Julien Reboud

Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system.

An electrical biosensor for the detection of circulating tumor cells

In this report, we demonstrate a semi-integrated electrical biosensor for the detection of rare circulating tumor cells (CTCs) in blood. The sample was first enriched through a combination of immunomagnetic isolation and size filtration. The integration of both methods provided a high enrichment performance with a recovery rate above 70%, even for very low numbers of cancer cells present in the original sample (10 spiked MCF7 cells in 0.5 mL of blood). In the same system, the sample was then transferred to a microchip for further magnetic concentration, followed by immunochemical trapping and electronic detection by impedance spectroscopy. Three levels of spiked CTC number (30±2, 124±29, 273±23) in 10 μL of filtered blood sample were distinguished by monitoring the impedance change of the microelectrode array (MEA). The integration of different functions in a single system provided a methodology to process milliliter-sized blood samples at the macroscale and interface with the microdimensions of a highly sensitive electronic detector. The results showed that the whole system was able to detect different levels of spiked cancer cells without the use of time- and cost-intensive fluorescence labeling and image analysis. This has the potential to provide clinicians with a standalone system to monitor changes in CTC numbers throughout therapy conveniently and frequently for efficient cancer treatments.

Tuneable surface acoustic waves for fluid and particle manipulations on disposable chips

We establish a powerful new acoustic technique to programme complex fluidic functions such as droplet movement, merging, mixing and concentration, on a disposable superstrate.

Phononic Crystals for Shaping Fluids

Surface acoustic waves (SAWs) generated on piezoelectric materials have been used as a convenient method for microfl uidic manipulation, where microliter volumes of liquids are actuated by their interaction with sound waves. A wide range of fundamental fl uid actuations, including droplet movement, mixing, splitting, nebulization, and centrifugation, have been performed on such a piezoelectric surface. [ 1 , 2 ] Mention of the jetting of a sessile drop from a piezoelectric substrate have been made in reports on other phenomenon, where the propagating SAW is refracted into the liquid and jets a droplet in a direction known as the Rayleigh angle. [ 3 , 4 ] More signifi cantly, it has also been reported that, by using two circular single-phase unidirectional transducers to radiate the SAW, the energy can be focused to deform a drop into an interfacial jet perpendicular to the piezoelectric surface. [ 5 ]

Palm-sized biodetection system based on localized surface plasmon resonance.

We present a complete palm-sized, battery-operated biodetection system based on highly sensitive localized surface plasmon resonance (LSPR). We have replaced the spectrum analyzer by four pulse-powered light-emitting diodes (LED), each with different emission spectra. The reflected light beams from all LEDs were detected by a single photodiode. Its composite output current was demultiplexed by a four-channel lock-in amplifier. Device performance was demonstrated using an LSPR chip covered with a mixture of ethanol/water and 2-propanol/water at different concentrations. The miniaturized system does not require any external power supply or personal computer and it is therefore suitable for point-of-care and field applications.

Label-free impedance detection of low levels of circulating endothelial progenitor cells for point-of-care diagnosis.

This paper presents a novel microfluidic system for rapid label-free detection of endothelial progenitor cells (EPCs) from small volumes of white blood cells samples, to obtain a bedside cardiovascular diagnostic solution. The system was built on a single 1 cm(2) microelectrode array silicon chip, integrated with negative dielectrophoresis for cell trapping, surface immunochemistry for selective cell capture, and fluidics for cell washing and impedance detection. The level of circulating EPC level in blood is a biomarker of clinical interest, linked to the assessment of risk factors in cardiovascular diseases which are a major global concern. Rare EPCs are usually detected through in vitro culture or flow cytometry, which are too time-consuming to bring timely reports in acute diseases. Although microfluidics approaches have enabled reduced processing time and enhanced portability, their sensitivity and processing volumes are still inadequate for rare cell detection at a bedside setting. Using small highly sensitive microelectrodes, our novel integrated system achieved the detection of 720 EPCs in a small 12 microl sample of 72,000 peripheral blood mononuclear cells (PBMC), i.e. equivalent to a concentration of EPCs of 0.1% of 100 microl blood. This demonstrated that clinically significant level of EPCs (<0.5% of PBMC) could be detected for the first time on a detection system at bedside set-up, showing great potential in applications for point-of-care diagnosis.

Paper‐Origami‐Based Multiplexed Malaria Diagnostics from Whole Blood

Abstract We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.

Toxicity Assays in Nanodrops Combining Bioassay and Morphometric Endpoints

Background Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. Methodology This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl2), Sodium Arsenate (NaAsO2) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl2 and NaAsO2, in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses. Conclusions Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework.

Cell chip array for microfluidic proteomics enabling rapid in situ assessment of intracellular protein phosphorylation

We discuss the ability to perform fluorescent immunocytochemistry, following cell fixation, using a microfluidic array of primary, nonadherent, single CD34+ stem cells. The technique requires small cell samples and proceeds with no cell loss, making it well-suited to monitoring these rare patient-derived cells. The chip allows us to correlate live cell dynamics across arrays of individual cells with post-translational modifications of intracellular proteins, following their exposure to drug treatments. Results also show that due to the microfluidic environment, the time scale of cell fixation was significantly reduced compared to conventional methods, leading to greater confidence in the status of the protein modifications studied.

From chip-in-a-lab to lab-on-a-chip: towards a single handheld electronic system for multiple application-specific lab-on-a-chip (ASLOC)

We present a portable, battery-operated and application-specific lab-on-a-chip (ASLOC) system that can be easily configured for a wide range of lab-on-a-chip applications. It is based on multiplexed electrical current detection that serves as the sensing system. We demonstrate different configurations to perform most detection schemes currently in use in LOC systems, including some of the most advanced such as nanowire-based biosensing, surface plasmon resonance sensing, electrochemical detection and real-time PCR. The complete system is controlled by a single chip and the collected information is stored in situ, with the option of transferring the data to an external display by using a USB interface. In addition to providing a framework for truly portable real-life developments of LOC systems, we envisage that this system will have a significant impact on education, especially since it can easily demonstrate the benefits of integrated microanalytical systems.