M. Koets

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

AbstractCarbon 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. FigureScheme of a NALFIA-, NAMIA, or NALMIA. Neutravidin adsorbed onto CNPs detects biotin-labelled amplicons; the discriminating tag is recognized by its respective antibody, which is immobilized onto nitrocellulose membranes or pads

Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted skimmed milk. Four aminoglycosides (gentamicin, neomycine, kanamycin and a streptomycin derivative) were immobilised onto the sensor surface of a biosensor chip (CM5) in the four Fcs of the biosensor system by amine coupling. In the Biacore, milk (reconstituted from skimmed milk powder) was 10 times diluted with a mixture of the four specific antibodies and injected through the four serially connected Fcs (1 min at a flow rate of 20 μl min -1 ). The responses measured just prior to the injection (20 μl at a flow rate of 20 μl min -1 ) of the regeneration solution (0.2 M NaOH + 20% acetonitril) were indicative for the presence or absence of the aminoglycosides in reconstituted milk. The limits of detection were between 15 and 60 ng ml -1 , which was far below the maximum residue limits (MRLs) (varying from 100 to 500 ng ml -1 ) and the total run time between samples was 7 min.

Rapid one-step assays for on-site monitoring of mouse and rat urinary allergens

Allergy to rodent proteins is common among laboratory animal workers. Sensitive methods to measure exposure to these allergens have been developed. These assays are, however, expensive, time-consuming, and require a laboratory facility and methodological expertise. A simple method to screen for allergen spread, or to test whether hygiene standards are maintained, would be useful. Lateral flow immunoassays (LFIAs) are especially suited for field settings; the tests are simple and results are visible within minutes. LFIAs were developed for detection of the rodent urinary allergens Mus m 1 and Rat n 1. Pilot studies were performed in animal facilities in three countries using both extracts from airborne dust samples and samples collected by wiping surfaces. For comparison and determination of sensitivity, the concentrations of rodent urinary allergens in the samples were also measured using enzyme immunoassays (EIAs). The LFIAs for rat and mouse urinary allergens had a detection limit of 31 pg allergen per mL in a buffer system with purified allergen standards. Results of environmental dust extracts tested in LFIAs correlated well with levels obtained using EIAs. Spread of rodent allergens, or non-adherence to hygiene around laboratory animal facilities, may aggravate rodent allergy. Using a simple, sensitive one-step assay, allergens can be detected to prevent allergen exposure. The results reveal that the rapid assays are suited for on-site demonstration of exposure to rodent allergens, and thus, useful in occupational hygiene practice.

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