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-μm-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°C, and flow velocities of the cells over the capture areas were 100-280 μm~s−1, resulting in a ∼0.1-0.3 s residency time of the cells on each of the eleven 30 × 30 μm 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°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 surface

Suivi rhéologique du processus d'agrégation de la protéine tau

International audienceAlzheimer's disease is one of thirty pathology called conformational disease that are characterized by errors folding and assembly of protein. In Alzheimer's disease, tau protein and amyloid beta peptide are responsible of the neuronal degeneration. Understanding these mechanisms and diagnosis are closely linked to the availability of an efficient analytical concept for monitoring ex vivo self-assembly of proteins. To understand the aggregation's mechanism, a microsystem is developed for the detection of protein tau based on the micro-rheological followed by high frequency ultrasonic waves. The sensor used is a TSM (Thickness Shear Mode) resonator operating in shear mode at a fundamental frequency of 5MHz. In contact with the fluid to be characterized, the sensor generate shear waves, which are measured in reflection through instrumentation developed in the laboratory. From the measurement of the complex impedance of quartz with a network analyzer, the extraction of G' (elastic modulus) and G'' (viscous modulus) is possible. Initial results indicate that our sensor can quantify, by viscosity, concentration variations of a peptide sequence of tau protein, and can differentiate, by plasticity, the conformational state.  </p

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 application

Methods for immobilizing receptors in microfluidic devices: A review

In this review article, we discuss state-of-the-art methods for immobilizing functional receptors in microfluidic devices. Strategies used to immobilize receptors in such devices are essential for the development of specific, sensitive (bio)chemical assays that can be used for a wide range of applications. In the first section, we review the principles and the chemistry of immobilization techniques that are the most commonly used in microfluidics. We afterward describe immobilization methods on static surfaces from microchannel surfaces to electrode surfaces with a particular attention to opportunities offered by hydrogel surfaces. Finally, we discuss immobilization methods on mobile surfaces with an emphasis on both magnetic and non-magnetic microbeads, and finally, we highlight recent developments of new types of mobile supports

Microscale Interfacial Polymerization on a Chip

Forming hydrogels with precise geometries is challenging and mostly done using photopolymerization, which involves toxic chemicals, rinsing steps, solvents, and bulky optical equipment. Here, we introduce a new method for in situ formation of hydrogels with a well-defined geometry in a sealed microfluidic chip by interfacial polymerization. The geometry of the hydrogel is programmed by microfluidic design using capillary pinning structures and bringing into contact solutions containing hydrogel precursors from vicinal channels. The characteristics of the hydrogel (mesh size, molecular weight cut-off) can be readily adjusted. This method is compatible with capillary-driven microfluidics, fast, uses small volumes of reagents and samples, and does not require specific laboratory equipment. Our approach creates opportunities for filtration, hydrogel functionalization, and hydrogel-based assays, as exemplified by a rapid, compact competitive immunoassay that does not require a rinsing step

Complex Nucleic Acid Hybridization Reactions inside Capillary-Driven Microfluidic Chips

Nucleic acid hybridization reactions play an important role in many (bio)chemical fields, for example, for the development of portable point‐of‐care diagnostics, and often such applications require nucleic acid‐based reaction systems that ideally run without enzymes under isothermal conditions. The use of novel capillary‐driven microfluidic chips to perform two isothermal nucleic acid hybridization reactions, the simple opening of molecular beacon structures and the complex reaction cascade of a clamped‐hybridization chain reaction (C‐HCR), is reported here. For this purpose, reagents are arranged in a self‐coalescence module (SCM) of a passive silicon microfluidic chip using inkjet spotting. The SCM occupies a footprint of ≈7 mm$^{2}$ of a ≈0.4 × 2 cm$^{2}$ microfluidic chip. By means of fluorophore‐labeled DNA probes, the hybridization reactions can be analyzed in just ≈2 min and using only ≈3 µL of the sample. Furthermore, the SCM chip offers a variety of reagent delivery options, allowing, for example, the influence of the initiator concentration on the kinetics of C‐HCR to be investigated systematically with minimal sample and time requirements. These results suggest that self‐powered microfluidic chips equipped with a SCM provide a powerful platform for performing and investigating complex reaction systems

Rapid quantitative assays for glucose-6-phosphate dehydrogenase (G6PD) and hemoglobin combined on a capillary-driven microfluidic chip

Rapid tests for glucose-6-phosphate dehydrogenase (G6PD) are extremely important for determining G6PD deficiency, a widespread metabolic disorder which triggers hemolytic anemia in response to primaquine and tafenoquine medication, the most effective drugs for the radical cure of malaria caused by Plasmodium parasites. Current point-of-care diagnostic devices for G6PD are either qualitative, do not normalize G6PD activity to the hemoglobin concentration, or are very expensive. In this work we developed a capillary-driven microfluidic chip to perform a quantitative G6PD test and a hemoglobin measurement within 2 minutes and using less than 2 μL of sample. We used a powerful microfluidic module to integrate and resuspend locally the reagents needed for the G6PD assay and controls. We also developed a theoretical model that successfully predicts the enzymatic reactions on-chip, guides on-chip reagent spotting and allows efficient integration of multiple assays in miniaturized formats with only a few nanograms of reagents

Malaria and the 'last' parasite : how can technology help?

Malaria, together with HIV/AIDS, tuberculosis and hepatitis are the four most deadly infectious diseases globally. Progress in eliminating malaria has saved millions of lives, but also creates new challenges in detecting the 'last parasite'. Effective and accurate detection of malaria infections, both in symptomatic and asymptomatic individuals are needed. In this review, the current progress in developing new diagnostic tools to fight malaria is presented. An ideal rapid test for malaria elimination is envisioned with examples to demonstrate how innovative technologies can assist the global defeat against this disease. Diagnostic gaps where technology can bring an impact to the elimination campaign for malaria are identified. Finally, how a combination of microfluidic-based technologies and smartphone-based read-outs could potentially represent the next generation of rapid diagnostic tests is discussed