Procalcitonin detection in human plasma specimens using a fast version of proximity extension assay.

An exciting trend in clinical diagnostics is the development of easy-to-use, minimally invasive assays for screening and prevention of disease at the point of care. Proximity Extension Assay (PEA), an homogeneous, dual-recognition immunoassay, has proven to be sensitive, specific and convenient for detection or quantitation of one or multiple analytes in human plasma. In this paper, the PEA principle was applied to the detection of procalcitonin (PCT), a widely used biomarker for the identification of bacterial infection. A simple, short PEA protocol, with an assay time suitable for point-of-care diagnostics, is presented here as a proof of concept. Pairs of oligonucleotides and monoclonal antibodies were selected to generate tools specifically adapted to the development of an efficient PEA for PCT detection. The assay time was reduced by more than 13-fold compared to published versions of PEA, without significantly affecting assay performance. It was also demonstrated that T4 DNA polymerase could advantageously be replaced by other polymerases having strong 3'>5' exonuclease activity. The sensitivity of this improved assay was determined to be about 0.1 ng/mL of PCT in plasma specimen. The potential use of such an assay in an integrated system for the low-plex detection of biomarkers in human specimen at the point of care was discussed.

Paper-based point-of-care testing for cost-effective diagnosis of acute flavivirus infections.

Flavivirus infections are a serious healthcare concern in tropical and subtropical countries. Although well-established laboratory tests can provide early diagnosis of acute dengue or Zika infections, access to these tests is limited in developing countries, presenting an urgent need to develop simple, rapid, and robust diagnostic tools. Microfluidic Paper-based Analytical Devices (µPAD), are typically rapid, cost-effective, user-friendly, and they can be used as diagnostic tools for the diagnosis of these infections at Point of Care settings. Early and prompt diagnosis is crucial to improve patient management and reduce the risk of complications. In the present study, we developed and evaluated a wax-printed paper-based device for the detection of the dengue and Zika non-structural NS1 viral protein in blood and plasma. Experiments have been carried out to increase specificity, while maintaining the required sensitivity. As a consequence, the quality of the raw materials and the washing steps were proved to be crucial. The µPAD was able to detect specifically in 6-8 min 10 ng/mL of protein in various sample types. A prototype for the differential detection of dengue and/or Zika NS1 protein was developed. The reading of the results was simplified by using a dedicated application on a smartphone.

Block copolymer-oligonucleotide conjugates for genotyping on microarrays.

Polymer-oligonucleotide conjugates were synthesized from the amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-co-N-acryloxysuccinimide)) using an original solid-phase DNA synthesis strategy. This method provided conjugates highly functionalized with oligonucleotides throughout the polymer chain. After purification, block copolymer-oligonucleotide conjugates were spotted on a multidetection microarray system developed by Apibio using a standard nanodroplet piezo inkjet spotting technique to develop the oligosorbent assay (OLISA). Two genotyping models (HLA-DQB1 and platelet glycoproteins [GPs]), which are particularly difficult to study with standard systems, were evaluated. For both models, block copolymer-oligonucleotide conjugates used as capture probes amplified the responses of in vitro diagnostic assays. The detection limit reached by using conjugates was estimated at 15 pM for a 219-bp DNA target (HLA-DQB1 model). Moreover, single nucleotide polymorphism was detected in the platelet GPs genotyping model. The use of polymer conjugates led to a significant improvement in both sensitivity and specificity of standard hybridization assays even when applied to complex biological models.

Polymer-oligonucleotide conjugate synthesis from an amphiphilic block copolymer. Applications to DNA detection on microarray.

An amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-N-acryloxysuccinimide)) (poly(TBAm-b-(NAM/NAS)) and a random copolymer poly(NAM/NAS), synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization process, have been used as support for oligonucleotide (ODN) synthesis, to elaborate polymer-oligonucleotide conjugates. In a first step, starters of ODN solid-phase synthesis were coupled to activated ester functions of polymers, and second, resulting functionalized polymers were covalently grafted onto hydroxylated controlled pore glass (CPG) support to further accomplish ODN synthesis. An efficient capping of residual hydroxyl functions of CPG was performed before synthesis, with both acetic anhydride and diethoxy-N,N-diisopropyl-phosphoramidite reagents, to suppress parasite-free ODN population present in conjugate crude material and resulting from syntheses directly initiated on silica beads. After purification, conjugates were evaluated in a DNA hybridization assay on a microarray, as macromolecules being able to favor capture of the target. Conjugate coating conditions were studied on the dT25/dA25 model. The role of the hydrophobic part (poly(TBAm)) of the conjugate synthesized with the block copolymer in the orientation of the conjugate after coating was revealed by spotting experiments achieved in a mixed solvent (DMF/H(2)O). The use of block copolymer-dT25 conjugate afforded a significant sensitivity improvement of the hybridization assay.