Real-time PCR melting analysis with fiber optic SPR enables multiplex DNA identification of bacteria.

A fiber optic surface plasmon resonance (FO-SPR) technology was developed that enables simultaneous quantification and identification of multiple DNA targets on the same platform. The bioassay was based on the hybridization/melting of DNA-coated Au nanoparticles on the FO-SPR sensor when targets are present. The multiplex concept was successfully demonstrated on two related bacteria and for detection of multiple mutations in sequences. In conclusion, FO-SPR technology shows a great potential as a next generation in vitro diagnostics tool.

Visible and near-infrared bulk optical properties of raw milk.

The implementation of optical sensor technology to monitor the milk quality on dairy farms and milk processing plants would support the early detection of altering production processes. Basic visible and near-infrared spectroscopy is already widely used to measure the composition of agricultural and food products. However, to obtain maximal performance, the design of such optical sensors should be optimized with regard to the optical properties of the samples to be measured. Therefore, the aim of this study was to determine the visible and near-infrared bulk absorption coefficient, bulk scattering coefficient, and scattering anisotropy spectra for a diverse set of raw milk samples originating from individual cow milkings, representing the milk variability present on dairy farms. Accordingly, this database of bulk optical properties can be used in future simulation studies to efficiently optimize and validate the design of an optical milk quality sensor. In a next step of the current study, the relation between the obtained bulk optical properties and milk quality properties was analyzed in detail. The bulk absorption coefficient spectra were found to mainly contain information on the water, fat, and casein content, whereas the bulk scattering coefficient spectra were found to be primarily influenced by the quantity and the size of the fat globules. Moreover, a strong positive correlation (r ≥ 0.975) was found between the fat content in raw milk and the measured bulk scattering coefficients in the 1,300 to 1,400 nm wavelength range. Relative to the bulk scattering coefficient, the variability on the scattering anisotropy factor was found to be limited. This is because the milk scattering anisotropy is nearly independent of the fat globule and casein micelle quantity, while it is mainly determined by the size of the fat globules. As this study shows high correlations between the sample's bulk optical properties and the milk composition and fat globule size, a sensor that allows for robust separation between the absorption and scattering properties would enable accurate prediction of the raw milk quality parameters.

Enabling fiber optic serotyping of pathogenic bacteria through improved anti-fouling functional surfaces.

Significant research efforts are continually being directed towards the development of sensitive and accurate surface plasmon resonance biosensors for sequence specific DNA detection. These sensors hold great potential for applications in healthcare and diagnostics. However, the performance of these sensors in practical usage scenarios is often limited due to interference from the sample matrix. This work shows how the co-immobilization of glycol(PEG) diluents or 'back filling' of the DNA sensing layer can successfully address these problems. A novel SPR based melting assay is used for the analysis of a synthetic oligomer target as well as PCR amplified genomic DNA extracted from Legionella pneumophila. The benefits of sensing layer back filling on the assay performance are first demonstrated through melting analysis of the oligomer target and it is shown how back filling enables accurate discrimination of Legionella pneumophila serogroups directly from the PCR reaction product with complete suppression of sensor fouling.

Exploratory study on domain-specific determinants of opiate-dependent individuals' quality of life.

BACKGROUND/AIMS: Studies on determinants of quality of life (QoL) among opiate-dependent individuals are scarce. Moreover, findings concerning the role of severity of drug use are inconsistent. This exploratory study investigates the association between domain-specific QoL and demographic, social, person, health and drug-related variables, and potential indirect effects of current heroin use on opiate-dependent individuals' QoL. METHODS: A cohort of opiate-dependent individuals who started outpatient methadone treatment at least 5 years previously (n = 159) were interviewed about their current QoL, psychological distress, satisfaction with methadone treatment and the severity of drug-related problems using the Lancashire Quality of Life Profile, the Brief Symptom Inventory, the Verona Service Satisfaction Scale for Methadone Treatment and the EuropASI. RESULTS: None of the QoL domains were defined by the same compilation of determinants. No direct effect of current heroin use on QoL was retained, but path analyses demonstrated its indirect effects on the domains of 'living situation', 'finances' and 'leisure and social participation'. CONCLUSION: These findings illustrate the particularity of each QoL domain and the need for a multidimensional approach to the concept. The relationship between current heroin use and various domains of opiate-dependent individuals' QoL is complex, indirect and mediated by psychosocial and treatment-related variables.

Visible and near-infrared spectroscopic analysis of raw milk for cow health monitoring: reflectance or transmittance?

The composition of produced milk has great value for the dairy farmer. It determines the economic value of the milk and provides valuable information about the metabolism of the corresponding cow. Therefore, online measurement of milk components during milking 2 or more times per day would provide knowledge about the current health and nutritional status of each cow individually. This information provides a solid basis for optimizing cow management. The potential of visible and near-infrared (Vis/NIR) spectroscopy for predicting the fat, crude protein, lactose, and urea content of raw milk online during milking was, therefore, investigated in this study. Two measurement modes (reflectance and transmittance) and different wavelength ranges for Vis/NIR spectroscopy were evaluated and their ability to measure the milk composition online was compared. The Vis/NIR reflectance measurements allowed for very accurate monitoring of the fat and crude protein content in raw milk (R(2)>0.95), but resulted in poor lactose predictions (R(2)<0.75). In contrast, Vis/NIR transmittance spectra of the milk samples gave accurate fat and crude protein predictions (R(2)>0.90) and useful lactose predictions (R(2)=0.88). Neither Vis/NIR reflectance nor transmittance spectroscopy lead to an acceptable prediction of the milk urea content. Transmittance spectroscopy can thus be used to predict the 3 major milk components, but with lower accuracy for fat and crude protein than the reflectance mode. Moreover, the small sample thickness (1mm) required for NIR transmittance measurement considerably complicates its online use.

A VersaTile-driven platform for rapid hit-to-lead development of engineered lysins.

Health care authorities are calling for new antibacterial therapies to cope with the global emergence of antibiotic-resistant bacteria. Bacteriophage-encoded lysins are a unique class of antibacterials with promising (pre)clinical progress. Custom engineering of lysins allows for the creation of variants against potentially any bacterial pathogen. We here present a high-throughput hit-to-lead development platform for engineered lysins. The platform is driven by VersaTile, a new DNA assembly method for the rapid construction of combinatorial libraries of engineered lysins. We constructed approximately 10,000 lysin variants. Using an iterative screening procedure, we identified a lead variant with high antibacterial activity against Acinetobacter baumannii in human serum and an ex vivo pig burn wound model. This generic platform could offer new opportunities to populate the preclinical pipeline with engineered lysins for diverse (therapeutic) applications.

High-throughput microplate enzymatic assays for fast sugar and acid quantification in apple and tomato.

In this article, we report on the use of miniaturized and automated enzymatic assays as an alternative technology for fast sugar and acid quantification in apples and tomatoes. Enzymatic assays for d-glucose, d-fructose, sucrose, D-sorbitol/xylitol, L-malic acid, citric acid, succinic acid, and L-glutamic acid were miniaturized from the standard 3 mL assays in cuvettes into assays of 200 microL or lower in 96 or 384 well microplates. The miniaturization and the automation were achieved with a four channel automatic liquid handling system in order to reduce the dispensing errors and to obtain an increased sample throughput. Performance factors (limit of detection, linearity of calibration curve, and repeatability) of the assays with standard solutions were proven to be satisfactory. The automated and miniaturized assays were validated with high-pressure liquid chromatography (HPLC) analyses for the quantification of sugars and acids in tomato and apple extracts. The high correlation between the two techniques for the different components indicates that the high-throughput microplate enzymatic assays can serve as a fast, reliable, and inexpensive alternative for HPLC as the standard analysis technique in the taste characterization of fruit and vegetables. In addition to the analysis of extracts, the high-throughput microplate enzymatic assays were used for the direct analysis of centrifuged and filtered tomato juice with an additional advantage that the sample preparation time and analysis costs are reduced significantly.

Competitive inhibition assay for the detection of progesterone in dairy milk using a fiber optic SPR biosensor.

Analytical methods that are often used for the quantification of progesterone in bovine milk include immunoassays and chromatographic techniques. Depending on the selected method, the main disadvantages are the cost, time-to-result, labor intensity and usability as an automated at-line device. This paper reports for the first time on a robust and practical method to quantify small molecules, such as progesterone, in complex biological samples using an automated fiber optic surface plasmon resonance (FO-SPR) biosensor. A FO-SPR competitive inhibition assay was developed to determine biologically relevant concentrations of progesterone in bovine milk (1-10 ng/mL), after optimizing the immobilization of progesterone-bovine serum albumin (P4-BSA) conjugate, the specific detection with anti-progesterone antibody and the signal amplification with goat anti-mouse gold nanoparticles (GAM-Au NPs). The progesterone was detected in a bovine milk sample with minimal sample preparation, namely ½ dilution of the sample. Furthermore, the developed bioassay was benchmarked against a commercially available ELISA, showing excellent agreement (R2 = 0.95). Therefore, it is concluded that the automated FO-SPR platform can combine the advantages of the different existing methods for quantification of progesterone: sensitivity, accuracy, cost, time-to-result and ease-of-use.

Bioinspired seeding of biomaterials using three dimensional microtissues induces chondrogenic stem cell differentiation and cartilage formation under growth factor free conditions.

Cell laden biomaterials are archetypically seeded with individual cells and steered into the desired behavior using exogenous stimuli to control growth and differentiation. In contrast, direct cell-cell contact is instructive and even essential for natural tissue formation. Namely, microaggregation and condensation of mesenchymal progenitor cells triggers chondrogenesis and thereby drives limb formation. Yet a biomimetic strategy translating this approach into a cell laden biomaterial-based therapy has remained largely unexplored. Here, we integrate the microenvironment of cellular condensation into biomaterials by encapsulating microaggregates of a hundred human periosteum-derived stem cells. This resulted in decreased stemness-related markers, up regulation of chondrogenic genes and improved in vivo cartilage tissue formation, as compared to single cell seeded biomaterials. Importantly, even in the absence of exogenous growth factors, the microaggregate laden hydrogels outperformed conventional single cell laden hydrogels containing supraphysiological levels of the chondrogenic growth factor TGFB. Overall, the bioinspired seeding strategy described herein represents an efficient and growth factor-free approach to efficiently steer cell fate and drive tissue formation for biomaterial-based tissue engineering strategies.

Spectroscopic evaluation of the surface quality of apple.

Different spectroscopic techniques based on infrared and Raman were used to evaluate the natural wax and related surface quality of apple fruit. Transmission near-infrared (NIR) spectroscopy was applied to solutions of single wax components and extracted apple wax. Fourier transform infrared (FTIR) spectroscopy was used for transmission measurements of wax films on NaCl crystals, diffuse reflectance spectroscopy (DRIFTS) was used to analyze wax powders, and FT-Raman spectroscopy was explored to examine intact wax layers on whole fruit. The natural wax layers of apple fruit from a maximum of three different cultivars (Jonagold, Jonagored, and Elshof) from three picking dates (early, commercial, and late), three controlled atmosphere storage durations (0, 4, and 8 months), and three shelf life periods (0, 1, and 2 weeks) within each storage duration were examined. Canonical discriminant analysis was carried out on the first derivative NIR and FTIR spectra to describe the information contained in the spectra. Discrimination between cultivars and between storage duration based on wax layer properties was achieved with reasonable accuracy from both of the techniques. Information contained in the spectra of apples from different picking dates and shelf life periods was not significant. Differences between cultivars and storage periods in this analysis mostly related to differences in the number of aliphatic chains (e.g., alkanes and esters) and the presence of alpha-farnesene. No satisfactory results were obtained by means of Raman spectroscopy and DRIFTS.

Digital microfluidics for time-resolved cytotoxicity studies on single non-adherent yeast cells.

Single cell analysis (SCA) has gained increased popularity for elucidating cellular heterogeneity at genomic, proteomic and cellular levels. Flow cytometry is considered as one of the most widely used techniques to characterize single cell responses; however, its inability to analyse cells with spatio-temporal resolution poses a major drawback. Here, we introduce a digital microfluidic (DMF) platform as a useful tool for conducting studies on isolated yeast cells in a high-throughput fashion. The reported system exhibits (i) a microwell array for trapping single non-adherent cells by shuttling a cell-containing droplet over the array, and allows (ii) implementation of high-throughput cytotoxicity assays with enhanced spatio-temporal resolution. The system was tested for five different concentrations of the antifungal drug Amphotericin B, and the cell responses were monitored over time by time lapse fluorescence microscopy. The DMF platform was validated by bulk experiments, which mimicked the DMF experimental design. A correlation analysis revealed that the results obtained on the DMF platform are not significantly different from those obtained in bulk; hence, the DMF platform can be used as a tool to perform SCA on non-adherent cells, with spatio-temporal resolution. In addition, no external forces, other than the physical forces generated by moving the droplet, were used to capture single cells, thereby avoiding cell damage. As such, the information on cellular behaviour during treatment could be obtained for every single cell over time making this platform noteworthy in the field of SCA.

Ara h 1 protein-antibody dissociation study: evidence for binding inhomogeneities on a molecular scale.

The characterization of biomolecular interactions is essential when designing novel biosensors, since the interaction between the bioreceptor and the ligand determines important biosensing parameters such as sensitivity and selectivity. In this paper we study the interaction of the trimeric Ara h 1 protein with a monoclonal anti-Ara h 1 antibody by means of magnetic force-induced dissociation. The proteins were bound to magnetic particles and polystyrene surfaces by EDC/NHS reaction chemistry and by physisorption, respectively. Two different molecular configurations have been investigated, with either the Ara h 1 protein on the particles or the Ara h 1 protein on the polystyrene surface. A model with a Gaussian distribution of energy barriers for dissociation gives an adequate description for the measured multi-exponential decays. We hypothesize that distributions of molecular orientations as well as experimentally induced variations may underlay the observed distributions. The two molecular configurations show a different peak value of the energy distribution. Similarly, SPR experiments for two distinct configurations (either Ara h 1 protein on the surface, or anti-Ara h 1 antibody on the surface) also show clear differences in dissociation behavior. We hypothesize that the multivalency of the involved molecules leads to different modes of binding. The results of this work highlight the importance of molecular inhomogeneities when studying the interaction processes of biomolecular complexes.

Irrelevant digits affect feature-based attention depending on the overlap of neural circuits.

Feature-based attention was investigated by examining the effect of irrelevant information on the processing of relevant information. In all experiments, irrelevant information consisted of digits whose semantic information is known to be processed in parietal areas. Between experiments we varied the degree of parietal involvement in the processing of the relevant feature. The influence of the irrelevant digit on the binary manual response task on the relevant feature was measured by the SNARC effect, a spatial numerical association of response codes demonstrating faster left than right hand responses for small numbers and faster right than left hand responses for large numbers. When processing of the relevant feature depended on parietal cortex, as is the case for orientation processing (exps. 1 and 4), there was an effect of the digit's semantic value on response times. Conversely, there was no effect of the irrelevant digit on the processing of color (exps. 2 and 3) or shape (exp. 5), which rely only minimally on parietal resources. After ruling out alternative explanations we conclude that the efficiency of feature-based attention is determined by the degree of neural overlap of structures dedicated to process relevant and irrelevant information.

Inactivation of conidia of Botrytis cinerea and Monilinia fructigena using UV-C and heat treatment.

The effect of UV-C (lambda = 254 nm) and heat treatment was investigated on the inactivation of conidia of Botrytis cinerea and Monilinia fructigena, two major postharvest spoilage fungi of strawberries and cherries, respectively. Both fungi were grown at 21 degrees C in the dark and conidia were isolated after 1 week by washing the mycelium with a mild detergent solution. After filtration and resuspension in phosphate buffer to a titer of 10(5) to 10(6) cfu/ml, the conidia were subjected to different treatments. The applied UV-C doses varied from 0.01 to 1.50 J/cm2, and the conditions for the thermal treatment were 3, 5, 10, 15 and 20 min at temperatures ranging from 35 to 48 degrees C. Both techniques were applied individually and in combination. Spore inactivation increased with increasing intensity of single treatments. No surviving spores of B. cinerea were observed after 15 min at 45 degrees C or an UV-C treatment of 1.00 J/cm2. M. fructigena was more sensitive and a thermal treatment of 3 min at 45 degrees C or an UV-C treatment of 0.50 J/cm2 resulted in complete spore inactivation. Combination of both techniques reduced the required intensity of the treatment for inactivation of both fungi. The order of the applications had a significant effect on the degree of inactivation. The inactivation of B. cinerea conidia was greater when the heat treatment came first, and for M. fructigena, most inactivation was achieved when the heat treatment was preceded with an UV-C irradiation.

Combinations of pulsed white light and UV-C or mild heat treatment to inactivate conidia of Botrytis cinerea and Monilia fructigena.

The use of pulses of intense white light to inactivate conidia of the fungi Botrytis cinerea and Monilia fructigena, responsible for important economical losses during postharvest storage and transport of strawberries and sweet cherries, was investigated in this study. In the first stage, a light treatment applying pulses of 30 micros at a frequency of 15 Hz was investigated, resulting in a treatment duration varying from 1 to 250 s. The conidia of both fungi showed similar behaviour to pulsed light, with a maximal inactivation of 3 and 4 log units for B. cinerea and M. fructigena, respectively. The inactivation of the conidia increased with increasing treatment intensity, but no complete inactivation was achieved. The sigmoidal inactivation pattern obtained by the pulsed light treatment was described using a modification of the model of Geeraerd et al. [Int. J. Food Microbiol. 59 (2000) 185]. Hereto, the shoulder length was incorporated explicitly and relative values for the microbial populations were used. In the second stage, combinations of light pulses and ultraviolet-C or heat were applied. The UV light used in the experiments is the short-wave band or UV-C, running from 180 to 280 nm with a peak at 254 nm (UV-B runs from 280 to 320 nm and UV-A from 320 to 380 nm). The UV-C doses were 0.025, 0.05 and 0.10 J/cm(2), and the temperatures for the thermal treatment ranged from 35 to 45 degrees C during 3-15 min. When combining UV-C and light pulses, there was an increase in inactivation for both B. cinerea and M. fructigena, and synergism was observed. There was no effect of the order of the treatments. For the heat-light pulses combination, there was a difference between both fungi. The order of the treatments was highly significant for B. cinerea, but not for M. fructigena. Combining heat and light treatments improved the inactivation, and synergism between both methods was again observed. Complete inactivation of M. fructigena conidia was obtained after, e.g., a 40-s pulsed light treatment and 15 min at 41 degrees C, or after an 80-s light treatment and 10 min at 41 degrees C.

MRI and x-ray CT study of spatial distribution of core breakdown in 'Conference' pears.

Two non-destructive tomographic techniques, X-ray CT imaging and magnetic resonance imaging (MRI), were applied to study the development of core breakdown disorder in 'Conference' pears (Pyrus communis cv. Conference). This disorder, which is characterized by brown discoloration of the tissue and development of cavities, is induced by elevated CO(2) and decreased O(2) levels during controlled atmosphere storage. Tomographic images of pears stored for 10 months under disorder inducing conditions, were acquired with both techniques and compared to the actual slices. Both X-ray and MRI were able to differentiate between unaffected tissue, brown tissue and cavities. A simple image-processing program, based on threshold values, was developed to determine the area percentage of affected and unaffected tissue as well as the cavity and core area per slice. For all three imaging techniques the area percentage brown tissue per slice increased with the diameter of the pear, but was systematically underestimated by 12% and 6% for, respectively, X-ray and MRI, compared to the actual slices. The area percentage cavity corresponded very well for all techniques. It was also found that the contours of the brown tissue were parallel to the fruit boundaries, suggesting a relation between the disorder symptoms and gas diffusion properties of the fruit. It was concluded that MRI is the most appropriate technique to study the development of core breakdown disorder during postharvest storage in future experiments.

Fast and accurate peanut allergen detection with nanobead enhanced optical fiber SPR biosensor.

This paper is the first report of a fiber optic SPR biosensor with nanobead signal enhancement. We evaluated the system with a bioassay for the fast and accurate detection of peanut allergens in complex food matrices. Three approaches of an immunoassay to detect Ara h1 peanut allergens in chocolate candy bars were compared; a label-free assay, a secondary antibody sandwich assay and a nanobead enhanced assay. Although label-free detection is the most convenient, our results illustrate that functionalized nanobeads can offer a refined solution to improve the fiber SPR detection limit. By applying magnetite nanoparticles as a secondary label, the detection limit of the SPR bioassay for Ara h1 was improved by two orders of magnitude from 9 to 0.09 µg/mL. The super paramagnetic character of the nanoparticles ensured easy handling. The SPR fibers could be regenerated easily and one fiber could be reused for up to 35 times without loss of sensitivity. The results were benchmarked against a commercially available polyclonal ELISA kit. An excellent correlation was found between the Ara h1 concentrations obtained with the ELISA and the concentrations measured with the SPR fiber assay. In addition, with the SPR fiber we could measure the samples twice as fast as compared to the fastest ELISA protocol. Since the dipstick fiber has no need for microchannels that can become clogged, time consuming rinsing step could be avoided. The linear dynamic range of the presented sensor was between 0.1 and 2 µg/mL, which is considerably larger than the ELISA benchmark.