Amorphous carbon nanoparticles: a versatile label for rapid diagnostic (immuno)assays

Carbon nanoparticles (CNPs) labeled with reporter molecules can serve as signaling labels in rapid diagnostic assays as an alternative to gold, colored latex, silica, quantum dots, or up-converting phosphor nanoparticles. Detailed here is the preparation of biomolecule-labeled CNPs and examples of their use as a versatile label. CNPs can be loaded with a range of biomolecules, such as DNA, antibodies, and proteins (e.g., neutravidin or a fusion protein of neutravidin with an enzyme), and the resulting conjugates can be used to detect analytes of high or low molecular mass

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"

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 di¿erent Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes speci¿c for STEC (vt1,vt2, eae, andehxA) and the gene forE. coli16S (hui). Optimization was performed using a Box Behnken design, andthe limit of detection for each virulence factor was established. Finally,this NAMIA using CNPs was tested with DNA from 48 ¿eld 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 speci¿city values higher than 0.80 and e¿ciency values higher than 0.92. Kappa coe¿cients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used forvt1, eae, and ehxA, and a perfect agreement (k = 1) forvt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed ¿t for purpose, i.e., it is valuable for fast screening of ampli¿ed genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.This work was partially supported by the Generalitat Valenciana (BEST/2009/026), the Universidad Politecnica de Valencia (PAID-00-09-2837), and the Dutch Ministry of Agriculture, Nature and Food Quality (Strategic research program Food Safety, Monitoring and Detection KB-06-005). The authors thank Dr. Eva Moller Nielsen at the Danish Veterinary Institute (Copenhagen, Denmark) for providing E. coli control strains and Dr. Lutz Geue (Friedrich-Loeffler-Institut, Wusterhausen, Germany) and Dr. Dorte Dopfer (School of Veterinary Medicine, University of Wisconsin, Madison, WI) for field isolates.Noguera Murray, PS.; Posthuma-Trumpie, GA.; Van Tuil, M.; Van Der Wal, FJ.; De Boer, A.; Moers, APHA.; Van Amerongen, A. (2011). Carbon nanoparticles as detection labels in antibody microarrays.Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli. Analytical Chemistry. 83:8531-8536. https://doi.org/10.1021/ac201823v853185368

Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone

The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50 10%), reconstitutable dimers (20 10%) and irreversibly aggregated oligomers (30 20%). Incubation of monomeric apoGOx with FAD or N-6-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N-6-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25-50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD. (c) 2007 Elsevier B.V. All rights reserved

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