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.

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.

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.

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.

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.

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.

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.

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.

Evaluation and optimization of high-throughput enzymatic assays for fast l-ascorbic acid quantification in fruit and vegetables.

In this paper, we compare and evaluate the applicability of three UV-VIS absorbance based assays for high-throughput quantification of ascorbic acid in horticultural products. All the methods involve the use of a common enzyme (ascorbate oxidase) in combination with a different indicator molecule. The three methods were retrieved from literature: a direct oxidase-method, an OPDA coupled oxidase-method and a PMS-method, which is commercially available. The analysis in high-throughput context involved the analysis in microplates in combination with the use of an automated liquid handling system. We checked (i) the performance factors of the selected methods on standard solutions, (ii) the applicability of the defined methods in high-throughput context, and, (iii) the accuracy of the methods on real samples using HPLC as a reference technique. The OPDA-method was found to be the most appropriate method for the quantification of ascorbic acid in high-throughput context with a linear range between 7.0 and 950 mgL(-1) and excellent correlation parameters (slopes close to 1, intercepts close to 0, R(2)>0.91) with the reference technique when real samples were analyzed. Finally, this method was optimized for assay cost and assay time. Hereto the enzymatic reaction was mathematically described using a model for enzyme kinetics, which was then used to calculate the optimal concentrations of ascorbate oxidase and OPDA. As a result of the modeling the amount of enzyme in the assay could be reduced with a factor 2.5 without affecting significantly the reaction time. In a last step the optimal concentrations were used for a successful validation with the HPLC-reference technique.

Microplate differential calorimetric biosensor for ascorbic acid analysis in food and pharmaceuticals.

In this paper we report on the development of a label-free low-volume (12.5 microL), high-throughput microplate calorimetric biosensor for fast ascorbic acid quantification in food and pharmaceutical products. The sensor is based on microplate differential calorimetry (MiDiCal) technology in which the heat generation, due to the exothermic reaction between ascorbic acid and ascorbate oxidase, is differentially monitored between two neighboring wells of an IC-built wafer. A severe discrepancy is found between expected and observed sensor readings. To investigate the underlying mechanisms of these findings a mathematical model, taking into account the biochemical reactions and diffusion properties of oxygen, ascorbic acid, and ascorbate oxidase, is developed. This model shows that oxygen depletion in the microliter reaction volumes, immediately after injection of sample (ascorbic acid) into the well, causes the enzymatic reaction to slow down. Calibration experiments show that the sensor's signal is linearly correlated to the area under the output versus time profile for the ascorbic acid concentration range from 2.4 to 350 mM with a limit of detection of 0.8 mM. Validation experiments on fruit juice samples, food supplements, and a pain reliever supplemented with ascorbic acid reveal that the designed method correlates well with HPLC reference measurements. The main advantages of the presented biosensor are the low analysis cost due to the low amounts of enzyme and reagents required and the possibility to integrate the device in fully automated laboratory analysis systems for high-throughput screening and analysis.

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.