Biomedical subjects

Marc Wolf

Publications and source records attributed to Marc Wolf.

7 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↗

PubMed2005

Coordinates, DNA content and heterogeneity of cell nuclei and segments of the Caenorhabditis elegans intestine.

A specific protocol allowed to selectively stain and image the cell nuclei of the intestine of the nematode Caenorhabditis elegans. Digital processing of these images yielded quantitative information on the coordinates and the relative DNA content of these cell nuclei. With this technique, the exact locations (in relation to the length of worm or intestine) of the 20 intestinal cell nuclei of larval stage L1 and of the 34 intestinal cell nuclei of larval stages L2-4 and adults were determined. Tracking the DNA content of an individual intestinal cell nuclei allowed to study nuclear DNA endoreduplications during larval growth. The evaluation of the DNA content of all intestinal cell nuclei revealed a strong heterogeneity between the cell nuclei and intestinal segments with a maximum in segments int4-int5 in the L1 stage and int5-int6 in the L2-L4 stages. Minimum values were detected in the segments int2 and int8-int9 of all larval stages. Heterogeneity between the intestinal segments of C. elegans does not only concern the DNA content of cell nuclei, but also other cellular features like the quantity of intracellular vesicles or the absorption of particles out of the intestinal lumen.

Animals↗

PubMed2005

The ABC transporter PGP-2 from Caenorhabditis elegans is expressed in the sensory neuron pair AWA and contributes to lysosome formation and lipid storage within the intestine.

The functional role of the ABC transporter PGP-2 from the nematode Caenorhabditis elegans has been studied by combining phenotype analyses of pgp-2 deletion mutants or pgp-2 RNAi treated worms with reporter gene studies using a pgp-2::GFP construct. pgp-2 mutants showed a strong reduction of lipid stores. In addition, we found that in the case of the pgp-2 mutant or after pgp-2 RNAi the worms were unable to perform pinocytosis and to acidify intestinal lysosomes. Especially under cholesterol-restricted conditions, the viability of the mutant was reduced. Surprisingly, the chemosensory AWA neurons in the head region were identified as expression sites by reporter gene studies. These neurons are known to be involved in attraction behaviour towards odorants associated with potential food bacteria. Our results imply that PGP-2 is involved in a signalling process that connects sensory inputs to intestinal functions, possibly by influencing acidification of intestinal lysosomes, which in turn may affect pinocytosis and lipid storage.

ATP Binding Cassette Transporter, Subfamily B↗

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↗

PubMed2005

Sequencing of hemoglobin gene 4 (dmhb4) and Southern blot analysis provide evidence of more than four members of the Daphnia magna globin family.

A genomic fragment containing the hemoglobin gene dmhb4 of Daphnia magna was cloned and its nucleotide sequence determined. Concerning induction under hypoxic conditions, dmhb4 was found to be expressed constitutively with similar mRNA quantities in D. magna bred in either normoxic or hypoxic medium. Southern blot analysis revealed at least six hemoglobin-like sequences in the genome of Daphnia magna.

Amino Acid Sequence↗

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↗