The inverted open microwell is a novel microstructure supporting isolation and trapping of cells,... more The inverted open microwell is a novel microstructure supporting isolation and trapping of cells, analysis of cell-cell and cell-molecule interactions and functional cell sorting. This work introduces the inverted open microwell concept, demonstrating successful isolation of K562 cells in 75 μm microwells fabricated on a flexible printed circuit board substrate, and recovery of viable cells onto standard microtiter plates after analysis and manipulation. Dielectrophoresis (DEP) was used during the delivery phase to control cell access to the microwell and force the formation of cell aggregates so as to ensure cell-cell contact and interaction. Cells were trapped at the air-fluid interface at the bottom edge of the open microwell. Once trapped, cells were retained on the meniscus even after DEP de-activation and fluid was exchanged to enable perfusion of nutrients and delivery of molecules to the microwell, as demonstrated by a calcein-staining protocol performed in the microsystem. Finally, cell viability was assessed on trapped cells by a calcein release assay and cell proliferation was demonstrated after multiple cells had been recovered in parallel onto standard microtiter plates.
We present a novel method for the isolation, functional analysis and sorting of single cells or s... more We present a novel method for the isolation, functional analysis and sorting of single cells or small cellular aggregates. Inverted open microwells featuring a microchannel on top, an open air-fluid interface on bottom side and embedded electrodes were implemented on flexible-PCB technology. Microwell arrays in pitch with standard microtiter plates were implemented. K562 cells were delivered to the microwells and trapped at the air-flow interface. Cell delivery and aggregate formation were controlled by dielectrophoresis. Continuous fluid replacement around the cells during the experiment was demonstrated. After analysis, live cells were easily recovered to standard plates where cell growth was demonstrated.
Particle detection in microstructures is a key procedure required by modern lab-on-a-chip devices... more Particle detection in microstructures is a key procedure required by modern lab-on-a-chip devices. Unfortunately, state of the art approaches to impedance measuring as applied to cell detection do not perform well in regions characterized by non-homogeneous physical parameters due, for example, to the presence of air-liquid interfaces or when the particle-electrode distance is relatively high. This paper presents a robust impedance measurement technique and a circuit for detecting cells flowing in microstructures such as microchannels and microwells. Our solution makes use of an innovative three-electrode measurement scheme with asymmetric polarization in order to increase cell detection ability in microstructures featuring large electrode distances of up to 100 μm as well as to limit signal loss due to cell position relative to the electrodes. Compared to standard techniques, numerical simulations show that, with the proposed approach, the cell detection sensitivity is increased by more than 40%. In addition, we propose a custom circuit based on division instead of difference between signals, as in standard differential circuits, so as to reduce the baseline signal drift induced by non-homogeneous conductivity. A simplified analytical model shows an increase in the signal-to-noise-ratio comprised in the range 3.9-5.9. Experimental results, carried out using an open-microwell device made with flexible printed circuit board technology, are in agreement with simulations, suggesting a six-fold increase of the signal-to-noise ratio compared to the differential measurement technique. We were thus able to successfully monitor the process of isolating K562 leukemia cells inside open-microwells determining all single-cell events with no false positive detection.
We present here a lab-on-a-chip to perform cell centrifugation and re-suspension at single-cell l... more We present here a lab-on-a-chip to perform cell centrifugation and re-suspension at single-cell level. Microchannels are used to isolate cells inside microwells where fluid replacement can be obtained thanks to microfluidic mixing. Electrodes integrated into each microwell were used to detect conductivity variation of the infused fluids allowing also for a monitored and automated execution of biological protocols which require supernatant substitution. A protocol involving a precise replacement of the supernatants as required by a cell viability assay was implemented demonstrating single-cell microcentrifugation and re-suspension capabilities as confirmed by calcein uptake shown by fluorescence microscopy.
The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of re... more The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of researchers and engineers on standard process of the electronics industry for low production costs and large scale development, necessary for disposable applications. The printed circuit board technology could be used for this purpose, in particular for the wide range of materials available. In this paper, assays on biocompatibility of materials used for Lab-on-a-chip fabrication has been carried out using two tumor cell lines growing in suspension, the human chronic myelogenous leukemia K562 cell line, able to undergo erythroid differentiation when cultured with chemical inducers, and the lymphoblastoid cell line (LCL), extensively used for screening of cytotoxic T-lymphocytes (CTLs). We have demonstrated that some materials strongly inhibit cell proliferation of both the two cell lines to an extent higher that 70-75%, but only after a prolonged exposure of 3-6 days (Copper, Gold over Nickel, Aramid fiber filled epoxy uncured, b-stage epoxy die attach film, Tesa 4985 adhesive tape, Pyralux uncured, Copper + 1-octodecanethiol). However, when experiments were performed with short incubation time (1 h), only Aramid fiber filled epoxy uncured was cytotoxic. Variation of the results concerning the other materials was appreciable when the experiments performed on two cell lines were compared together. Furthermore, the effects of the materials on erythroid differentiation and CTL-mediated LCL lysis confirmed, in most of the cases, the data obtained in cytotoxic and antiproliferative tests.
In this work we present a novel concept of active microwells based on cylindrical wells able to v... more In this work we present a novel concept of active microwells based on cylindrical wells able to vertically trap and control single particles by means of negative dielectrophoresis. The device is fabricated by drilling through holes on a polyimide substrate with copper–gold or aluminum metals, forming three annular electrodes within the well. A channel under the device provides a fluid flow filling the microwell by capillarity. Particles are delivered from the top by a microdispenser and applying sinusoidal signals to the electrodes at frequencies ranging from 100 kHz to 1.5 MHz and amplitudes between 2 V and 7 V they are successfully trapped and levitated at the level of the central electrode in the middle of microwells with a diameter of 125 μm. By changing signal phases, other configurations are also enabled to load particles in the well or eject them from the bottom. The extension to an array of microwells is presented and design rules are described for routing electrode connections and setting signal parameters. K562 cells cultured with Ara-C 1000 nM were successfully trapped and controlled in physiological media. Polystyrene beads were also levitated in water and were used for experimental measurements on minimum amplitudes and phase differences in the signals required to levitate beads, confirming the results obtained by simulation.
This review paper summarizes some of the most challenging issues regarding the development of ele... more This review paper summarizes some of the most challenging issues regarding the development of electronic microsystems for the isolation, manipulation, and characterization of rare cells. Two relevant areas, namely, immunology for cancer therapy and monitoring of microbial contaminants in water, are presented as example applications which require handling of rare cells. Starting from these applications, the state of the art in electronic microsystem research is presented, and various solutions are discussed, highlighting the advantages and disadvantages of each technology and suggesting the need to integrate multiple solutions to meet the application requirements.
We present the structure of an open microwell, i.e. a microwell open at both the top and bottom e... more We present the structure of an open microwell, i.e. a microwell open at both the top and bottom ends, which enables single-cells to be handled, processed and recovered after the experiment. The microwell has a novel architecture which allows particles to be trapped and forced to interact by means of a cylindrical negative dielectrophoretic cage. Particles are aligned along a horizontal axis where the electric field minimum is placed. Arrays of open microwells are fabricated using flexible printed circuit board (PCB) technology providing cheap and disposable devices. Levitation and precise positioning of both polystyrene beads and K562 cells were experimented, confirming the results of physical simulations. Assessment of cell viability after 20 min exposure to the electric field was performed through a standard calcein-release assay.
Printed circuit board (PCB) technology can be used for producing lab-on-a-chip (LOAC) devices. PC... more Printed circuit board (PCB) technology can be used for producing lab-on-a-chip (LOAC) devices. PCBs are characterized by low production costs and large-scale development, both essential elements in the frame of disposable applications. LOAC platforms have been employed not only for diagnostic and/or analytical purposes, but also for identification and isolation of eukaryotic cells, including cancer and stem cells. Accordingly, the compatibility of the employed materials with the biological system under analysis is critical for the development of LOAC devices to be proposed for efficient and safe cell isolation. In this study, we analyzed the in-vitro compatibility of a large set of materials and surface treatments used for LOAC development and evaluation with quasi-standard PCB processes. Biocompatibility was analyzed on hippocampal primary cells (a model of attached cell cultures), in comparison with the reference K562 cell line (a model of cells growing in suspension). We demonstrate here that some of the materials under study alter survival, organization, morphology and adhesion capacity of hippocampal cells, and inhibit growth and differentiation of K562 cells. Nonetheless, a subset of the materials tested did not negatively affect these functions, thus demonstrating that PCB technology, with some limitations, is suitable for the realization of LOAC devices well compatible at least with these preparations.
Reconfigurable processor architectures (RAs) have been proving as an effective way to couple sign... more Reconfigurable processor architectures (RAs) have been proving as an effective way to couple significant performance improvements with severe energy constraints, such as those imposed by modern portable real-time applications. XiRisc is a VLIW RISC processor architecture featuring a reconfigurable dataflow-oriented functional unit, the so-called PiCoGA, allowing run-time dynamic extension of the instruction set. In this paper, we propose a LISA-based instruction set simulator (ISS) for the reconfigurable processor, retargetable through a dynamically linked library that emulates instruction set extension. The ISS comprises a SystemC system-level model with embedded bus architecture and memory hierarchy (on-chip and off-chip) to provide a reconfigurable system-on-chip performance evaluator.
This paper introduces a novel heterogeneous shared memory multiprocessor architecture based on a ... more This paper introduces a novel heterogeneous shared memory multiprocessor architecture based on a reconfigurable processor and a standard RISC processor. The work aims at increasing further the computational density of a reconfigurable device by the integration of the reconfigurable hardware into a heterogeneous multi-core architecture. Though a RISC processor has a lower inner computational density than a reconfigurable processor, this
ARCES (DEIS) University of Bologna, Bologna, Italy email: {andrea.lodi,cmucci}@deis.unibo. it {ac... more ARCES (DEIS) University of Bologna, Bologna, Italy email: {andrea.lodi,cmucci}@deis.unibo. it {acappelli,mbocchi}@deis.unibo.it ... STMicroelectronics FTM Agrate Brianza, Italy email: [email protected] ... The integration of a reconfigurable device into complex SoCs is ...
In this work we present a method for delivering small fluid volumes possibly containing cells and... more In this work we present a method for delivering small fluid volumes possibly containing cells and particles over a flexible large area substrate. Particles are deposited by a dispensing device that performs serial delivery of liquid volumes in the range of 200 pL. Due to the required precision, the alignment and the calibration of the spotting system are crucial for the proper performance of the delivery process. The system provides automated motion of mechanical parts with micrometric precision. To address the problem of the device deformation caused by the flexibility of the substrate, the 3D coordinates of each site need to be known, but the serial acquisition of all the coordinates would be highly time consuming for high-throughput arrays and large devices. We solved this problem by sampling the coordinates of a small subset and interpolating the positions of the remaining sites by means of a custom algorithm. System calibration for a device with 1536 sites was achieved in 150 s vs. 9630 s required for full sampling. Measurements performed with fluorescent microbeads showed that 100% of the particles were deposited within a spot with a radius of 50 μm.
This paper presents a local buffer memory in the form of a Stream Register File (SRF), that was d... more This paper presents a local buffer memory in the form of a Stream Register File (SRF), that was developed in order to connect, in a compiler-friendly pattern, large-bandwidth run-time configurable logic units in processor-based SOCs. The proposed SRF offers to the host SOC system performance speedups in the range of 4x, with area/power overhead in the order of 6%. The described hardware and algorithm mapping strategy was implemented on silicon in a SOC based on the PiCoGA reconfigurable architecture. The SOC provides an average 450 MOPS (Mega Operations per Second) in STM CMOS090 technology running at 100MHZ.
The inverted open microwell is a novel microstructure supporting isolation and trapping of cells,... more The inverted open microwell is a novel microstructure supporting isolation and trapping of cells, analysis of cell-cell and cell-molecule interactions and functional cell sorting. This work introduces the inverted open microwell concept, demonstrating successful isolation of K562 cells in 75 μm microwells fabricated on a flexible printed circuit board substrate, and recovery of viable cells onto standard microtiter plates after analysis and manipulation. Dielectrophoresis (DEP) was used during the delivery phase to control cell access to the microwell and force the formation of cell aggregates so as to ensure cell-cell contact and interaction. Cells were trapped at the air-fluid interface at the bottom edge of the open microwell. Once trapped, cells were retained on the meniscus even after DEP de-activation and fluid was exchanged to enable perfusion of nutrients and delivery of molecules to the microwell, as demonstrated by a calcein-staining protocol performed in the microsystem. Finally, cell viability was assessed on trapped cells by a calcein release assay and cell proliferation was demonstrated after multiple cells had been recovered in parallel onto standard microtiter plates.
We present a novel method for the isolation, functional analysis and sorting of single cells or s... more We present a novel method for the isolation, functional analysis and sorting of single cells or small cellular aggregates. Inverted open microwells featuring a microchannel on top, an open air-fluid interface on bottom side and embedded electrodes were implemented on flexible-PCB technology. Microwell arrays in pitch with standard microtiter plates were implemented. K562 cells were delivered to the microwells and trapped at the air-flow interface. Cell delivery and aggregate formation were controlled by dielectrophoresis. Continuous fluid replacement around the cells during the experiment was demonstrated. After analysis, live cells were easily recovered to standard plates where cell growth was demonstrated.
Particle detection in microstructures is a key procedure required by modern lab-on-a-chip devices... more Particle detection in microstructures is a key procedure required by modern lab-on-a-chip devices. Unfortunately, state of the art approaches to impedance measuring as applied to cell detection do not perform well in regions characterized by non-homogeneous physical parameters due, for example, to the presence of air-liquid interfaces or when the particle-electrode distance is relatively high. This paper presents a robust impedance measurement technique and a circuit for detecting cells flowing in microstructures such as microchannels and microwells. Our solution makes use of an innovative three-electrode measurement scheme with asymmetric polarization in order to increase cell detection ability in microstructures featuring large electrode distances of up to 100 μm as well as to limit signal loss due to cell position relative to the electrodes. Compared to standard techniques, numerical simulations show that, with the proposed approach, the cell detection sensitivity is increased by more than 40%. In addition, we propose a custom circuit based on division instead of difference between signals, as in standard differential circuits, so as to reduce the baseline signal drift induced by non-homogeneous conductivity. A simplified analytical model shows an increase in the signal-to-noise-ratio comprised in the range 3.9-5.9. Experimental results, carried out using an open-microwell device made with flexible printed circuit board technology, are in agreement with simulations, suggesting a six-fold increase of the signal-to-noise ratio compared to the differential measurement technique. We were thus able to successfully monitor the process of isolating K562 leukemia cells inside open-microwells determining all single-cell events with no false positive detection.
We present here a lab-on-a-chip to perform cell centrifugation and re-suspension at single-cell l... more We present here a lab-on-a-chip to perform cell centrifugation and re-suspension at single-cell level. Microchannels are used to isolate cells inside microwells where fluid replacement can be obtained thanks to microfluidic mixing. Electrodes integrated into each microwell were used to detect conductivity variation of the infused fluids allowing also for a monitored and automated execution of biological protocols which require supernatant substitution. A protocol involving a precise replacement of the supernatants as required by a cell viability assay was implemented demonstrating single-cell microcentrifugation and re-suspension capabilities as confirmed by calcein uptake shown by fluorescence microscopy.
The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of re... more The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of researchers and engineers on standard process of the electronics industry for low production costs and large scale development, necessary for disposable applications. The printed circuit board technology could be used for this purpose, in particular for the wide range of materials available. In this paper, assays on biocompatibility of materials used for Lab-on-a-chip fabrication has been carried out using two tumor cell lines growing in suspension, the human chronic myelogenous leukemia K562 cell line, able to undergo erythroid differentiation when cultured with chemical inducers, and the lymphoblastoid cell line (LCL), extensively used for screening of cytotoxic T-lymphocytes (CTLs). We have demonstrated that some materials strongly inhibit cell proliferation of both the two cell lines to an extent higher that 70-75%, but only after a prolonged exposure of 3-6 days (Copper, Gold over Nickel, Aramid fiber filled epoxy uncured, b-stage epoxy die attach film, Tesa 4985 adhesive tape, Pyralux uncured, Copper + 1-octodecanethiol). However, when experiments were performed with short incubation time (1 h), only Aramid fiber filled epoxy uncured was cytotoxic. Variation of the results concerning the other materials was appreciable when the experiments performed on two cell lines were compared together. Furthermore, the effects of the materials on erythroid differentiation and CTL-mediated LCL lysis confirmed, in most of the cases, the data obtained in cytotoxic and antiproliferative tests.
In this work we present a novel concept of active microwells based on cylindrical wells able to v... more In this work we present a novel concept of active microwells based on cylindrical wells able to vertically trap and control single particles by means of negative dielectrophoresis. The device is fabricated by drilling through holes on a polyimide substrate with copper–gold or aluminum metals, forming three annular electrodes within the well. A channel under the device provides a fluid flow filling the microwell by capillarity. Particles are delivered from the top by a microdispenser and applying sinusoidal signals to the electrodes at frequencies ranging from 100 kHz to 1.5 MHz and amplitudes between 2 V and 7 V they are successfully trapped and levitated at the level of the central electrode in the middle of microwells with a diameter of 125 μm. By changing signal phases, other configurations are also enabled to load particles in the well or eject them from the bottom. The extension to an array of microwells is presented and design rules are described for routing electrode connections and setting signal parameters. K562 cells cultured with Ara-C 1000 nM were successfully trapped and controlled in physiological media. Polystyrene beads were also levitated in water and were used for experimental measurements on minimum amplitudes and phase differences in the signals required to levitate beads, confirming the results obtained by simulation.
This review paper summarizes some of the most challenging issues regarding the development of ele... more This review paper summarizes some of the most challenging issues regarding the development of electronic microsystems for the isolation, manipulation, and characterization of rare cells. Two relevant areas, namely, immunology for cancer therapy and monitoring of microbial contaminants in water, are presented as example applications which require handling of rare cells. Starting from these applications, the state of the art in electronic microsystem research is presented, and various solutions are discussed, highlighting the advantages and disadvantages of each technology and suggesting the need to integrate multiple solutions to meet the application requirements.
We present the structure of an open microwell, i.e. a microwell open at both the top and bottom e... more We present the structure of an open microwell, i.e. a microwell open at both the top and bottom ends, which enables single-cells to be handled, processed and recovered after the experiment. The microwell has a novel architecture which allows particles to be trapped and forced to interact by means of a cylindrical negative dielectrophoretic cage. Particles are aligned along a horizontal axis where the electric field minimum is placed. Arrays of open microwells are fabricated using flexible printed circuit board (PCB) technology providing cheap and disposable devices. Levitation and precise positioning of both polystyrene beads and K562 cells were experimented, confirming the results of physical simulations. Assessment of cell viability after 20 min exposure to the electric field was performed through a standard calcein-release assay.
Printed circuit board (PCB) technology can be used for producing lab-on-a-chip (LOAC) devices. PC... more Printed circuit board (PCB) technology can be used for producing lab-on-a-chip (LOAC) devices. PCBs are characterized by low production costs and large-scale development, both essential elements in the frame of disposable applications. LOAC platforms have been employed not only for diagnostic and/or analytical purposes, but also for identification and isolation of eukaryotic cells, including cancer and stem cells. Accordingly, the compatibility of the employed materials with the biological system under analysis is critical for the development of LOAC devices to be proposed for efficient and safe cell isolation. In this study, we analyzed the in-vitro compatibility of a large set of materials and surface treatments used for LOAC development and evaluation with quasi-standard PCB processes. Biocompatibility was analyzed on hippocampal primary cells (a model of attached cell cultures), in comparison with the reference K562 cell line (a model of cells growing in suspension). We demonstrate here that some of the materials under study alter survival, organization, morphology and adhesion capacity of hippocampal cells, and inhibit growth and differentiation of K562 cells. Nonetheless, a subset of the materials tested did not negatively affect these functions, thus demonstrating that PCB technology, with some limitations, is suitable for the realization of LOAC devices well compatible at least with these preparations.
Reconfigurable processor architectures (RAs) have been proving as an effective way to couple sign... more Reconfigurable processor architectures (RAs) have been proving as an effective way to couple significant performance improvements with severe energy constraints, such as those imposed by modern portable real-time applications. XiRisc is a VLIW RISC processor architecture featuring a reconfigurable dataflow-oriented functional unit, the so-called PiCoGA, allowing run-time dynamic extension of the instruction set. In this paper, we propose a LISA-based instruction set simulator (ISS) for the reconfigurable processor, retargetable through a dynamically linked library that emulates instruction set extension. The ISS comprises a SystemC system-level model with embedded bus architecture and memory hierarchy (on-chip and off-chip) to provide a reconfigurable system-on-chip performance evaluator.
This paper introduces a novel heterogeneous shared memory multiprocessor architecture based on a ... more This paper introduces a novel heterogeneous shared memory multiprocessor architecture based on a reconfigurable processor and a standard RISC processor. The work aims at increasing further the computational density of a reconfigurable device by the integration of the reconfigurable hardware into a heterogeneous multi-core architecture. Though a RISC processor has a lower inner computational density than a reconfigurable processor, this
ARCES (DEIS) University of Bologna, Bologna, Italy email: {andrea.lodi,cmucci}@deis.unibo. it {ac... more ARCES (DEIS) University of Bologna, Bologna, Italy email: {andrea.lodi,cmucci}@deis.unibo. it {acappelli,mbocchi}@deis.unibo.it ... STMicroelectronics FTM Agrate Brianza, Italy email: [email protected] ... The integration of a reconfigurable device into complex SoCs is ...
In this work we present a method for delivering small fluid volumes possibly containing cells and... more In this work we present a method for delivering small fluid volumes possibly containing cells and particles over a flexible large area substrate. Particles are deposited by a dispensing device that performs serial delivery of liquid volumes in the range of 200 pL. Due to the required precision, the alignment and the calibration of the spotting system are crucial for the proper performance of the delivery process. The system provides automated motion of mechanical parts with micrometric precision. To address the problem of the device deformation caused by the flexibility of the substrate, the 3D coordinates of each site need to be known, but the serial acquisition of all the coordinates would be highly time consuming for high-throughput arrays and large devices. We solved this problem by sampling the coordinates of a small subset and interpolating the positions of the remaining sites by means of a custom algorithm. System calibration for a device with 1536 sites was achieved in 150 s vs. 9630 s required for full sampling. Measurements performed with fluorescent microbeads showed that 100% of the particles were deposited within a spot with a radius of 50 μm.
This paper presents a local buffer memory in the form of a Stream Register File (SRF), that was d... more This paper presents a local buffer memory in the form of a Stream Register File (SRF), that was developed in order to connect, in a compiler-friendly pattern, large-bandwidth run-time configurable logic units in processor-based SOCs. The proposed SRF offers to the host SOC system performance speedups in the range of 4x, with area/power overhead in the order of 6%. The described hardware and algorithm mapping strategy was implemented on silicon in a SOC based on the PiCoGA reconfigurable architecture. The SOC provides an average 450 MOPS (Mega Operations per Second) in STM CMOS090 technology running at 100MHZ.
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Papers by Massimo Bocchi