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Biomedical subjects

Emmanuel Delamarche

Publications and source records attributed to Emmanuel Delamarche.

13 recordsLinked to original sources
PubMed2007

Screening cell surface receptors using micromosaic immunoassays.

This report presents a general method for screening cell surface receptors using so-called micromosaic immunoassays. This method employs a microfluidic chip having n (n = 11) independent flow paths to move cells over m (m = 11) lines of surface-patterned antibodies for screening individual cells in a parallel, combinatorial, fast and flexible manner. The antibodies are patterned as 30-microm-wide lines on a poly(dimethylsiloxane) layer used to seal the area of the chip in which screening is being monitored. Mouse hybridoma cells having CD44 cell surface receptors and anti-CD44 antibodies were used to establish a proof-of-concept for this method. Both the capture antibodies and the cells were fluorescently labelled to allow the position of the cells to be accurately tracked over the binding sites using an inverted fluorescence microscope. The chips and cells were maintained at a constant temperature between 20 to 37 degrees C, and flow velocities of the cells over the capture areas were 100-280 microm s(-1), resulting in a approximately 0.1-0.3 s residency time of the cells on each of the eleven 30 x 30 microm s2 capture areas. Binding of the cells appeared to be specific to the capture areas, with a yield of 30% when the assay was performed at a temperature of 37 degrees C and with a slow flow velocity. We suggest that this proof-of-concept is broadly applicable to the screening of cells for medical/diagnostic purposes as well as for basic research on the interaction of cells with surfaces.

Animals↗
PubMed2006

Capillary pumps for autonomous capillary systems.

Autonomous capillary systems (CSs), where liquids are displaced by means of capillarity, are efficient, fast and convenient platforms for many bioanalytical applications. The proper functioning of these microfluidic devices requires displacing accurate volumes of liquids with precise flow rates. In this work, we show how to design capillary pumps for controlling the flow properties of CSs. The capillary pumps comprise microstructures of various shapes with dimensions from 15-250 microm, which are positioned in the capillary pumps to encode a desired capillary pressure. The capillary pumps are designed to have a small flow resistance and are preceded by a constricted microchannel, which acts as a flow resistance. Therefore, both the capillary pump and the flow resistance define the flow rate in the CS, and flow rates from 0.2-3.7 nL s(-1) were achieved. The placement and the shape of the microstructures in the capillary pumps are used to tailor the filling front of liquids in the capillary pumps to obtain a reliable filling behaviour and to minimize the risk of entrapping air. The filling front can, for example, be oriented vertically or tilted to the main axis of the capillary pump. We also show how capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in a CS.

Journal Article↗
PubMed2005

Microcontact printing of proteins inside microstructures.

Microfluidic devices are well suited for the miniaturization of biological assays, in particular when only small volumes of samples and reagents are available, short time to results is desirable, and multiple analytes are to be detected. Microfluidic networks (MFNs), which fill by means of capillary forces, have already been used to detect important biological analytes with high sensitivity and in a combinatorial fashion. These MFNs were coated with Au, onto which a hydrophilic, protein-repellent monolayer of thiolated poly(ethyleneglycol) (HS-PEG) was self-assembled, and the binding sites for analytes were present on a poly(dimethylsiloxane) (PDMS) sealing cover. We report here a set of simple methods to extend previous work on MFNs by integrating binding sites for analytes inside the microstructures of MFNs using microcontact printing (muCP). First, fluorescently labeled antibodies (Abs) were microcontact-printed from stamps onto planar model surfaces such as glass, Si, Si/SiO2, Au, and Au derivatized with HS-PEG to investigate how much candidate materials for MFNs would quench the fluorescence of printed, labeled Abs. Au coated with HS-PEG led to a fluorescence signal that was approximately 65% weaker than that of glass but provided a convenient surface for printing Abs and for rendering the microstructures of the MFNs wettable. Then, proteins were inked from solution onto the surface of PDMS (Sylgard 184) stamps having continuous or discontinuous micropatterns or locally inked onto planar stamps to investigate how the aspect ratio (depth:width) of microstructures and the printing conditions affected the transfer of protein and the accuracy of the resulting patterns. By applying a controlled pressure to the back of the stamp, Abs were accurately microcontact-printed into the recessed regions of MFNs if the aspect ratio of the MFN microstructures was lower than approximately 1:6. Finally, the realization of a simple assay between Abs (used as antigens) microcontact-printed in microchannels and Abs from solution suggests that this method could become useful to pattern proteins in microstructures for advanced bioanalytical purposes.

Antibodies↗
PubMed2005

Continuous flow in open microfluidics using controlled evaporation.

This paper presents a method for programming the flow rate of liquids inside open microfluidic networks (MFNs). A MFN comprises a number of independent flow paths, each of which starts with an open filling port, has a sealed microchannel in which assays can be performed, and an open capillary pump (CP). The MFN is placed over Peltier elements and its flow paths initially fill owing to capillary forces when liquids are added to the filling ports. A cooling Peltier element underneath the filling ports dynamically prevents evaporation in all filling ports using the ambient temperature and relative humidity as inputs. Another Peltier element underneath the CPs heats the pumps thereby inducing evaporation in the CPs and setting the flow rate in the microchannels. This method achieves flow rates in the microchannels ranging from approximately 1.2 nL s(-1) to approximately 30 pL s(-1), and is able to keep 90% of a 0.6 microL solution placed in an open filling port for 60 min. This simple and efficient method should be applicable to numerous assays or chemical reactions that require small and precise flow of liquids and reagents inside microfluidics.

Capillary Action↗
PubMed2005

Multipurpose microfluidic probe.

Microfluidic systems allow (bio)chemical processes to be miniaturized with the benefit of shorter time-to-result, parallelism, reduced sample consumption, laminar flow, and increased control and efficiency. However, such miniaturization inherently limits the size of the solid objects that can be processed and entails new challenges such as the interfacing of macroscopic samples with microscopic conduits. Here, we report a microfluidic probe (MFP) that overcomes these problems by combining the concepts of 'microfluidics' and of 'scanning probes'. Here, liquid boundaries formed by hydrodynamic forces underneath the MFP confine a flow of processing solution and replace the solid walls of closed microchannels. The MFP is therefore mobile and can be used to process large surfaces and objects by scanning across them. We illustrate the versatility of this concept with several examples including protein microarraying, complex gradient-formation, multiphase laminar-flow patterning, erasing, localized staining of cells and the contact-free detachment of a single cell. Many constraints imposed by the monolithic construction of microfluidic channels can now be circumvented using an MFP, opening up new avenues for microfluidic processing.

Journal Article↗
PubMed2005

Diffusion of alkanethiols in PDMS and its implications on microcontact printing (muCP).

n-Alkanethiols HS-(CH2)n-CH3 such as hexadecanethiol (HDT, n = 15), octadecanethiol (ODT, n = 17), and eicosanethiol (ECT, n = 19) have been shown to provide highly protective etch resists on microcontact-printed noble metals. As the quality of the printed pattern strongly depends on the mobility of the ink compound, we focused on understanding the diffusion behavior of HDT, ODT, and ECT in poly(dimethylsiloxane) (PDMS) stamps. We used a commercial PDMS material (Sylgard184), which is commonly used for microcontact printing (muCP), and a custom-synthesized one with a higher modulus. On the basis of linear-diffusion experiments, which maintained realistic printing conditions, we showed that the ink transport in the stamp follows Fick's law of diffusion. We then determined the diffusion coefficient by analytical and numerical modeling of the diffusion experiments. Numerical calculations were carried out with the finite-difference method applying more realistic boundary conditions (ink adsorption). Values for the diffusion coefficients of the three ink compounds in the two different PDMS materials all are on the order of (4-7) x 10(-7) cm2 s(-1). The scope and limits of the mathematical models are discussed. To demonstrate the potential of such models for microcontact printing, we simulate multiple printing cycles of an inked stamp and compare the results with experimental data.

Journal Article↗
PubMed2005

Modeling and optimization of high-sensitivity, low-volume microfluidic-based surface immunoassays.

Microfluidics are emerging as a promising technology for miniaturizing biological assays for applications in diagnostics and research in life sciences because they enable the parallel analysis of multiple analytes with economy of samples and in short time. We have previously developed microfluidic networks for surface immunoassays where antibodies that are immobilized on one wall of a microchannel capture analytes flowing in the microchannel. This technology is capable of detecting analytes with picomolar sensitivity and from sub-microliter volume of sample within 45 min. This paper presents the theoretical modeling of these immunoassays where a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We then derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance, which we think will allow further development of microfluidic networks for immunoassay applications.

Antigen-Antibody Complex↗
PubMed2004

High-sensitivity miniaturized immunoassays for tumor necrosis factor alpha using microfluidic systems.

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).

Antibodies↗
PubMed2004

Simultaneous detection of C-reactive protein and other cardiac markers in human plasma using micromosaic immunoassays and self-regulating microfluidic networks.

We show a proof-of-concept in which we combine our previously published concepts of micromosaic immunoassays (microMIAs) with self-regulating microfluidic networks (microFNs) to detect C-reactive protein (CRP) and other cardiac markers such as myoglobin (Mb) and cardiac Troponin I (cTnI). The microFNs are microfabricated in Si, have a well-defined surface chemistry, and are affixed to a bibulous material so as to self-regulate the displacement of an aliquot of liquid through the microFNs using capillary forces. An open section of the channels of the microFNs is covered with a hydrophobic poly(dimethylsiloxane) (PDMS) slab that acts as the substrate for a solid-phase immunoassay. Here, individual assays are conducted using independent channels. These assays are "sequential": series of samples, reagents, and buffers are displaced one after the other over the PDMS surface, and, as these assays are conducted under "microfluidic" conditions, they are fast to perform, very economical in their use of reagents, extremely integrated, and yield high-quality signals. The combinatorial character of microMIAs is exploited to optimize the assay parameters for detecting CRP. In particular, we found it optimal to deposit the capture antibody for CRP on PDMS at a concentration between 20 and 500 microg ml(-1) in PBS in 1 min and to detect captured CRP in 2 min using a detection antibody having a concentration in PBS of 120 microg ml(-1). With this method, CRP is quantitatively detected within 10 min in one microliter of human plasma down to concentrations of 30 ng ml(-1), which suggests the possibility to detect CRP at clinically relevant concentrations for the management of coronary heart disease (CHD) and systemic inflammation.

Antibodies↗
PubMed2002

Autonomous microfluidic capillary system.

The transport of minute amounts of liquids using microfluidic systems has opened avenues for higher throughput and parallelization of miniaturized bio/chemical processes combined with a great economy of reagents. In this report, we present a microfluidic capillary system (CS) that autonomously transports aliquots of different liquids in sequence: liquids pipetted into the service port of the CS flow unidirectionally through the various sections of the CS, which comprises a 15-pL reaction chamber, into the capillary pump. A CS can thus be operated by simply delivering the different samples to its service port. The liquid transport concept presented here is advantageous because the pumping and valving functions are integrated into the device by means of capillary phenomena, and it therefore does not require any external power supply or control device. Thus, arrays of CSs can easily be formed by cloning a functional CS. Alternatively, the flow of liquids in CSs can also be interactively tuned if desired by (i) forcing the evaporating of liquid out of the capillary pumps and (ii) by contacting a secondary, removable capillary pump to the embedded ones. We illustrate the possibilities of CSs by conducting a surface immunoassay for a cardiac marker, within 25 min, on an area of 100 x 100 microm2, using 16 sequential filling steps.

Journal Article↗
PubMed2002

Positive microcontact printing.

Microcontact printing alkanethiols from an inked, microstructured stamp onto Au or Cu results in the formation of a self-assembled monolayer, which can locally protect the substrate from wet etching. This lithographic technique resembles a negative-type of lithography but can be inverted to the positive process. This is done by printing a type of oligothiol that does not protect the substrate from etching but prevents the adsorption of a protective monolayer from solution.

Journal Article↗