Development and Validation of a Fully Automated Platform for Extended Blood Group Genotyping.

Thirty-five blood group systems, containing >300 antigens, are listed by the International Society of Blood Transfusion. Most of these antigens result from a single nucleotide polymorphism. Blood group typing is conventionally performed by serology. However, this technique has some limitations and cannot respond to the growing demand of blood products typed for a large number of antigens. The knowledge of the molecular basis of these red blood cell systems allowed the implementation of molecular biology methods in immunohematology laboratories. Here, we describe a blood group genotyping assay based on the use of TKL immobilization support and microarray-based HIFI technology that takes approximately 4 hours and 30 minutes from whole-blood samples to results analysis. Targets amplified by multiplex PCR were hybridized on the chip, and a revelation step allowed the simultaneous identification of up to 24 blood group antigens, leading to the determination of extended genotypes. Two panels of multiplex PCR were developed: Panel 1 (KEL1/2, KEL3/4; JK1/2; FY1/2; MNS1/2, MNS3/4, FY*Fy et FY*X) and Panel 2 (YT1/2; CO1/2; DO1/2, HY+, Jo(a+); LU1/2; DI1/2). We present the results of the evaluation of our platform on a panel of 583 and 190 blood donor samples for Panel 1 and 2, respectively. Good correlations (99% to 100%) with reference were obtained.

Hydrogel microparticles for biosensing.

Due to their hydrophilic, biocompatible, and highly tunable nature, hydrogel materials have attracted strong interest in the recent years for numerous biotechnological applications. In particular, their solution-like environment and non-fouling nature in complex biological samples render hydrogels as ideal substrates for biosensing applications. Hydrogel coatings, and later, gel dot surface microarrays, were successfully used in sensitive nucleic acid assays and immunoassays. More recently, new microfabrication techniques for synthesizing encoded particles from hydrogel materials have enabled the development of hydrogel-based suspension arrays. Lithography processes and droplet-based microfluidic techniques enable generation of libraries of particles with unique spectral or graphical codes, for multiplexed sensing in biological samples. In this review, we discuss the key questions arising when designing hydrogel particles dedicated to biosensing. How can the hydrogel material be engineered in order to tune its properties and immobilize bioprobes inside? What are the strategies to fabricate and encode gel particles, and how can particles be processed and decoded after the assay? Finally, we review the bioassays reported so far in the literature that have used hydrogel particle arrays and give an outlook of further developments of the field.

High-Throughput Contact Flow Lithography.

High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

Shrinking hydrogel-DNA spots generates 3D microdots arrays.

This report describes a straightforward approach for the achievement of sub-100 micrometers size hydrogel dots supporting DNA immobilization. Hydrogel-DNA spots are arrayed and UV-crosslinked on PolyShrink, an innovative polymer material having the remarkable property of isotropically shrinking under high temperature. Curing the microarray enables then spot miniaturization, resulting in 6 µm thick and 60 µm wide hydrogel dots in which oligonucleotides are immobilized in a 3D hydrophilic environment. The probe immobilization within the hydrogel network and its capacity to detect targets specifically and quantitatively is demonstrated using chemiluminescent as well as colorimetric detection techniques. The hydrogel material improves probe accessibility within the spot, leading to an enhanced sensitivity.

Polymer adhesive surface as flexible generic platform for multiplexed assays biochip production.

The present report describes the integration and application possibilities of a new microarray concept based on adhesive surface. The method was shown to enable the straightforward production of 384 and 1536-well plates modified with 100 and 25 spots per well, respectively. Such in-well densities were only possible thanks to the fabrication process which implies first the deposition of the microarray on a flat adhesive surface and then its assembly with bottomless 384 or 1536-well plates. The concept was also confronted to various applications such as oligonucleotide detection, localised cell culture onto spotted adhesion proteins and immobilisation of peptide or active antibodies for immunoassays. In the particular case of immunotesting, the study focused on liver diseases diagnosis and more particularly on the detection of either one liver cancer marker, the alpha-fetoprotein, or the detection of Hepatitis C Virus infection. In every cases, interesting performances were obtained directly in crude patient serum, proof of the robust and generic aspect of the platform.

Impact of immobilization support on colorimetric microarrays performances.

We report here a comparison of support materials for colorimetric hybridization assays on microarrays. Four surfaces with various chemistries and architectures (roughness and porosity) were evaluated: (i) bare and (ii) activated polystyrene surfaces classically used for ELISA; (iii) a double-sided adhesive support; and (iv) a porous nitrocellulose/cellulose acetate membrane. Each substrate was functionalized with a microarray of probes and subjected to an enzymatic colorimetric DNA hybridization test. Tests were carried out in a 96-well assembly suitable for automated high-throughput analysis. Colorimetry results, microscopy observations and a chemiluminescence study showed that the test efficiency not only depends on the surface probe density but that the capacity of the material to retain the colored enzymatic product is also a critical parameter. All parameters being considered, the adhesive coated surface proposes the best surface properties for efficient colorimetric microarrays.

Adhesive microarrays for multipurpose diagnostic tools.

We are reporting here a new technology for the straightforward production of integrated microarrays. The approach is based on the use of adhesive supports enabling (i) the immobilization of biomolecules as microarrays (up to 2500 spots per cm(2)) and (ii) the easy assembly of these microarrays with complex 3D structures such as 96-well bottomless microplates or polymer and glass microfluidic networks. The analytical performances of the system were demonstrated for sandwich protein detection (C-reactive protein) and hybridization assays, both in classical 96-well microplate format and microfluidic environment.

Enhanced colorimetric detection on porous microarrays using in situ substrate production.

A new technique is reported for the enhanced colorimetric detection of multiplexed hybridization onto porous membrane-based microarrays. This approach combines the use of horseradish peroxidase (HRP) as a label together with a chromogen substrate and a local production of the hydrogen peroxide required for substrate oxidation. This in situ production of coreagent is obtained using glucose oxidase (GOx) directly immobilized within the microarray porous membrane mesh. The oxidation of glucose by the immobilized GOx produces hydrogen peroxide which itself enables the oxidation of TMB (3,3',5,5'-tetramethylbenzidine) by HRP and yields a blue precipitate on positive spots. Thanks to a coreagent overconcentration within the membrane, this design drastically surpasses the performances of the standard TMB/H(2)O(2) kit used for peroxidase label detection. The obtained target limit of detection is then 50 times lower (20 pM) than the one obtained with the standard kit approach, and the dynamic range expands at least one decade. Furthermore, the developed method was shown to compete well with the widely used alkaline phosphatase-BCIP (5-bromo-4-chloro-3-indolyl phosphate)/NBT (nitro blue tetrazolium chloride) readout while minimizing background signal. The method was finally successfully applied to the multiplex detection of single nucleotide polymorphisms (SNPs) in complex PCR samples. The background lowering was impacted here positively on the SNPs' detection by increasing the complementary/noncomplementary signal ratio.

Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays.

We are reporting here a low cost colorimetric device for high-throughput multiplexed blood group genotyping and allergy diagnosis, displayed as an automated 96-well microtiter plate format. A porous polymeric membrane sealed at the bottom of each well accounts for the sensor support. For each sensing unit, a 6×6 matrix of specific probes is spotted on the external surface of the membrane resulting in 5 mm(2) microarrays. Thanks to the membrane porosity, reagents dispensed into the well can be eliminated through vacuum soaking. This unusual design drastically reduces the assay background signal. The system was first validated on robust models composed of either two complementary oligonucleotide sequences or one allergen/specific rabbit IgG pair. The quality of both oligonucleotide and protein immobilisation on the membrane substrate was then demonstrated together with the capacity to use the arrayed biomolecules as probes for the quantitative detection of specific targets (respectively complementary oligonucleotide and specific antibody). On the basis of these good results, two multiplex assays were developed for crude biological samples testing, focussing on two human in vitro diagnosis applications: a hybridisation assay for multiplex blood group genotyping and a multiparametric immunoassay for allergy diagnosis. In both cases, the transfer to crude biological samples testing was successful i.e. high signal to noise ratio of the stained membranes, reproducibility and good correlation with results obtained using routine testing procedures.

Robust, high-throughput solution for blood group genotyping.

With the concomitant increase of blood transfusions and safety rules, there is a growing need to integrate high-throughput and multiparametric assays within blood qualification centers. Using a robust and automated solution, we describe a new method for extended blood group genotyping (HiFi-Blood 96) bringing together the throughput possibilities of complete automation and the microarray multiplexed analysis potential. Our approach provides a useful resource for upgrading blood qualification center facilities. A set of six single-nucleotide polymorphisms (SNPs) associated with clinically important blood group antigens (Kell, Kidd, Duffy, and MNS systems) were selected and the corresponding genotyping assays developed. A panel of 293 blood samples was used to validate the approach. The resulting genotypes were compared to phenotypes previously determined by standard serologic techniques, and excellent correlations were found for five SNPs out of six. For the Kell, Kidd, Duffy, and MNS3/MNS4 systems, high matching percentages of 100%, 98.9%, 97.7%, and 97.4% were obtained, respectively, whereas a concordance percentage of 83.3% only was attained for the MNS1/MNS2 polymorphism.