particles by phaseguide-controlled laminar flow

The selective recovery of particles is demonstrated after separation with dielectrophoretic (DEP) forces. Particles are separated based on their size using the so-called cage-speed separation protocol. A two-lane laminar flow enables the selective recovery of the particles. In order to prevent hydrostatic pressure flow, liquid reservoirs are absent. The sample liquid is thus replaced with air during recovery. Phaseguides are introduced in the system to control the liquid–air interface, so that the two-lane laminar flow profile is preserved.

Lab on a Chip for Live-Cell Manipulation

Precisely manipulating and sorting live cells on a lab on a chip is still a major challenge. This article shows how to use dielectrophoresis for cell sorting. The authors also describe a prototype CMOS chip with a sensor-actuator array, row-column addressing logic and readout circuitry. In this article, we examine the new microelectronic technology that gives scientists the ability to monitor, sort, and analyze vast populations of cells and interact with each cell individually. A microelectronic platform called a lab on a chip (LoC) allows precise manipulation of cells with no effect on their phenotypes. The motivation for developing this technology is that investigations in recent years have shown that a few cells changing their behavior unexpectedly can induce deadly diseases such as cancer. Current LoC design and manufacturing techniques are spawning new biotechnology methods with potential for research, diagnosis, and therapy.

Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing.

Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.

Programmable interactions of functionalized single bioparticles in a dielectrophoresis-based microarray chip

Manipulating single biological objects is a major unmet challenge of biomedicine. Herein, we describe a lab-on-a-chip platform based on dielectrophoresis (DEP). The DEParray is a prototypal version consisting of 320 × 320 arrayed electrodes generating >10,000 spherical DEP cages. It allows the capture and software-guided movement to predetermined spatial coordinates of single biological objects. With the DEParray we demonstrate (a) forced interaction between a single, preselected target cell and a programmable number of either microspheres or natural killer (NK) cells, (b) on-chip immunophenotypic discrimination of individual cells based on differential rosetting with microspheres functionalized with monoclonal antibodies to an inhibitory NK cell ligand (HLA-G), (c) on-chip, real-time (few minutes) assessment of immune lysis by either visual inspection or semiautomated, time-lapse reading of a fluorescent dye released from NK cell-sensitive targets, and (d) manipulation and immunophenotyping with limiting amounts (about 500) cells. To our knowledge, this is the first report describing a DEP-based lab-on-a-chip platform for the quick, arrayed, software-guided binding of individually moved biological objects, the targeting of single cells with microspheres, and the real-time characterization of immunophenotypes. The DEParray candidates as a discovery tool for novel cell:cell interactions with no prior (immuno)phenotypic knowledge.

Lysis-on-chip of single target cells following forced interaction with CTLs or NK cells on a dielectrophoresis-based array.

Guiding the interaction of single cells acting as partners in heterotypic interactions (e.g., effectors and targets of immune lysis) and monitoring the outcome of these interactions are regarded as crucial biomedical achievements. In this study, taking advantage of a dielectrophoresis (DEP)-based Laboratory-on-a-chip platform (the DEPArray), we show that it is possible to generate closed DEP cages entrapping CTLs and NK cells as either single cells or clusters; reversibly immobilize a single virus-presenting or tumor cell within the chip at a selected position; move cages and their content to predetermined spatial coordinates by software-guided routing; force a cytotoxic effector to physically interact with a putative target within a secluded area by merging their respective cages; generate cages containing effector and target cells at predetermined E:T ratios; accurately assess cytotoxicity by real-time quantitation of the release kinetics of the fluorescent dye calcein from target cells (>50 lytic events may be tested simultaneously); estimate end points of calcein release within 16 min of initial E:T cell contact; simultaneously deliver Ab-based phenotyping and on-chip lysis assessment; and identify lytic and nonlytic E:T combinations and discriminate nonlytic effector phenotypes from target refractoriness to immune lysis. The proof of principle is provided that DEPArray technology, previously used to levitate and move single cells, can be used to identify highly lytic antiviral CTLs and tumor cells that are particularly refractory to NK cell lysis. These findings are of primary interest in targeted immunotherapy.

A system-on-a-programmable-chip for real-time control of massively parallel arrays of biosensors and actuators

The development of microelectronic lab-on-a-chip devices (LOACs) can now be pursued thanks to advances in silicon technology. As these kinds of devices may integrate different functional units, much care has to be put in the design of control units which have to provide real-time control capabilities in order to deal with complex systems composed of sensors, actuators, signal conditioning and processing circuits. Moreover, reconfigurability and expandability are key design features to get a flexible and reusable architecture. The FPGA implementation of a control system for an existing LOAC is presented as a case study with emphasis on the advantages of a programmable device approach. The presented system has been implemented on an Altera EPF10K200S device and the achieved operating frequency is 20MHz. The device was successfully tested and experimental results are hereby shown.