Geertruida A. Posthuma-Trumpie

Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey.

Lateral flow (immuno)assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food and environmental settings. We describe principles of current formats, applications, limitations and perspectives for quantitative monitoring. We illustrate the potentials and limitations of analysis with lateral flow (immuno)assays using a literature survey and a SWOT analysis (acronym for “strengths, weaknesses, opportunities, threats”). Articles referred to in this survey were searched for on MEDLINE, Scopus and in references of reviewed papers. Search terms included “immunochromatography”, “sol particle immunoassay”, “lateral flow immunoassay” and “dipstick assay”.

Patterning of peptide nucleic acids using reactive microcontact printing.

PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

Carbon Nanoparticles as Detection Labels in Antibody Microarrays. Detection of Genes Encoding Virulence Factors in Shiga Toxin-Producing Escherichia coli

The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA) and the gene for E. coli 16S (hui). Optimization was performed using a Box-Behnken design, and the limit of detection for each virulence factor was established. Finally, this NAMIA using CNPs was tested with DNA from 48 field strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and specificity values higher than 0.80 and efficiency values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed fit for purpose, i.e., it is valuable for fast screening of amplified genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.

Perspectives for on-site monitoring of progesterone

The steroid hormone progesterone is the primary biomarker of the reproductive status of female mammals. Current techniques of monitoring progesterone are based predominantly on (enzyme) immunoassays, but these are too expensive to be affordable in daily screening programmes because of their associated labour costs and the need for laboratory facilities and/or equipment. Here, we discuss existing methods as well as new perspectives for (automated) application at point of care/need, e.g. the milking parlour. These make it apparent that a low-cost, fully automated progesterone assay system to monitor the reproductive status is far from being realised at present. Timely ovulation prediction techniques for artificial insemination and reproductive cycling are thus urgently needed, and promising perspectives will be highlighted.

Ultraslow microdialysis and microfiltration for in-line, on-line and off-line monitoring

In medicine and biotechnology, close monitoring of molecular processes might assist to optimise therapeutic interventions and production of biochemicals, respectively. Here, we summarize the current status of two automatic and continuous sampling technologies, microdialysis and microfiltration, which facilitate both in vivo and in vitro monitoring of nearly any analyte, because they can be combined easily with many analytical techniques. Conventional microdialysis and microfiltration, which require collecting relatively large samples, are however often impractical and semi-quantitative; hence, we focus on ultraslow sampling to circumvent such limitations. Ultraslow microdialysis and microfiltration already have been used successfully for quantitative pharmacokinetics, glucose metabolism (e.g. of the brain), cytokines and proteomics (e.g. tumour secretomes), both in vivo and in vitro.

Carbon Nanoparticles as Detection Label for Diagnostic Antibody Microarrrays

Aart van Amerongen1,2,*, Geert A.J. Besselink3, Martina Blazkova4, Geertruida A. Posthuma-Trumpie1, Marjo Koets1 and Brigit Beelen-Thomissen1 1Wageningen University and Research Centre, Food and Biobased Research – Biomolecular Sensing and Diagnostics, 2Laboratory of Organic Chemistry, Wageningen University, 3MESA+ Institute for Nanotechnology, University of Twente, 4Institute of Chemical Technology, University of Prague, 1,2,3The Netherlands, 4Czech Republic