Biochip Spray: Simplified Coupling of Surface Plasmon Resonance Biosensing and Mass Spectrometry.

A simplified coupling of surface plasmon resonance (SPR) immuno-biosensing with ambient ionization mass spectrometry (MS) was developed. It combines two orthogonal analysis techniques: the biosensing capability of SPR and the chemical identification power of high resolution MS. As a proof-of-principle, deoxynivalenol (DON), an important mycotoxin, was captured using an SPR gold chip containing an antifouling layer and monoclonal antibodies against the toxin and, after washing, the chip could be taken out and analyzed by direct spray MS of the biosensor chip to confirm the identity of DON. Furthermore, cross-reacting conjugates of DON present in a naturally contaminated beer could be successfully identified, thus showing the potential of rapid identification of (un)expected cross-reacting molecules.

Analysis of Mycotoxins in Beer Using a Portable Nanostructured Imaging Surface Plasmon Resonance Biosensor.

A competitive inhibition immunoassay is described for the mycotoxins deoxynivalenol (DON) and ochratoxin A (OTA) in beer using a portable nanostructured imaging surface plasmon resonance (iSPR) biosensor, also referred to as imaging nanoplasmonics. The toxins were directly and covalently immobilized on a 3-dimensional carboxymethylated dextran (CMD) layer on a nanostructured iSPR chip. The assay is based on competition between the immobilized mycotoxins and free mycotoxins in the solution for binding to specific antibodies. The chip surface was regenerated after each cycle, and the combination of CMD and direct immobilization of toxins allowed the chips to be used for more than 450 cycles. The limits of detection (LODs) in beer were 17 ng/mL for DON and 7 ng/mL for OTA (or 0.09 ng/mL after 75 times enrichment). These LODs allowed detection of even less than 10% depletion of the tolerable daily intake of DON and OTA by beer. Significant cross-reactivity of anti-DON was observed toward DON-3-glucoside and 3-acetyl-DON, while no cross-reactivity was seen for 15-acetyl-DON. A preliminary in-house validation with 20 different batches of beer showed that both toxins can be detected at the considered theoretical safe level for beer. The assay can be used for in-field or at-line detection of DON in beer and also in barley without preconcentration, while OTA in beer requires an additional enrichment step, thus making the latter in its present form less suitable for field applications.

Multiplex surface plasmon resonance biosensing and its transferability towards imaging nanoplasmonics for detection of mycotoxins in barley.

A 6-plex competitive inhibition immunoassay for mycotoxins in barley was developed on a prototype portable nanostructured imaging surface plasmon resonance (iSPR) instrument, also referred to as imaging nanoplasmonics. As a benchmark for the prototype nanoplasmonics instrument, first a double 3-plex assay was developed for the detection of deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin (T-2), ochratoxin A (OTA), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) using a well-established benchtop SPR instrument and two biosensor chips. To this end, ovalbumin (OVA) conjugates of mycotoxins were immobilized on the chip via amine coupling. The SPR response was then recorded upon injection of a mixture of antibodies at a fixed concentration and the sample (or matrix-matched standard) over a chip with immobilized mycotoxin-OVA conjugates. The chips were regenerated after each sample using 10 mM HCl and 20 mM NaOH and could be used for at least 60 cycles. The limits of detection in barley (in µg kg(-1)) were determined to be 26 for DON, 6 for ZEA, 0.6 for T-2, 3 for OTA, 2 for FB1 and 0.6 for AFB1. Preliminary in-house validation showed that DON, T-2, ZEA and FB1 can be detected at the European Union regulatory limits, while for OTA and AFB1 sensitivities should be improved. Furthermore, measurement of naturally contaminated barley showed that the assay can be used as a semi-quantitative screening method for mycotoxins prior to liquid chromatography tandem mass spectrometry (LC-MS/MS). Finally, using the same bio-reagents the assay was transferred to a 6-plex format in the nanoplasmonics instrument and subsequently the two assays were compared. Although less sensitive, the 6-plex portable iSPR assay still allowed detection of DON, T-2, ZEA and FB1 at relevant levels. Therefore, the prototype iSPR shows potential for future applications in rapid in-field and at-line screening of multiple mycotoxins.

Ambient surface analysis of organic monolayers using direct analysis in real time Orbitrap mass spectrometry.

A better characterization of nanometer-thick organic layers (monolayers) as used for engineering surface properties, biosensing, nanomedicine, and smart materials will widen their application. The aim of this study was to develop direct analysis in real time high-resolution mass spectrometry (DART-HRMS) into a new and complementary analytical tool for characterizing organic monolayers. To assess the scope and formulate general interpretation rules, DART-HRMS was used to analyze a diverse set of monolayers having different chemistries (amides, esters, amines, acids, alcohols, alkanes, ethers, thioethers, polymers, sugars) on five different substrates (Si, Si3N4, glass, Al2O3, Au). The substrate did not play a major role except in the case of gold, for which breaking of the weak Au-S bond that tethers the monolayer to the surface, was observed. For monolayers with stronger covalent interfacial bonds, fragmentation around terminal groups was found. For ester and amide-terminated monolayers, in situ hydrolysis during DART resulted in the detection of ions characteristic of the terminal groups (alcohol, amine, carboxylic acid). For ether and thioether-terminated layers, scission of C-O or C-S bonds also led to the release of the terminal part of the monolayer in a predictable manner. Only the spectra of alkane monolayers could not be interpreted. DART-HRMS allowed for the analysis of and distinction between monolayers containing biologically relevant mono or disaccharides. Overall, DART-HRMS is a promising surface analysis technique that combines detailed structural information on nanomaterials and ultrathin films with fast analyses under ambient conditions.

Interactions of amino acids and polypeptides with metal oxide nanoparticles probed by fluorescent indicator adsorption and displacement.

The adsorption of polypeptides containing an N-terminal tryptophan (Trp) residue attached to a hexa-backbone of alanine, serine, lysine, histidine, and aspartate was investigated by monitoring the fluorescence response of the Trp chromophore upon titration with metal oxide nanoparticles (MOx-NPs: CuO, Co(3)O(4), TiO(2), MgO, and CeO(2)). After correction for light-scattering effects, a strong static fluorescence quenching was observed upon addition of CuO and Co(3)O(4) to the peptides. The interaction of MOx-NPs with the peptides was assigned to an adsorption of the peptide backbone on the nanoparticle surface. The method was refined using a derivatized amino acid, 5-fluoro-Trp (5F-Trp), which resulted in a stronger fluorescence response. The use of the fluorescent amino acid labels allowed the direct assessment of the adsorption propensities of Trp-containing peptides in dependence on the backbone, which was verified by zeta-potential measurements. Moreover, upon addition of different analytes to nanoparticles with preadsorbed Trp-containing polypeptides, adsorption propensities of the analytes were assessed by an indicator displacement strategy; that is, addition of increasing amounts of analyte resulted in a continuous fluorescence enhancement/recovery. This method afforded adsorption propensities for several analytes. The relative binding constants for the MOx-NPs, obtained from the competitive titrations, varied by more than 6 orders of magnitude for CuO (5F-TrpHis(6)-NH(2) > TrpAsp(6)-NH(2), TrpSer(6)-NH(2) > TrpLys(6)-NH(2), Trp, 5F-Trp > TrpAla(6)-NH(2)) but only 4 for Co(3)O(4) (TrpHis(6)-NH(2), TrpAsp(6)-NH(2) ≫ TrpLys(6)-NH(2), TrpAla(6)-NH(2), TrpSer(6)-NH(2), Trp, 5F-Trp). The study reveals that MOx-NPs adsorb biomolecular analytes with high selectivity, which has immediate implications for their applications in protein purification, drug delivery, and, potentially, for the assessment of their toxicology.