A portable smartphone-based imaging surface plasmon resonance biosensor for allergen detection in plant-based milks.

Food allergies are hypersensitivity immune responses triggered by (traces of) allergenic compounds in foods and drinks. The recent trend towards plant-based and lactose-free diets has driven an increased consumption of plant-based milks (PBMs) with the risk of cross-contamination of various allergenic plant-based proteins during the food manufacturing process. Conventional allergen screening is usually performed in the laboratory, but portable biosensors for on-site screening of food allergens at the production site could improve quality control and food safety. Here, we developed a portable smartphone imaging surface plasmon resonance (iSPR) biosensor composed of a 3D-printed microfluidic SPR chip for the detection of total hazelnut protein (THP) in commercial PBMs and compared its instrumentation and analytical performance with a conventional benchtop SPR. The smartphone iSPR shows similar characteristic sensorgrams compared with the benchtop SPR and enables the detection of trace levels of THP in spiked PBMs with the lowest tested concentration of 0.625 µg/mL THP. The smartphone iSPR achieved LoDs of 0.53, 0.16, 0.14, 0.06, and 0.04 µg/mL THP in 10x-diluted soy, oat, rice, coconut, and almond PBMs, respectively, with good correlation with the conventional benchtop SPR system (R2 0.950-0.991). The portability and miniaturized characteristics of the smartphone iSPR biosensor platform make it promising for the future on-site detection of food allergens by food producers.

Rapid Single Particle Atmospheric Solids Analysis Probe-Mass Spectrometry for Multimodal Analysis of Microplastics.

Despite mass spectrometry (MS) being proven powerful for the characterization of synthetic polymers, its potential for the analysis of single particle microplastics (MPs) is yet to be fully disclosed. To date, MPs are regarded as ubiquitous contaminants, but the limited availability of techniques that enable full characterizations of MPs results in a lack of systematic data regarding their occurrence. In this study, an atmospheric solid analysis probe (ASAP) coupled to a compact quadrupole MS is proposed for the chemical analysis of single particle microplastics, while maintaining full compatibility with complementary staining and image analysis approaches. A two-stage ASAP probe temperature program was optimized for the removal of additives and surface contaminants followed by the actual polymer characterization. The method showed specific mass spectra for a wide range of single particle MPs, including polyolefins, polyaromatics, polyacrylates, (bio)polyesters, polyamides, polycarbonates, and polyacrylonitriles. The single particle size detection limits for polystyrene MPs were found to be 30 and 5 µm in full scan and selected ion recording mode, respectively. Moreover, results are presented of a multimodal microplastic analysis approach in which filtered particles are first characterized by staining and fluorescence microscopy, followed by simple probe picking of individual particles for subsequent analysis by ASAP-MS. The method provides a full characterization of MP contamination, including particle number, particle size, particle shape, and chemical identity. The applicability of the developed multimodal method was successfully demonstrated by the analysis of MPs in bioplastic bottled water.

Immunoaffinity Plastic Blade Spray Mass Spectrometry for Rapid Confirmatory Analysis of Food Contaminants.

The lack of chromatographic separation in ambient and direct mass spectrometry (MS) ionization techniques jeopardizes the overall selectivity of the developed methods. Incorporating a biosensing element at the ionization source could compensate for that inherent lack of selectivity. Thus, a simplified immunoaffinity-direct MS technique was developed, immunoaffinity blade spray (iBS), featuring a conductive polystyrene blade material. In iBS, the generic coating used in conventional coated blade spray is replaced with a layer of highly specific monoclonal antibodies (mAbs), while the stainless steel is replaced with conductive polystyrene to allow for simple ELISA platelike hydrophobic immobilization of mAbs. Because of its high relevance for climate change-induced food safety issues, the mycotoxin deoxynivalenol (DON) was chosen as a model substance. Following a rapid extraction from wheat flour, DON is immuno-captured, and the blade is positioned in front of the MS for direct iBS-MS/MS analysis. The method's applicability was demonstrated by analyzing spiked and incurred wheat flour samples, omitting the need for time-consuming chromatographic separation. Apart from DON, cross-reacting DON conjugates could be successfully analyzed as well. The direct iBS-MS/MS method is generic and adaptable to detecting any analyte in sample extracts, provided that specific mAbs are available.

Microsphere Peptide-Based Immunoassay for the Detection of Recombinant Bovine Somatotropin in Injection Preparations.

The use of peptides in immunoassays can be favored over the use of the full protein when more cost effective or less toxic approaches are needed, or when access to the full protein is lacking. Due to restricted access to recombinant bovine somatotropin (rbST), a protein enhancing growth and lactating performances of livestock, which use has been banned in the EU, Canada and Australia (amongst others), we developed a peptide-based biorecognition assay on an imaging planar array analyzer. For this, we identified the rbST epitope that is responsible for binding to the rbST-targeting monoclonal antibody 4H12 (MAb 4H12) to be 115DLEEGILALMR125. This linear peptide was synthesized and coupled to microspheres, after which it was tested in a biorecognition competitive inhibition assay format. We observed IC50 values of approximately 0.11 µg mL-1, which are lower than observed for the full rbST protein (IC50 = 0.20 µg mL-1). Importantly, there was no binding with the scrambled peptide. Preliminary results of directly coupled peptides in a microsphere biorecognition assay for detection of rbST are presented. Real-life applicability for detection of somatotropins (STs) in injection preparations of bovine-, porcine- and equine ST are shown. This newly developed immunoassay strongly supports future developments of peptide-based immunoassays to circumvent the limited access to the full protein.

Simplified screening approach of anabolic steroid esters using a compact atmospheric solid analysis probe mass spectrometric system.

Due to the absence of chromatographic separation, ambient ionization mass spectrometry had the potential to improve the throughput of control laboratories in the last decades and will soon be an excellent approach for on-site use as well. In this study, an atmospheric solids analysis probe (ASAP) with a single quadrupole mass analyzer has been evaluated to identify anabolic steroid esters rapidly. Sample introduction, applied scan time, and probe temperature were optimized for sensitivity. The in-source fragmentations of seventeen selected steroid esters, commonly found in illicit samples, were determined by applying different cone voltages (12, 20, 30, and 40 V). A spectral library was created for these steroid esters based on the four stages of in-source fragmentation spectra. The applicability of this method was demonstrated for the rapid identification of steroid esters in oily injection solutions, providing test results in less than 2 min.

Immuno-Enriched Microspheres - Magnetic Blade Spray-Tandem Mass Spectrometry for Domoic Acid in Mussels.

Paramagnetic microspheres can be used in planar array fluorescence immunoassays for single or multiplex screening of food contaminants. However, no confirmation of the molecular identity is obtained. Coated blade spray (CBS) is a direct ionization mass spectrometry (MS) technique, and when combined with triple quadrupole MS/MS, it allows for rapid confirmation of food contaminants. The lack of chromatography in CBS, though, compromises the specificity of the measurement for unequivocal identification of contaminants, based on the European Union (EU) regulation. Therefore, a rapid and easy-to-use immuno-magnetic blade spray (iMBS) method was developed in which immuno-enriched paramagnetic microspheres replace the coating of CBS. The iMBS-MS/MS method was fully optimized, validated in-house following the EU 2021/808 regulation, and benchmarked against a commercial lateral flow immunoassay (LFIA) for on-site screening of DA. The applicability of iMBS-MS/MS was further demonstrated by analyzing incurred mussel samples. The combination of immunorecognition and MS/MS detection in iMBS-MS/MS enhances the measurement's selectivity, which is demonstrated by the rapid differentiation between the marine toxin domoic acid (DA) and its structural analog kainic acid (KA), which cannot be achieved with the LFIA alone. Interestingly, this first-ever reported iMBS-MS/MS method is generic and can be adapted to include any other immuno-captured food contaminant, provided that monoclonal antibodies are available, thus offering a complementary confirmatory analysis approach to multiplex immunoassay screening methods. Moreover, thanks to its speed of analysis, iMBS-MS/MS can bridge the logistics gap between future large-scale on-site testings using LFIAs and classical time-consuming confirmatory MS analysis performed in official control laboratories.

ASSURED Point-of-Need Food Safety Screening: A Critical Assessment of Portable Food Analyzers.

Standard methods for chemical food safety testing in official laboratories rely largely on liquid or gas chromatography coupled with mass spectrometry. Although these methods are considered the gold standard for quantitative confirmatory analysis, they require sampling, transferring the samples to a central laboratory to be tested by highly trained personnel, and the use of expensive equipment. Therefore, there is an increasing demand for portable and handheld devices to provide rapid, efficient, and on-site screening of food contaminants. Recent technological advancements in the field include smartphone-based, microfluidic chip-based, and paper-based devices integrated with electrochemical and optical biosensing platforms. Furthermore, the potential application of portable mass spectrometers in food testing might bring the confirmatory analysis from the laboratory to the field in the future. Although such systems open new promising possibilities for portable food testing, few of these devices are commercially available. To understand why barriers remain, portable food analyzers reported in the literature over the last ten years were reviewed. To this end, the analytical performance of these devices and the extent they match the World Health Organization benchmark for diagnostic tests, i.e., the Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end-users (ASSURED) criteria, was evaluated critically. A five-star scoring system was used to assess their potential to be implemented as food safety testing systems. The main findings highlight the need for concentrated efforts towards combining the best features of different technologies, to bridge technological gaps and meet commercialization requirements.

Controlled Production of Zearalenone-Glucopyranoside Standards with Cunninghamella Strains Using Sulphate-Depleted Media.

In recent years, conjugated mycotoxins have gained increasing interest in food safety, as their hydrolysis in human and animal intestines leads to an increase in toxicity. For the production of zearalenone (ZEN) glycosides reference standards, we applied Cunninghamellaelegans and Cunninghamella echinulata fungal strains. A sulphate-depleted medium was designed for the preferred production of ZEN glycosides. Both Cunninghamella strains were able to produce zearalenone-14-ß-D-glucopyranoside (Z14G), zearalenone-16-ß-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S). In a rich medium, Cunninghamellaelegans preferably produced Z14S, while Cunninghamellaechinulata preferably produced Z14G. In the sulphate-depleted medium a dramatic change was observed for Cunninghamellaelegans, showing preferred production of Z14G and Z16G. From 2 mg of ZEN in sulphate-depleted medium, 1.94 mg of Z14G and 0.45 mg of Z16G were produced. Following preparative Liquid Chromatography-Mass Spectrometry (LC-MS) purification, both fractions were submitted to 1H and 13C NMR and High-Resolution Mass Spectrometry (HRMS). These analyses confirmed that the purified fractions were indeed Z14G and Z16G. In conclusion, the presented research shows that a single Cunninghamella strain can be an effective and efficient tool for the controlled biotransformation of ZEN glycosides and other ZEN metabolites. Additionally, the biotransformation method was extended to zearalanone, ß-zearalenol and other mycotoxins.

Hand-Held Diode Laser for On-Site Analysis Using Transportable Mass Spectrometry.

A hand-held laser diode thermal desorption electrospray ionization (LDTD-ESI) mass spectrometry (MS) method was developed for rapid screening of illegal substances in solid samples. To achieve that, a simple, inexpensive, battery-powered surgical laser diode at 940 nm was employed to ablate the solid samples. The potential of using a black polytetrafluoroethylene substrate to enhance the analytes' desorption to the gas phase was investigated and demonstrated. Among the optimized ESI parameters, the solvent (methanol/water, 50:50, v/v) and the flow rate (50 µL h-1) were critical to obtain the best sensitivity. The applicability was demonstrated for the rapid identification of selective androgen receptor modulators (SARMs) in pills and powders based on accurate mass measurements by time-of-flight MS. Also, the hand-held LDTD-ESI was combined with a transportable single quadrupole MS. The same SARMs samples were analyzed, and identifications were based on in-source cone voltage fragmentation patterns observed. These initial results demonstrate the applicability of the developed simplified LDTD-ESI MS method for future on-site testing of organic compounds in solid samples.

From Smartphone Lateral Flow Immunoassay Screening to Direct MS Analysis: Development and Validation of a Semi-Quantitative Direct Analysis in Real-Time Mass Spectrometric (DART-MS) Approach to the Analysis of Deoxynivalenol.

In current food safety monitoring, lateral flow immunoassays (LFIAs) are widely used for rapid food contaminant screening. Recent advances include smartphone readouts, offering semi-quantitative analysis of LFIAs with time, location, and data transfer in case of on-site testing. Following the screening, the next step in the EU regulations is confirmation by, e.g., liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this work, using direct analysis in real time ambient ionization and triple quadrupole MS/MS (DART-QqQ-MS/MS), we achieved rapid confirmation of the identity of the substance(s) causing the LFIA result. In the workflow proposed, an individual performs the (on-site) smartphone LFIA screening, and when the result is suspect, an identification LFIA (ID-LFIA) strip is developed with the same sample extract. The ID-LFIA can be dissociated and rapidly analyzed in a control laboratory with DART-QqQ-MS/MS. The ID-LFIA consists of multiple lines of monoclonal antibodies against the mycotoxin deoxynivalenol, acting as a bioaffinity trap. The ID-LFIA/DART-QqQ-MS/MS approach has been developed and validated, along with the screening smartphone LFIA, and has demonstrated its applicability by analyzing incurred and spiked samples. The developed approach has been critically compared with our previous direct electrospray ionization MS method and was found to provide highly complementary information on the total deoxynivalenol contamination in the sample.