Luigi Altomare

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.

Electronic sorting and recovery of single live cells from microlitre sized samples

Sorting and recovering specific live cells from samples containing less than a few thousand cells have become major hurdles in rare cell exploration such as stem cell research, cell therapy and cell based diagnostics. We describe here a new technology based on a microelectronic chip integrating an array of over 100,000 independent electrodes and sensors which allow individual and parallel single cell manipulation of up to 10,000 cells while maintaining viability and proliferation capabilities. Manipulation is carried out using dynamic dielectrophoretic traps controlled by an electronic interface. We also demonstrate the capabilities of the chip by sorting and recovering individual live fluorescent cells from an unlabeled population.

Capacitive sensor array for localization of bioparticles in CMOS lab-on-a-chip

Fully-electronic detection of cells and microbeads is achieved on a 320x320 array of capacitive sensors in 0.35/spl mu/m 2P3M CMOS technology that also integrates particle actuation by dielectrophoresis. Particle-associated equivalent input capacitance variations larger than 0.42fF are measured with 39dB SNR. Output noise is /spl les/1.6mV, the resolution of the 12b ADC.

Specific detection of bifidobacterium strains in a pharmaceutical probiotic product and in human feces by polymerase chain reaction.

For PCR specific detection of the strains Bifidobacterium longum Y 10, B. infantis Y 1 and B. breve Y 8 used in a new probiotic product (VSL-3), strains-specific rDNA primers have been developed. Spacer regions between the 16S and 23S rRNA genes (ITS) of the three strains were amplified by PCR with conserved primers and the nucleotide sequence of these ITSs were determined. On the basis of their comparison with the rDNA sequences retrieved from GenBank, we designed new primers which specifically recognize the species B. breve and the two strains B. infantis Y 1 and B. breve Y 8. Specificity of these primers was confirmed through the analysis of 60 bifidobacteria strains belonging to the more representative human species. The feasibility of this PCR method was investigated in commercial VSL-3 product and fecal samples collected from 4 patients affected by inflammatory bowel deseases and two healthy subjects before and after the VSL-3 administration. By PCR analysis of different VSL-3 commercial batches we were successful in differentiating and quantifying the strains B. longum Y 10, B. infantis Y 1 and B. breve Y 8. B. infantis Y 1 and B. breve Y 8 could be detected at high concentration in fecal specimens of both patients and subjects treated with the probiotic preparation, showing a different colonization behaviour. Seven days after the VSL-3 treatment suspension, no patients and subjects harbored B. infantis Y 1 and B. breve Y 8, indicating a transient presence of these exogenous strains.

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.

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.

Dielectrophoretic cage-speed separation of bio-particles

A novel approach to the size characterization and separation of bio-particles is presented. Fluid-flow is not required to activate the separation since moving dielectrophoretic cages (MDC) (G. Medoro et al., IEDM, pp. 415-418, 2000) are used. By sequencing a set of cage patterns in a micro-fabricated chip, a movement can be induced or not on bio-particles depending on the ratio between the dielectrophoretic (DEP) mobility (H. Morgan and N.G. Green, Research Studies Press, 2003) and the cage-speed, causing selective particle transport. The effectiveness of the approach has been proven by experimental results with different sized beads. This approach can be easily combined with optical or impedance measurement for on-chip quantification of the mixture constituents for a miniaturized, automatic detection system.