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Journal for Biophysical Chemistry

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A virus biosensor with single virus-particle sensitivity based on fluorescent vesicle labels and equilibrium fluctuation analysis

Biosensors allowing for the rapid and sensitive detection of viral pathogens in environmental or clinical samples are urgently needed to prevent disease outbreaks and spreading. We present a bioanalytical assay for the detection of whole viral particles with single virus sensitivity. Specifically, we focus on the detection of human norovirus, a highly infectious virus causing gastroenteritis. In our assay configuration, virus-like particles are captured onto a supported lipid bilayer containing a virus-specific glycolipid and detected after recognition by a glycolipid-containing fluorescent vesicle. Read-out is performed after illumination of the vesicle labels by total internal reflection fluorescence microscopy. This allows for visualization of individual vesicles and for recording of their binding kinetics under equilibrium conditions (equilibrium fluctuation analysis), as demonstrated previously. In this work we extend the concept and demonstrate that this simple assay setup can be used as a bioanalytical assay for the detection of virus particles at a limit of detection of 16 fM. Furthermore, we demonstrate how the analysis of the single vesicle-virus-like particle interaction dynamics can contribute to increase the accuracy and sensitivity of the assay by discriminating specific from non-specific binding events. This method is suggested to be generally applicable, provided that these events display different interaction kinetics.

Over the last decades, increasing effort has been put into the development of bioanalytical devices allowing for the detection of biological compounds in environmental or human samples. Such biosensors commonly take advantage of specific binding between a molecule of interest (target) and a biological counterpart (e.g. receptor-ligand, antigen-antibody or complementary oligonucleotides) to detect the presence of the target with high specificity. Generally speaking, one can distinguish between solution-based assays usually combined with beads or nanoparticles, and heterogeneous surface-based assays [13]. In the latter case, a variety of biosensors relying on the generation of e.g. optical, electrical or gravimetric signals has been described, of which optical detection in conjunction with biomolecule labeling is nowadays the most widespread [4, 5]. In this approach, a detectable fluorescent, colorimetric or chemiluminescent signal is generated by a label attached to a reporter biomolecule that binds specifically to the target, which is in turn bound to the sensor surface via a capture probe (e.g. receptor, antibody, oligonucleotide…) [6, 7].

In the context of environmental monitoring and medical diagnostics, biosensors for the detection of viral pathogens have recently attracted considerable interest: rapid identification of the presence of a virus in e.g. contaminated food or water or in a patient’s sample is a prerequisite to efficiently counteract viral outbreaks, epidemics or bioterrorism. Today, the highest sensitivity for virus detection is achieved with assays relying on polymerase chain reaction (PCR) for multiplication and detection of viral DNA and RNA [8]. Major drawbacks of this technique are however the long processing time (typically 24 hours), the need for advanced laboratory equipment and trained personnel, as well as lack of real-time monitoring and rapid on-site pathogen detection. This calls for the development of simple field-oriented devices capable of detecting the pathogen with high sensitivity and accuracy. In particular, efforts have been directed at the implementation of assays allowing for the detection of whole viral particles in a variety of sensor formats. Optical detection often relies on virus capture and detection in a sandwich assay format in which a molecule immobilized on the sensor surface, captures the target analyte (e.g. a viral particle). A detectable signal is then generated after subsequent binding of a reporter molecule carrying a suitable label. Read-out has been performed with a variety of methods, including fluorescence [911], enzyme-linked immunosorbent assays [8, 12] and using gold colloids for visual [13] or Surface Enhanced Raman Scattering (SERS) [14] detection. In view of their potential for miniaturization and their relatively simple read-out schemes, optical sandwich assays have been combined to dipstick-like sensors (lateral flow assays) [13] or microfluidic setups which make it possible to engineer assays exhibiting a reduced sample consumption or including a sample enrichment step [9, 10]. Together with detection limits in the 10–100 fM (106–107 particles/ml) range [9], this makes such sensors promising candidates for field-oriented applications. As an alternative to such optical sandwich-based assays, approaches proposed in the context of viral particle detection rely, for example, on direct label-free optical detection [15], on the use of functionalized microcantilevers [16], on the detection of virus-induced assembly of magnetic nanoparticles [17] or on electrical [18] and electrochemical [19] transduction.

The growing need for biosensors combining high sensitivity and short processing time, as required e.g. for the detection of virus particles in environmental samples, has also stimulated the development of assays with single-molecule sensitivity, i.e. assays capable of detecting the signal generated by individual target-bound reporter molecules at a sensing interface. In such assays, the targets can be individually counted and the limit of detection (LOD) of the biosensor is not limited by the sensitivity of the transducer, but rather by other limiting factors connected with the bioanalytical assay itself. A relatively simple approach to reach single-molecule sensitivity relies on the use of micro- or nanoscale labels generating a strong signal (via e.g. scattering or fluorescence). Instead of depending on the transducer sensitivity, the LOD is, in this case, rather related to non-specific binding events, leading to an increased background signal, but also to the affinity between the ligand and the receptor, which determines the surface coverage, as well as the target incubation time, which depends on diffusion limitations. A variety of biosensors making use of metallic or semi-conductor nanoparticles [13, 14, 20], polymeric microparticles [21], or lipid vesicles [9, 22, 23] for signal amplification has been developed [7], of which a few specifically take direct advantage of the individual visualization of the particles for read-out [21, 22, 2427].

In the context of nanoscale labels for biosensing applications, phospholipid vesicles – hollow water-filled shells self-assembled from lipids – are particularly interesting candidates: functional biomolecules such as proteins or glycolipids, but also fluorescent markers, can be easily incorporated into the bilayer membrane or attached to it. In addition, either their interior or the lipid bilayer membrane itself can contain reporter molecules for signal generation [7]. Importantly, phosphocholine-containing bilayers are also known to exhibit excellent anti-fouling properties, resulting in low non-specific adsorption of proteins [28]. Moreover, vesicles represent minimal cell membrane models making them well-suited for studies of biomolecular reactions occurring at the cell membrane surface [23, 29, 30].

Our group has recently presented a method to probe the interaction between a surface-immobilized target and functional fluorescent vesicles using total internal reflection fluorescence (TIRF) microscopy [26, 3134]. The method takes advantage of the surface confinement of the evanescent field of TIRF to visualize individual sensor-bound vesicles while filtering out the fluorescence background generated from out-of-focus vesicles. Beyond the use of vesicles as signal enhancing elements to reach single-molecule sensitivity, real-time monitoring of vesicle binding and release events under equilibrium binding conditions (equilibrium fluctuation analysis) makes it possible to extract quantitative information on the biomolecular reaction under investigation: recording of the number of newly arrived vesicles over time can be related to the association behavior and used to quantify the association rate constant [32]. In addition, simultaneous analysis of the residence time of the vesicles is related to the dissociation behavior and can yield information on the dissociation rate constant [33]. In particular, this method makes it possible to probe both weak (> μM) and strong (< pM) interactions, because binding events are visualized individually. For example, equilibrium fluctuation analysis has been used to probe and quantify the interaction kinetics between a ligand and cell-membrane bound receptors [33] (KD in the low nM range), weak glycosphingolipid-glycoshingolipid (GSL) interaction (Kunze et al. Equilibrium fluctuation analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions, submitted) (KD in the low mM range) as well as the interaction between virus-like particles (VLP) from the norovirus and GSL- containing membranes [31, 32].

The equilibrium fluctuation analysis method has so far primarily been used to extract kinetic and thermodynamic information, while its capacity to detect low amounts of target molecules has so far only been explored for DNA detection [26]. The reported fM detection limit suggests that the concept represents a promising alternative, also in the development of viral biosensors. Moreover, the possibility of discriminating interactions according to their kinetic behavior opens the possibility of developing alternative means to determine whether an interaction is of specific or non-specific type. This novel aspect of the concept was, in this work, explored for the detection of human norovirus (NoV). VLPs of the Ast/6139/01 strain from the most common norovirus II.4 genogroup were used as models for the pathogen. NoV is a small non-enveloped RNA virus of the Caliciviridae family with a high genetic diversity. It consists of an outer icosahedral shell assembled mainly from 180 capsid proteins (56 kDa) which protect the viral genome [35]. The acute gastroenteritis caused by the NoV, also called “winter vomiting disease”, is globally spread through pandemics, and is recognized to be responsible for over 200.000 yearly deaths mainly in children in developing countries. In other countries mainly institutionalized, elderly and immunosuppressed patients are specifically at risk, and the harm caused during outbreaks should not be underestimated. This enteric virus spreads globally mostly through the oral-fecal route and spontaneous outbreaks often occur after consumption of contaminated food or water. NoVs are environmentally very stable and extremely infectious which makes their surveillance e.g. in water supplies highly demanding: only very low particle numbers (<100) might be sufficient to generate disease outbreaks making its early detection particularly challenging [36]. In lack of in vitro culture systems, self-assembled capsid proteins recombinantly expressed in insect cells are often used to probe the binding behavior of the virus. These non-infectious VLPs exhibit a morphology and binding properties similar to those of real viruses [37] and are known to recognize with high specificity a variety of saliva and cell-surface glycoconjugates, including membrane bound histo-blood group active GSLs [38]. Such VLPs thus represent excellent models in work aimed at designing new biosensor principles for virus detection.

The assay explored in this work is based on a sandwich-type configuration where the VLPs are first captured onto a non-fouling supported lipid bilayer containing a NoV-specific GSL ligand. The firmly bound NoVs are then visualized by imaging individual fluorescently-labeled phospholipid vesicles containing the same NoV-specific ligand. A considerable amount of vesicles was found to interact reversibly with the VLPs, which is attributed to a vesicle-curvature dependent interaction strength [32]. This allows for real-time recording of binding kinetics under equilibrium conditions. We further make an attempt to take advantage of the vesicle association and dissociation dynamics to improve the accuracy and LOD of the assay, by discriminating specific and non-specific binding events according to their kinetic signatures.

1-Palmityol-2-Oleyol-sn-Glycero-3-Phosphocholine (POPC) was purchased from Avanti Polar Lipids (USA) while 1% Lissamine-Rhodamine B-1,2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine (rhodamine-DHPE) was obtained from Invitrogen (USA). Histo-blood group H type 1 GSL [38, 39] was purified from human meconia, pooled according to the ABO blood group and characterized by mass spectrometry and 1H-NMR spectroscopy [40, 41]. VLPs from the Spanish norovirus isolate Ast6139/01/Sp [42] (accession number: CAE47529) were produced as described previously [43]. The VLP concentration was determined from the total protein content using a molecular weight of 1.17*104 kDa (the VLP consists of 180 copies of a 56 kDa recombinant capsid protein). This value was in good agreement with the number of particles counted by nanoparticle tracking analysis (performed with Nanosight LM 120 instrument placed on an optical microscope at 20X magnification). All experiments were performed in filtered TRIS buffer (10 mM Tris(hydroxymethyl)-aminomethane, 100 mM NaCl, pH 7).

Lipid vesicle preparation

Lipid vesicles were prepared by the lipid film hydration and extrusion method. Briefly, POPC, H type 1 GSL, and if needed, rhodamine-DHPE lipids diluted in chlorophorm were mixed in appropriate amounts and dried first under a gentle nitrogen stream and then under vacuum for at least 1.5hours. The lipid film was then hydrated in TRIS buffer while vortexing. The so-obtained vesicle suspension was extruded by pushing the suspension a defined number of times through polycarbonate membranes (pore size: 30 nm or 100 nm). The vesicles were stored at 4°C until use.

Assay preparation

All experiments were performed in glass-bottom microtiter wells (96 well-plate) that were cleaned overnight in a 10 mM sodium dodecyl sulfate solution and rinsed thoroughly with MilliQ water. Supported lipid bilayers were formed at the bottom of the microwells by surface-induced vesicle fusion [44]. For this purpose, vesicles containing 10% H type 1 GSL (extruded 11 times at 30 nm) were added to the well (total lipid concentration 0.1 mg/ml). After incubation for 30 min, the total volume of the well was adjusted with buffer to 100 μl and rinsed carefully 8 times with 200 μl of buffer without drying the surface. After rinsing, 50 μl of VLP suspension were added (total volume in the well: 100 μl) and incubated for 1 hour. This was followed by rinsing and addition of POPC vesicles (final concentration: 0.05 mg/ml, extruded 21 times at 100 nm). After at least 10 min, fluorescent vesicles containing 5% H type 1 GSL and 3% rhodamine-DHPE (extruded 21 times, 100 nm) were added, yielding a final concentration of 0.25 μg/ml. Read-out was performed at least 30 min after addition of the fluorescent vesicles to make sure that equilibrium conditions were reached.

Data acquisition

Time-lapse movies were acquired on a Nikon Eclipse Ti-E inverted microscope using a 60X magnification (NA = 1.49) oil immersion objective (Nikon Corporation, Japan). The microscope was equipped with a mercury lamp (Intensilight C-HGFIE; Nikon Corporation), a TRITC filter cube (Nikon Corporation) and an Andor iXon + EMCCD camera (Andor Technology, Northern Ireland). Movies consisting of 1000 frames were taken at an acquisition rate of 5 frames/second. For each well, 7 different positions were imaged to ensure good statistics, as bilayer quality and VLP surface coverage may vary within one well.

Images were processed and analyzed with MatLab (MathWorks, Inc., USA) based on a method described in detail elsewhere [26]. Briefly, a fluorescent vesicle was counted if its intensity exceeded a pre-set threshold and if it was present on a pre-defined number of consecutive frames (minimum 7 frames). Association plots were generated by recording the number of newly arrived vesicles (n +) over time. The program further analyses the residence time (Δτ) of these vesicles. To ensure accurate statistics of the residence time analysis, only the vesicles that arrived in the first half of the movie were considered and the maximal residence time was set to half of the total measurement time. Moreover, bleached vesicles, which disappeared without exhibiting a sudden drop in intensity, were discarded from the analysis. Residence time analysis was used to reconstruct the dissociations plots which display the number of vesicles that are still bound as a function of time.

Movies of insufficient quality (due e.g. to the apparition of aggregates in the field of view) were discarded automatically by the software. To decide whether a movie should be taken into account for evaluation, the slope of the association signal was compared to the average signal of the other movies for the same sample. If this value differed more than two standard deviations (of the signal of the remaining movies) from the average signal, this movie was discarded. A minimum of 3 movies was used for the evaluation.

To generate association plots and for the residence time analysis, as well as to estimate the surface coverage, the data obtained from all movies taken within the same microwell (3–7 movies/well) was summed up and normalized to the total area imaged. To make up for inconsistencies in the final volume associated with the numerous pipetting steps, all signals were normalized by multiplying with the final well content (determined by weighting). The average image intensity was measured with Image J (Image processing and analysis in Java, National Institute of Health, USA); each data point was the average of all the frames taken within a well. To determine the equilibrium surface coverage (neq), only the vesicles bound for more than 50 frames (10 s) were counted. The slope of association (δn+ /δt) was determined by fitting the data with a linear fit y = a*x. The area under the dissociation curve (Adiss) was determined after fitting the data with a double exponential function y = a*exp(bx) + c*exp(dx).

Evaluation of the limit of detection

To determine the LOD of the assay a 4-fold VLP dilution series was prepared. At least 8 VLP concentrations were included in each experiment and added to a microwell which was imaged at 7 different locations. The VLP concentration in the wells ranged from 125 ng/ml (12.5 pM) to 0.122 ng/ml (12.1 fM). The signal for a well where no VLPs were added was used as a negative control. The LOD was determined from the mean signal of the negative control incremented with the 3-fold standard deviation of three independent experiments. In this case, the procedure mentioned above was reproduced independently on different days using freshly prepared VLP dilutions.

The assay designed for the fluorescence-based detection of unlabeled NoV VLPs using TIRF microscopy utilizes a microwell functionalized with a POPC supported lipid bilayer containing 10% H type 1 GSL. POPC bilayer coatings have been shown to render glass surfaces highly resistant against non-specific protein and vesicle binding [28] while the GSLs ensure a high capture efficiency and specificity to the NoV VLPs [32, 39]. Fluorescently labeled vesicles containing H type 1 GSL for specific recognition were used to detect firmly bound VLPs by TIRF microscopy (Figure 1a). The vesicles contained a large amount of fluorophores (~3000 fluorophores / vesicle). This enables the visualization of each vesicle individually (Figure 1b), and confers the single VLP sensitivity of the method. As visible in Figure 1b, the sensor exhibited a good specificity with little non-specific binding on the negative controls performed in the absence of VLPs.

Figure 1

a) Detection of norovirus virus-like particles (VLP) by total internal reflection fluorescence (TIRF). a) Schematic representation of the sandwich assay setup. The particles are captured onto a bilayer containing 10% H type 1 glycosphingolipids (GSL) recognizing the VLPs with high specificity. The fluorescent signal is generated by TIRF illumination of the sensor-bound rhodamine-labeled vesicles containing 5% H type 1 GSL. b) A representative microscopy image of surface-bound vesicles i) on a bilayer incubated with 12.5 pM VLPs and ii) on a negative control performed in absence of VLPs. Scale bars: 50 μm.

Taking advantage of the evanescent field generated by the TIRF illumination to discriminate surface-bound vesicles from the ones in solution, vesicle binding and release events at the sensor interface were recorded under steady-state, i.e. equilibrium binding conditions. The equilibrium coverage (n eq), the rate of arrival of new vesicles at steady state (δn +t) and the residence times (Δτ) of the individual vesicles were extracted after analysis of the time-lapse movies and used to quantify the sensor signal. All results presented were recorded at least 30 min after adding the vesicles to make sure that the system had enough time to relax towards equilibrium. In these conditions, the average number of vesicles bound to the substrate did not vary over time (Figure 2a) while the number of newly arrived vesicles increased linearly (Figure 2b). Both observations confirm that the measurement is indeed performed under steady-state conditions.

Figure 2

Equilibrium fluctuation analysis of vesicle – virus-like particle (VLP) interactions. a) Equilibrium surface coverage (n eq) vs. time. b) number of newly arrived vesicles (n +) vs. time. The insert illustrates that individual binding events are recorded. c) Residence time (Δτ) distribution of the newly arrived vesicles. d) Dissociation plot displaying the number of vesicles still bound vs. time. In all cases a positive signal recorded on a substrate incubated with 12.5 pM VLPs (red) is compared to a negative control performed in absence of the VLPs (blue).

The distribution of the residence time of the vesicles (Figure 2c) can be converted into dissociation plots displaying the number of vesicles still bound over time (Figure 2d). The latter curves reveal that over the duration of the measurement (Δτ is analyzed for 100 s) a major fraction of vesicles was released (~ 80%, for the case shown in Figure 2d) while the remaining fraction was considered as irreversibly bound on the time scale of the measurement. At this VLP concentration (12.5 pM), the positive signal was clearly distinguishable from the negative background signal (experiment performed in absence of VLPs), further illustrating the sensor specificity (red vs. blue curves in Figure 2). Note in particular, that in Figure 2c most of the non-specific reversible events had Δτ < 10 s (blue curve) while for the specific signal (red curve) a significant number of events had a residence time 10 s < Δτ < 100 s, indicating that a discrimination of both types of interactions according to their kinetic signature could be possible.

In analogy to conventional end-point measurements, the sensor signal can be evaluated by counting the individual surface-bound vesicles under equilibrium conditions and using n eq as a measure for the sensor signal. A typical response curve, displayed as signal-to-background (s/b) vs. concentration for a sample dataset, is shown in Figure 3a (green squares). Here, the number of bound vesicles counted (n eq) obtained for positive (in the presence of VLPs) and negative (in the absence of VLPs) samples are taken as the signal and the background, respectively. As an alternative to n eq, the signal can be evaluated by measuring the average frame intensity (I frame), in analogy to fluorescence-based assays lacking single-molecule sensitivity. As shown in Figure 3a (light blue dots) no contrast was observed at any concentration, illustrating the advantage of an evaluation based on individual label counts.

Figure 3

Response curves displaying the signal-to-background (s/b) vs. concentration for a chosen data set analyzed with different strategies. a) After evaluation of the surface-bound vesicles at equilibrium. Evaluation was performed either by vesicle counting (green squares) or by measuring the average image intensity (light blue dots). b) Using the vesicle arrival rate (δn +t) as a signal. c) Using the area under the dissociation curve (A diss). Evaluation was performed taking all events into account (black triangles), for Δτ > 10 s, i.e. after removing of all events with a short residence time (red dots) or for 10 s < Δτ <100 s, i.e. after removing the events with a short residence time and the irreversibly bound vesicles (blue stars). In all cases, the background signal was the signal measured on the negative control.

We also specifically investigated whether real-time equilibrium fluctuation analysis of the recognition reaction can further contribute to increase the accuracy of the assay. The vesicle arrival rate (δn +t) is expected to correlate with the VLP surface concentration [32]. For analysis, δn +t was therefore used as a measure for the sensor signal and obtained by linear fit of the association curve (Figure 2b). To optimize the s/b, we investigated whether it is possible to take advantage of the differences in the kinetic signatures of specific and non-specific interactions (see e.g. Figure 2c) to further reduce the background, thus increasing the s/b ratio. A close look at the Δτ distribution for both cases (Figure 2c) reveals that, in absence of VLPs, the vesicles interact weakly with the supported lipid bilayer, and that most of them have residence times of less than 10 seconds (Δτ<10 s). To illustrate how an appropriate data analysis strategy can be used to maximize s/b values and hence to increase the sensor’s accuracy, we have compared different evaluation strategies using a representative dataset. For this dataset, the δn +t was evaluated by considering only the vesicles that had Δτ > 10s (Figure 3b red diamonds). Here, this evaluation strategy yields a ~3.5-fold increase in s/b compared to the case where all vesicles are taken into account and an increase by a factor ~2 compared to an evaluation of the vesicle equilibrium surface coverage (n eq).

In this context, it should be mentioned that the quality of the bilayer and its non-fouling properties can depend on the overall quality of the substrate. For bilayers of outstanding quality, less than 10% of the vesicles were irreversibly bound. However, an increase in the number of vesicles irreversibly attached to the bilayer was observed occasionally, most likely originating from non-specific binding due to the presence of defects in the coating. In such a case, the percentage of irreversible events could be as high as 70% of the total events (see Additional file 1: Figure S1). An additional data analysis was therefore performed by further discarding the irreversibly bound vesicles from the analysis, and taking therefore into consideration only the events that had a residence time of 10 s < Δτ < 100 s. For the data set presented in Figure 3, which had an irreversible fraction of 39% (see Additional file 1: Figure S1), this analysis yields an increase in s/b by a factor 3.5 (Figure 3b, blue stars) compared to the evaluation performed with Δτ> 10 s (Figure 3b red diamonds).

We thus conclude that for an analysis based on the use of δn +t as the signal, the highest s/b is obtained by analyzing the data for 10 s < Δτ < 100 s as the non-specific interactions appear to be predominantly either very transient (Δτ < 10 s) or irreversible (> 100 s).

An alternative evaluation strategy makes use of the contrasts in the dissociation curves (Figure 2d) to determine the sensor signal. In this case, the area under the curve (A diss) is measured after fitting the dissociation with a suitable analytical function, here a double exponential function. In analogy to the analysis performed above, all vesicles were taken into account (Δτ > 1.4 s, Figure 3c black triangles) or the vesicles were selected in order to discriminate specific and non-specific interaction (Δτ > 10 s, Figure 3c red dots; or 10 s < Δτ <100 s Figure 3c, blue stars). Generally, the s/b values obtained this way were lower than for the evaluation obtained using δn +t and the data were of inferior quality.

The LOD of the bioanalytical assay for the detection of NoV VLPs was determined from three independent measurements with irreversible fractions on the negative controls of 5.5%, 5.5% and 2% respectively. Based on the analysis above, all reversible events above 10 s (10 s < Δτ < 100 s) were taken into consideration, and δn +t was used as signal. For all three experiments the non-specific binding events were successfully filtered out and the background signal was indeed zero. In this case, the lowest detected signal was 12.1 fM, indicating that the assay sensitivity is in the low fM range (Figure 4). The signal depended linearly on the VLP concentration for a concentration range spanning over at least 3 orders of magnitude. Since for these experiments, the irreversibly bound fraction on the negative controls was low and therefore not expected to significantly influence the assay sensitivity, we further analyzed the data with Δτ > 10 s to provide a quantitative estimation of the LOD while taking the measurements standard deviations into account. This yields a detectable background signal (see Additional file 1: Figure S2 for the response curve) and the assay LOD estimated from the background signal incremented with its 3-fold standard deviation was 15.8 fM.

Figure 4

Response curve displaying the signal vs. virus-like particle (VLP) concentration. The signal is the slope of association curve (rate of arrival of new vesicles, δn + /δt) using a residence time interval 10 s < Δτ<100 s. Each data point is the average of three independent measurements (2 for the 12.5 pM data point). The error bars are the standard deviation of three independent experiments.

We have presented a biosensor for the fluorescence-based detection of NoV VLPs using TIRF illumination and fluorescently labeled vesicles to generate an optical contrast upon recognition of the VLP. The method exhibits single-molecule sensitivity, as individual vesicles can be readily visualized.

As compared to conventional fluorescence read-out based on average fluorescence intensity measurements of the sensing area, the possibility of imaging individual fluorescent reporter biomolecules – the vesicles in this case - and counting them one by one, represents a simple approach to increase the s/b ratio of a sensor, and hence to increase the reliability while maximizing the sensitivity. This is due to the fact that the vesicles generate localized signals which can be easily resolved and discriminated from the background noise, while contributing only scarcely to the average intensity of the imaged area. In essence, the intrinsic noise originating e.g. from the detector, from the substrate’s auto-fluorescence or from the presence of fluorescent molecules in solution is automatically suppressed from the evaluation. Also, variations in label brightness (e.g. associated with the distribution in size of the extruded vesicles) do not influence the signal, making quantitative estimates more accurate.

While background noise can be efficiently suppressed, a critical factor influencing the LOD of a bioanalytical assay exhibiting single-molecule sensitivity, such as the one reported here, is the background signal generated by non-specific binding events. Thus, an important aspect to be taken into consideration is the non-fouling character of the sensing interface which should, in this case, be virus but also vesicle repellent. The GSL-modified POPC supported lipid bilayers used in this work fulfilled these conditions, and the number of vesicles found on the negative controls varied between ~3 vesicles/frame (88 vesicles/mm2) and ~ 35 vesicles/frame (1027 vesicles/mm2), also if the bilayers were kept at room temperature overnight. Note in particular that 35 vesicles/frame corresponds to a coverage as low as 10-7% of full coverage.

In spite of the good non-fouling properties of the surface coating, we demonstrate that equilibrium fluctuation analysis has the potential of further pushing down the background signal of the biosensor and of increasing the s/b: we take advantage of the partially reversible character of the vesicle-VLP interaction to discriminate specific from non-specific events based on their affinity to the sensing interface. Although demonstrated at the specific example of a sensor for the detection of the norovirus using receptor-containing lipid vesicles with a curvature that reduces the affinity [32], the approach is in principle generic and applicable to both weak and strong binders. It can be applied to any biomolecular interaction provided that, for the experimental setup under consideration, the specific and non-specific interactions have a distinct characteristic kinetic behavior allowing for the determination of an optimal residence time window in which the equilibrium fluctuation analysis measurement should be performed. Besides the fact that the background signal can, in principle, be pressed down to zero as achieved for our platform, a major advantage of such an approach over conventional end-point measurements is that the performance of the biosensor is likely to be less dependent on the quality of the sensor’s chemical interface. This in turn reduces the need for high-performance protein repellent coatings, which have so far played a crucial role in the performance of sensors with single-molecule sensitivity. Moreover, the availability of high affinity binders might become less critical and effort can be put on utilizing weak but highly specific binders which were so far discarded from affinity-based biosensor applications.

To conclude, we have demonstrated that a relatively simple assay setup combined with single-molecule sensitivity and equilibrium fluctuation analysis can be used to detect viral particles with a LOD in the low fM regime (i.e. ~106 particles/ml). The LOD was therefore in the same range, if not slightly better than what has so far been reported by others, also in the context of biosensors for the detection of noroviruses [8, 9]. Without the need of any particle enrichment steps, these sensitivities are already sufficient to detect viral particles recovered from feces [8]. However, to meet the challenge of detecting norovirus particles in contaminated water, the focus of future development needs to be put on implementing particle enrichment steps to concentrate samples containing as little as 0.5–4 particles/ml [45]. Furthermore, to test the applicability of our platform for real diagnostic applications, efforts will now need to be directed at testing the sensor with biologically relevant samples which include stool, food and water sample as well as swabs from surfaces in restaurants and hospitals. Another aspect to be taken into consideration is the specificity of the assay. Histo-blood group antigens on glycosphingolipids, such as H type 1 have been shown to bind to noroviruses with high specificity although it cannot be excluded that they react with other species, in particular rotaviruses [46]. If needed, the specificity of the assay could be further optimized by using a combination of two different ligands in the sandwich assay format. In particular the glycolipids on the bilayer or on the vesicle could be replaced by an antibody against the virus which can provide additional genotype specificity. Such antibodies are commonly used in ELISA-based assays for NoV detection, but not in formats offering single virus sensitivity [8]. Additionally, real-time monitoring of binding reactions on a single particle level allows, in principle, for discrimination between different viral species according to their kinetic signatures. This aspect could further contribute to increase the assays specificity and will be the subject of further investigation.

Although optimization of the assay time was beyond the scope of this project, the whole assay could be performed within less than 2 hours. Currently, the major limiting factor towards shorter analysis time is the VLP incubation step (1 hour); read-out can be performed within minutes. A natural extension of the assay would be to skip the initial incubation step, and instead record transient binding events from a suspension containing a mixture of viruses and vesicles. This strategy would in fact work excellently for most sandwich assays, since the surface-bound probe and the secondary binder are typically directed to different (and single) epitopes on the target molecule. However, a virus typically contains a large number of identical epitopes, which means that the addition of vesicles can lead to aggregation. In this particular case, it is more relevant to stress that thanks to the simplicity of the fluorescence-based transduction, our assay is directly compatible with microfluidic devices that could, for example, integrate a sample pre-concentration step [9] or for optimized flow-based capture efficiency [47]. This could potentially increase the LOD by at least one order of magnitude. If assay time is not critical, the diffusion limitations inherent to assays performed under stagnant conditions could also be overcome by increasing the assay time (e.g. to overnight incubation) in order to maximize the VLP capture efficiency at the sensing interface without sacrificing the compatibility of our assay with the conventional micro-titer plates format. Finally, each vesicle, containing around 3% fluorescent lipids is easily detectable at an acquisition time of 100 ms. Although a relatively advanced imaging set up (a 60× oil immersion TIRF objective and a cooled CCD camera) was employed, this points towards the opportunity to significantly simplify the detection system without scarifying the single-molecule sensitivity.

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The work was supported by grants from Vinnova, the Swedish Research Council (8266 to GL), and t1he Swiss National Science Foundation (MB).

Author information

Corresponding authors

Correspondence to Marta Bally or Fredrik Höök.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

The project was formulated by FH and MB. MB wrote the manuscript. MG performed the experiments. FP provided the virus-like particles. Manuscript completion was performed by all authors. All authors read and approved the final manuscript.

Electronic supplementary material

Figure S2

Additional file 1: Figure S1: Dissociation behavior of the negative controls for bilayers of different quality. . Response curve displayed vs. time evaluated using a residence time interval Δτ > 10 s. (PDF 157 KB)

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Bally, M., Graule, M., Parra, F. et al. A virus biosensor with single virus-particle sensitivity based on fluorescent vesicle labels and equilibrium fluctuation analysis. Biointerphases 8, 4 (2013).

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  • Virus detection
  • Biosensor
  • Norovirus
  • Glycosphingolipids
  • Phospholipid vesicle
  • Nanoscale label
  • Fluorescence
  • Liposome
  • Virus-like particle


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I never thought I’d hear me say …

I never thought I’d hear me say …

This morning the mini Birksy that is Emelia had a toddler tantrum. Why? Our Amazon Echo didn’t understand her request to play ‘wheels on the bus’. Once her tears had dried and we’d settled down to watch Peppa Pig (again!), it got me thinking …. what else do I say today that wouldn’t have made any sense 10 years ago? Here are my top 10 …

Alexa, play ‘wheels on the bus’

Usually followed by “Alexa, turn volume down by 20%”.

I’ll just connect you to my hotspot

When setting up YouTube Kids on the ipad for a long journey.

Don’t forget to pack the chargers

When travelling anywhere.

I walked 10,001 steps today

Not said as often as I’d like anymore.

One tea please and what’s your wifi password?

Tea breaks are nothing without a good scroll.

Ahhh, my metabolic age is still 37

Said many times since investing in some smart scales.

Shall we just call an Uber?

Not said as much now that we are in North Wales.

Let’s just Google it

Said most often when trying to help my dad with the crossword.

I’ll WhatsApp you the pictures later

Following any social event – in particular playgroups!

What’s your handle, I’ll follow you

When written down this sounds so creepy.

Photo by Michael Discenza on Unsplash.

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text:
Will Bitcoin Reach $100K? – E-Crypto News

Bitcoin (BTC) has so far solved some of its biggest challenges. In its early days, the cryptocurrency had two key problems. Notably, its survival depended on them. After resolving these issues, analysts believe that the question should not be: will bitcoin reach $100K but instead when will it hit $100K.

It is possible to see bitcoin reach $100K. For the crypto to hit $100,000, it must have a market cap of $2.1 trillion which is about 20% the market cap of gold currently. Some analysts say that for the feat to happen, constant buying demand from retail investors should exist. One analyst said:

“At 100K per BTC, the market has to absorb a miner supply of $90 Million daily. Assuming that there are 10 million people worldwide buying BTC regularly. It would cost them each $9 daily to sustain those price levels. I’ve met people who spend more than $9 on coffee.”

Gold bitcoin coin one hundred dollars bills.

Data suggests that most of the bitcoin hodlers have not sold all through the last three all-time highs of the cryptocurrency.

“60% of BTC has not moved for more than 2 years. Even if that were to be true, the longtime supply of BTC will eventually equal the mining (new) supply.”

The current data from Coinmarketcap indicates that Bitcoin has a market cap of around $205.8 billion trading at $11,159.

Based on speculation and economic theory, there is a lot of issues to address the possibility of Bitcoin reaching six figures. To bring it into perspective, the long-standing economic theories and economists’ doubts play a key role in determining BTC price.

But, deeply-rooted market variables, forecasts, and global adoption are all bound to influence a change in the value of bitcoin in one way or the other.

Crypto Enthusiasts’ Projection

Many crypto enthusiasts have long forecasted the probability of Bitcoin reaching $100K. Evidence of that can be noted from renowned individuals like John McAfee and the Co-Founder and Partner of Morgan Creek Digital, Anthony Pompliano. Pompliano said:

“…I still think Bitcoin will hit $100,000 by the end of December 2021. Fixed supply. Increasing demand. Time will tell.”

In late 2019, Ethereum Co-Founder, Charles Hoskinson, tweeted:

The most recent actionable insight was rendered via Paul Tudor Jones’ “The Great Monetary Inflation.” He acquired bitcoin as a hedge against inflation amid the current COVID-19 health crisis.

While bitcoin enthusiasts rely on speculation in the cryptocurrency news, participants and investors formulate their assumptions upon tangible data acquired from macroeconomic theories and projection models.

Projection models like the M2 capitalization theory and the Bitcoin S2F Model forecast considerably high valuation for Bitcoin. Quantitative easing has been done by central banks around the world to combat financial ramifications.

M2 Money Stock

In the case of the Federal Reserve, it has been printing USD at an exponential rate since 1970. The effect of that printing is inflation with prices of goods and services surging to meet this money supply. A healthy economy needs depreciation in prices but that is not the case especially with the huge stimulus packages aiming to fight the aftermath of this pandemic.

The core point of the M2 Capitalization Theory suggests that if the Federal Reserve continues printing dollars at exponential scales; the US dollar price per bitcoin will continue rising exponentially. Hence, it is possible to see bitcoin reach $100K.

Bitcoin Stock-to-Flow (S2F) Cross Asset Model

The Bitcoin S2F model ratio was designed by @100trillionUSD who measures the effect of scarcity on Bitcoin price using the current market circulation and production rate. In 2019, he suggested through Modeling Bitcoin Value with Scarcity:

“The predicted market value for bitcoin after May 2020 halving is $1trn, which translates in a bitcoin price of $55,000. That is quite spectacular. I guess time will tell, and we will probably know one or two years after the halving, in 2020 or 2021. A great out of sample test of this hypothesis and model.”

Although it may have some flaws, this theory shows that many proponents are convinced that BTC price will continue surging in the coming years.

Economists Weigh In on Bitcoin Reaching $100K

When asked whether he expects to see bitcoin reach $100K, Kenneth Rogoff, an economist and Harvard University professor, said:

“I think bitcoin will be worth a tiny fraction of what it is now if we’re headed out 10 years from now … I would see $100 as being a lot more likely than $100,000 ten years from now. Basically, if you take away the possibility of money laundering and tax evasion, its actual uses as a transaction vehicle are very small.”

If speculations about Bitcoin’s future applications are the driving force behind its volatility, then the valuation of the crypto, in general, is crippled due to diminished cash flow.

Bitcoin Adoption

Mainstream adoption of bitcoin is one variable piece of the puzzle that can propel the crypto towards $100,000. If mass adoption happens, the general economic theory of supply and demand will automatically increase in price.

Bitcoin and dollar

Through an increase in demand comes value appreciation. Since the supply of Bitcoin is limited, that should further strengthen the ideology that an increase in BTC demand will increase the price of Bitcoin.

Based on the M2 capitalization theory and the principles of demand and supply, there is a possibility to see bitcoin reach $100K. But, it is impossible to determine the value of Bitcoin years from now. Nevertheless, history has taught us that anything is possible.

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Source: https://e-cryptonews.com/will-bitcoin-reach-100k/


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Goat Cheese and Bacon Biscuits

I’m not one of those people who is crazy for bacon in everything.  The idea of pairing it with chocolate still weirds me out a little, and I’ve never tried it in brownies or ice cream.  That being said, bacon is probably the top reason I would have trouble being a vegetarian.  Crisp, sandwiched with some dripping heirloom tomato slices and lettuce on toasted sourdough, and I’m dreamy happy.  Salty fatty fried chunks studding my bowl of baked beans, and my evening is made.
Food Blog April 2013-0990
What I am generally crazy for is breakfast.  But not at breakfast time.  I can’t handle a big savory meal early in the morning.  A fried egg sandwich, okay (and with a little sriracha in the mayonnaise?  Be still my heart!).  A pancake or three, maybe on occasion.  The big, multi-course breakfast is, for me, wasted on the morning.  I’m a breakfast-for-dinner kind of girl.
This week, in need of comfort as spring break drew to a close and allergy season burst wide open, we decided breakfast sandwiches were just what we needed.  Eggs, bacon, fluffy buttery biscuit, and why not, a little goat cheese?!  But layering these components together would not suffice.  Thick slices of bacon smashed against a cloud of scrambled egg and crumbles of goat cheese seemed like a mess waiting to happen.  I’ve incorporated cheese into biscuits before with great success, why not do the same with the bacon?
Food Blog April 2013-1002
The result: goat cheese bacon biscuits.  A simple revelation, but let me tell you, a spectacular base for a scrambled egg sandwich.  Crisp squares of bacon, cold cubes of butter, crumbles of chevre, and a healthy glug of buttermilk.
These are pretty cinchy to make, though thanks to the addition of the goat cheese your biscuit dough will be a little stickier than usual.  Try not to add too much flour – you don’t want them to get dense.  They bake up into lovely little puffs, and the bacon stays crisp against the soft dough.  The goat cheese wasn’t as strong a flavor as we were expecting, though after the biscuits cooled a bit we did pick up a pleasant tang from the larger crumbles.  Loaded up with a simple layer of scrambled egg, and you have a perfect, three-bite sandwich with all the right trimmings.  And because it’s only three little bites, you can have two or three without any guilt to speak of.  Or four… or…
 Food Blog April 2013-1013
The funny, blackened stakes lying in a pile in the background of this photo are roasted rainbow carrots.  They were incredible.  And don’t just take my word for it – I knew they were the real deal when N. carefully sampled one, turned to me, and said “wow.”  If you’ve been reading this blog for any length of time you will know that N. is not intentionally grudging when it comes to food praise; he’s just not particularly effusive about it.  A “wow” is like fireworks.
Breakfast-for-dinner slam dunk, then.  What’s your favorite?
 Food Blog April 2013-0993
Goat Cheese Bacon Biscuit Sandwiches
2 cups flour (All-Purpose is fine)
2 tsp sugar
1 tsp salt
2 TB baking powder
4 slices bacon, diced and fried until crisp, drained and cooled (do this a bit ahead of time so the bacon has time to cool off – if you toss it into the mixture hot, you’ll heat up the butter and your biscuits will be less fluffy)
6 TB cold butter, cut into chunks (chunking it isn’t absolutely necessary, but it does make it easier and quicker to incorporate)
½ cup crumbled goat cheese
6 oz. buttermilk
 Food Blog April 2013-0995
  • Preheat your oven to 400F and line a baking tray with parchment paper.
  • In a medium bowl, combine the flour, baking powder, sugar, and salt.  Whisk in the cooled, crisp bacon.
  • Add the butter and incorporate using a pastry blender or your hands.  When the chunks are about the size of lima beans, tumble in the goat cheese and blend it in until there are no more large pieces.  The pebbles of butter should be about the size of peas when you are done.
  • Pour in the buttermilk and fold it into the dry mixture.  I find using a fork works best for this – the tines pick up and jostle around the flour mixture better than a spatula or wooden spoon.  Don’t overmix, but be sure the buttermilk is well incorporated.
  • When your mixture is evenly damp, abandon the fork.  You can turn the whole mess out onto a floured board, or you can just reach in with flour dusted hands and knead the dough a few times in the bowl until it comes together.
Food Blog April 2013-1000
  • Pat the dough into a plump something-like-a-rectangle on a floured board.  The thickness and therefore the size of the rectangle is really to you, but mine was probably just under an inch thick.  Using a biscuit cutter or the floured top of a glass, punch out biscuit rounds by pushing straight down all the way through the dough.  Don’t twist your cutter as you go down; you’ll disrupt the craggy layers in the dough and the biscuit won’t rise as high or as evenly.
Food Blog April 2013-1001
  • When you’ve punched out as many rounds as the rectangle of dough will allow, place them on your parchment lined baking sheet at least an inch apart, gather the dough scraps, knead them together a bit, and pat them back into a new rectangle.  Continue punching out biscuits and reshaping the scraps until you run out of dough.  Given the small size of cutter I chose, I managed 16 sweet little biscuits.  You will have more or less depending on size and thickness.
Food Blog April 2013-1007
  • For a small biscuit (2 inch diameter), bake at 400F for 12-14 minutes, or until the layers have puffed and the top is golden.  Larger or extremely thick biscuits will take longer; try 15 minutes to start.
Food Blog April 2013-1011
If you just want to eat them as is – and I’d sympathize completely if you did – you’re all set to go.  Dig in.  If, however, you want them as sandwiches, split them down the middle of the puffy, buttery layers and insert a fold of softly scrambled egg.
Food Blog April 2013-1003
Food Blog April 2013-1004
These are best the day they are cooked, so I’d advise only baking as many as you and your dinner partner(s) are going to eat.  The remaining biscuits can be frozen, still unbaked, and enjoyed another day.  To freeze them, set them on a piece of wax paper or parchment in a single layer on a plate or baking tray.  Wait until they are frozen, then relocate them to a zip-top freezer bag.  They don’t even need to be defrosted, just pop them into a preheated oven for a few extra minutes (maybe 15-18 for a small biscuit) and dinner – or breakfast – is served!
Food Blog April 2013-1010


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Maybe going from Miss to Mrs. just got a little easier...

TGIF! Someone alerted me to this site a little while ago and I of course wanted to share with you guys. I figured anything to help make your life easier after saying “I Do” could be beneficial to Triple B. The Web site claims to be able to help you start your married life off with less paperwork and more time to enjoy the bliss of being a new wife.
A woman spends an average of 13 hours filling out forms and driving to various agencies just to go from a Miss to a Mrs. founder Danielle Tate has apparently found a way to streamline the process to a mere 30 minutes. The site has helped over 80,000 brides in the U.S. change their name securely with government, state and private agencies in just three steps.

So, have any of you used this site? Would you consider using it? Why or why not? Leave me a comment with your thoughts and have a great weekend!


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*This post is part of a Diner En Blanc series! Check out the rest of the series here

Yesterday, I attended Houston’s Diner En Blanc 2016.  Just like last year, I complained all the way up until the event, but once the tables were sat and the napkin wave began I was again in love with this event!Someone is probably thinking, DANG she’s writing about that event again?! I apologize in advance, but for whatever reason my views tell me that people love reading about Diner En Blanc SO I am just here to give the people what they want :).  Here is my event run down for Diner En Blanc 2016!

My HFBC friends and I at the event!

My HFBC friends and I at the event!

Table Decorations:

If you recall, in my last post I was at a loss for what I would decorate my table with, but I knew that it had to be better than last year.   I hit up Tuesday Morning, the Dollar Tree, Homegoods and some neighborhood boutiques looking for ideas.   Here is my rule, if I cannot use it for more than Diner En Blanc, I am not buying it!  At my first spot, I found a gorgeous white cake plate that I knew I would use a million times over.  This sparked my VISION, as my sister says.   I am food blogger.  Why not let my table decorations center around food?! With that in mind off I went.

My next biggest haul came from Tuesday Morning.  That place was a god send!  I found some heavier plastic plates that you can reuse and are light making for easy transport! I grabbed a couple of charger plates for like 3 bucks each to add a dash of fancy and then some napkin rings to really set it off.   Then I picked up two plastic wine glasses to minimize the amount of glass I had to carry with me.

Next I had an idea to do some branding at my table, so I bought a chalkboard meat and cheese plate from Whole Foods for about 12 bucks!  That was probably the cheapest thing I have ever purchased from Whole Foods!  Anywho, I wrote out Black Girls Who Brunch at my table for passer bys and pictures.  Genius, I know hahahaha!

This cheese plate was perfect! I picked it up from Whole Foods, but here are some similar ones from Target and Amazon.

This year I had time to do some pillaging through my own cabinets and there also was no prohibition on glass.  I found a tall mason jar that I received as a gift from a recent foodie event and threw some white hydrangeas from Trader Joes in that.  I also had a empty bottle which once housed some cheap HEB wine.  Well it came with a top and I always knew I would reuse it for water or lemonade one day! Today was the day! Finally, I grabbed a my silverware, square white bowl and a white glass pyrex bowl to display my food.

Long story short, I thought I purchased pre-packaged food from the hosts, but the morning of the event I could not find a receipt or proof that on my cards were charged.  Thankfully, I caught this mistake early enough for me to pick up some food myself.  Sadly this meant no wine (more on that later).  I set out to find a meal that featured courses and would make for great pictures. My food stops were Trader Joes, Whole Foods and Dessert Gallery!

First course!

First course!

Trader Joes is so budget friendly, so I always start there and get what I can.  I fill in the rest at their pricier neighbor, Whole Foods.  At Trader Joes, we grabbed meat, cheeses, fruits, lemonade, salad and water.   Sounds like a lot, but we knew we needed more dishes so we hit up Whole Foods too.  There, we bought some crackers, chicken and pasta salad.  My sister ordered a stuffed portbello mushroom for her entree.  I threw them in the oven right before we left with the hopes that they would be warm by the time we ate.  They really were not, but it was worth the try!

Finalllllly, we went to Dessert Gallery my favorite dessert spot! There we grabbed a mini all white cake for our dessert/center piece.  My sister tried to advocate for a chocolate cake, but when wearing all white, white food wins!  In the end we were stuffed! We would probably cut out some of those foods next year to save on space and money.

I loved that cake!

I loved that cake!

The Arrival & Setup

Before heading to the event I packaged all of my crap carefully in a suitcase for easing hauling.  We donned our all white, full makeup in 90 degree weather and white clothes and headed to our meet-up location near NRG.  There I met my fellow Houston Food Blogger Collective members including blogger Ryan (IG: DatFoodPorn).  Ryan was a life saver for me!  We negotiated a fair trade for the event.  He used my dolly, plates and silverware in exchange for hauling my table and chairs on said dolly.  This made the loading and unloading much less stressful than last year.

Once we loaded up on the shuttle bus, we headed to the undisclosed location.  As we neared downtown we all began to guess where the event could be.  City Hall? Discovery Green again??? Or maybe Minute Maid park?? Sure enough, the street and grassy area outside of Minute Maid park was the location.  The street was blocked off allowing for close bus access too.  Perfection!   The only thing that would have made this better would have been to set up inside the park.  We set in our assigned area and began to unload our stuff for setup.  Again, I was happy with how I planned stuff this year.  Other than my sister being in my way, my set up was perfect and I was ready when the ceremonial napkin wave began!

Ryan took this amazing panoramic picture of the event. There I am in the middle on the phone LOL

Ryan took this amazing panoramic picture of the event. There I am in the middle on the phone LOL

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Dinner and Entertainment

Once we cut the cake and were done eating, the party started. This year there was much better music and live entertainment.   There was string quartet playing pop music, a live band that started the evening off and a DJ to close it out.  I should note, that this year the crowd was majority black.  Black folks LOVE any excuse to dress in white!  Well the DJ played to the crowd and smartly went with songs like the Wobble and Frankie Beverly.  There was also a photo booth and plenty of great baseball themed backdrops around Minute Maid Park.   Just like last year we ubered back to avoid the rush.  I highly recommend doing the same.

The short of it is, we had SO much fun!! The planners this year were a New Orleans based group called the Spears Group. No shade to last years group of hosts, but this group planned a seamless event! I made sure to tell one of the main hosts that they needed to come back next year as well!

Quick Tips Before I Go

  • Last year I went straight from Trader Joes to the event. Bad idea.  Organize your food at home before you go! For example, a box of crackers takes up more space than necessary. Take out one roll, throw it in a zip log back and go.
  • Food based centerpieces are easy and functional, but still allow you to be creative.
  • I said this last year and it is worth repeating! Ladies go for the white dress over the white linen pants etc, you will feel underdressed compared to the rest of the crowd.  The ladies this year legit had on opera dresses and I even saw a guy with a white tuxedo.  Again, people especially black folks love any reason to get all dolled up in all white.
  • Quadruple check your order in the online store before the deadline or you will end up like me wine less!  This years event also did not allow outside wine or liquor.  The event hosts scared us with threats about security checking for unauthorized wine and liquor so I did not risk it.  I personally did not have my items searched from contraband, but that is a risk for others to take, not me the perpetual rule follower!
  • I did not bring a picnic basket and I still think that is a great way to cut costs if you do not have one just laying around.  People also took cute baskets and turned them into old school picnic baskets (Like this or this). It was super cute and cost efficient!
  • Go with a group! It is more fun that way! This year my group was my HFBC family.  These foodies were super creative with their dishes and decorations.  Loved seeing what they came up with.
  • The smartest thing I did was haul my decorations in a rollaway suitcase.  Other smart ways to haul your stuff, a wagon or a flatbed dolly.
  • AND I am still on the hunt for a good lightweight table.  The rollup camping table did not seem to be sturdy enough for heavy glass decorations, so the search continues.

Until next year :)!

2016 prep post

2015 review

2015 prep post


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Socrates' Defence

Socrates' Defence - Penguin Little Black Classics

Paperback (26 Feb 2015) | English

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'I'll stop doing it as soon as I understand what I'm doing.'

Somewhere between a historical account and work of philosophy, Socrates' Defence details the final plea of Plato's beloved mentor.

Introducing Little Black Classics: 80 books for Penguin's 80th birthday. Little Black Classics celebrate the huge range and diversity of Penguin Classics, with books from around the world and across many centuries. They take us from a balloon ride over Victorian London to a garden of blossom in Japan, from Tierra del Fuego to 16th-century California and the Russian steppe. Here are stories lyrical and savage; poems epic and intimate; essays satirical and inspirational; and ideas that have shaped the lives of millions.

Plato (474 BC-347 BC). Plato's works available in Penguin Classics are Republic, The Last Days of Socrates, The Laws, Phaedrus, Protagoras and Meno, Timaeus and Critias, Theaetetus, Early Socratic Dialogues, The Symposium and Gorgias.

About the Publisher

Penguin Classics

Penguin Classics

The first ten paperback Penguin books appeared in 1935 costing 6d each (the price of a packet of cigarettes). Since then the Penguin list has developed enormously, but still aims to bring the best writing to the widest possible audience. Penguin Paperbacks now range from Booker Prize-winning contemporary authors, to mass market bestsellers, with successful history, biography and general non-fiction as well.

Book information

ISBN: 9780141397641
Publisher: Penguin Books Ltd
Imprint: Penguin Classics
Pub date:
DEWEY: 184
DEWEY edition: 23
Language: English
Sales rank: 56534
Number of pages: 55
Weight: 54g
Height: 161mm
Width: 111mm
Spine width: 7mm


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Maintaining Your Concrete Floor Pt. 1

Concrete floors are easy to maintain and offer several advantages to high traffic and heavy duty facilities.  While it is extremely durable, there are a few things that concrete floors are susceptible to that should be attended to as they are easily avoidable.  Concrete floors can crack and those cracks can become potentially problematic if not quickly repaired. Mildew can also spread across the floor in environments with moisture present.  We will outline a few ways that you can augment the longevity of your concrete floor and keep your facility running clean and efficiently.  This is part 1, we will outline more methods next week in part 2. Without further ado, here is you the beginning of our list!

  1. Keep substances from building up on you floor: Keeping your floor swept and clear of dust buildup will keep your floor both safe and presentable. Dust is not the only substance that will build up on your floor, water and chemical spills can sit and erode at your floor and it’s coating if left unattended. Mopping and sweeping are important everyday steps that should be taken in order to ensure long lasting service from your concrete floor.
  2. Remove Stains: Certain substances can actually stain your concrete floor and leave it with an unattractive and glaring blemish. Some stains can be completely lifted with the proper cleaning materials, but the best way to prevent stains to clean all spills immediately. Removing stains that have set in can be painstaking and time consuming and the longer a substance sits, the more deeply set the stain will be in the pours of your concrete floor.

Next week we will return to briefly outline a couple more methods that you can deploy in order to keep your concrete floor in top condition and providing the service you desired when you initially made your investment.

Benefits of Polished Concrete Floors

Many facilities employ the use of concrete flooring as it is very durable and long lasting. However, there are ways you can make a concrete floor even more resilient which will cause it to last even longer. Polishing a concrete floor is a great way to extend and maximize your investment in your flooring.  Here are a few ways that a polished concrete floor is advantageous to have instead of an unpolished one.

Reduction of Necessary Maintenance = Saved $

Polished concrete floors are resistant to stains and the general wear and tear from the chemicals, tires, and everyday foot traffic that they may see. The necessity of periodically stripping and replacing wax and sealers disappears with polished concrete because the finish does not need to be reapplied.  Polished flooring also improves lighting with its reflective surface and is not abrasive to tires.

Dust Free

Unpolished concrete floors develop cracks and openings that house dust and also allow it to pass through to the floor’s surface. This leaves the floor with a coat of dust that can be both unsightly and unsafe. The dust from an unpolished floor will inevitably settle on everything in the room and create a scenario in which everything in your facility needs to be cleaned and maintained more frequently. Polishing a concrete floor eliminates this issue and presents a smooth, easy to clean surface that also will be more aesthetically pleasing.

Slip Resistance

Standard polishing is very slip resistant and meets OSHA’s standards for safety. The slip resistant qualities can be enhanced even further by adding special conditioners to the polish that augment the non-slip quality it will add to your flooring.

There are more benefits to having a polished concrete floor such as the fact that you can add designs to the floor to make your space more pleasing to the eyes. Whether focus on safety or visual appeal, a polished concrete floor is superior to an unpolished floor in every way. Call All Kote Lining Inc today and find out how a polished concrete floor can benefit you!

Finding A Full Service Cooling Tower and Flooring Maintenance Company.

If you are in the market for cooling tower services or flooring upgrades or repairs for your facility finding a company that can cover all aspects of cooling tower maintenance and flooring is ideal. Having the same entity handle all of your cooling tower maintenance and flooring needs is beneficial in many ways, the least of which is that over time the company will have intimate knowledge of your situation and that can greatly benefit and streamline the maintenance process. That is why we at All Kote Lining inc believe that taking a comprehensive approach to cooling tower and flooring maintenance is most beneficial to all involved, especially our customers.  In this blog we’ll outline some of the services we offer at All Kote Lining inc.

Coatings: We offer several fiberglass coating options for various applications. Over time the containment integrity of cooling towers and storage tanks can become compromised and will need some form of restoration.  All Kote is equipped to restore your fiberglass tanks, ceramic coating, steel tank, and sandblast your tanks and surfaces among several other services.

Cooling tower repairs: Cooling towers are large scale complex devices that can house many components that help them separate heat from water. Over time these components naturally wear down along with the overall structure of the tower. All Kote can repair your tower and its components and keep it operating at optimum levels.

Epoxy flooring: Keeping floors clean and conditioned is an important part of running any facility. Protective floor coatings wear down over time and lose much of their protective properties. Having your floor epoxy restored will improve both the look and performance of your facilities flooring.

Floor repairs: If your floor is cracked or damaged it can seriously effect your operation. Having your floor repaired and restored to ‘like new’ conditions will have your facility running on schedule and sporting the look that you want to portray to potential clientele and business partners.

All Kote is All You Need – Cooling Tower Company

All Kote believes in being a dynamic, full service cooling tower maintenance company that can cover any cooling tower related needs in the Phoenix, AZ area.  We strive to offer the very best service possible in order to increase our base of satisfied customers as well as remain the leader in cooling tower maintenance in our area.

All Kote’s goal is to achieve excellence in all aspects of our business, starting with our interactions with our clients and ending with their satisfaction with the results of the service we have provided. We employ top-quality, highly experienced technicians to ensure that All Kote is fully capable of handling anything in the range of possible cooling tower needs.  If your cooling tower is in need of new coating or if it is totally offline, our techs will be fully equipped and prepared to tackle the situation quickly and efficiently.  Emergency situations are no different; we will attend to your situation as quickly as possible with a plan of action to alleviate any urgent needs.

Cooling tower upgrades and upkeep are specialties of All kote and are our ‘bread and butter’ so to speak. Our staff’s level of expertise allows us to come up with solutions that are both efficient and cost effective. Top quality results that are economically friendly are a win-win for everyone involved. They keep our clients happy and our phones ringing with service requests. All Kote truly are your Valley of the Sun experts when it comes to cooling towers. Our region presents unique challenges and conditions can be tough on cooling towers. All Kote can maximize the longevity and efficiency of your cooling tower systems and save you money and valuable time over the long run. Trust us with your cooling tower needs and upkeep, you won’t be disappointed. We guarantee it because we believe in what we do. We think you will too.

Types of Cooling Towers

Cooling towers are used to extract heat from water and disperse it into the atmosphere, but not all towers do this the same way. There are many types of cooling towers out there because there are so  many various environments and situations that they need to perform in. We are going to cover a few of the common cooling tower types here:

Hyperbolic: Commonly used in nuclear facilities, hyperbolic cooling towers work due to their stack/chimney style design. This design is used to take advantage of the times when the air outside of the tower is cooler than the air inside the tower which causes the warmer, heavily humid air inside the tower to rise and eventually leave through the top of the tower. The upward draft effect cools the water in the tower.

Crossflow: These towers are designed to cool water by sending air across water descending from the distribution basin horizontally. Once the water has descended to the flowing collection basin it has been adequately cooled. While more these type of towers don’t usually cost as much as other types, they are more prone to freezing which could offset the benefit of the lower cost in the long run.

Counterflow: In these type of towers air moves vertically against water descending water and sends the extracted heat out of the top of the tower. It takes power to send the air upward so counterflow towers tend to cost a little more. They are also more compact and ergonomically efficient which is can offset some of the sting of the higher costs of construction and operation.

There are many, many more cooling tower types out there and the science behind the design of each is fascinating. Finding the right cooling tower to for a facility is a huge decision that will have major financial implications in the big picture. Knowledge is your best weapon against wasting valuable resources.

10 Tips to Maximize Your Cooling Towers

Here are 10 tips to help you get the most out of your cooling tower:

  1. Thoroughly check the condition of your units. Check for the presence of unfamiliar noises. Doings so routinely will give you a record of the behavior of your units so you have a better idea of when to schedule maintenance and repairs without minimal interruption to its everyday use.
  2. Make sure you follow protocol when it comes to beginning work on your unit. Make sure all lock out procedures are accurately followed and motors etc are disconnected. This is a safety measure and should be followed without exception.
  3. Clean all debris that may be near or in your cooling tower system to keep build ups from happening.
  4. Check the water distribution system to make sure the fill coil area is properly moisturized. If it is not uniformly moist, check nozzles for damage or clogging.
  5. Make sure debris and dirt in the cold water basin is washed through the tower drain. This is important because it allows for water filtration to remain effective and to keep dirt from starting to collect.
  6. Make sure the make-up water supply is at the appropriate level.
  7. Prevent solids from accumulating by making sure the bleed rate is adjusted to be in line with the quality of your water and the local evaporation rate regulations.
  8. Make sure the tension level of the belt is proper.
  9. The quality of the systems oil, its oil level, as well as the alignment of the shaft for the gear drive system of your tower.
  10. Make sure the bearings of the fan shafts are well lubricated at least every three months. You can even install automatic systems to grease your bearings.

These tips should help you maintain optimal operation of your cooling tower. Stay on top of your tower and it’s components by consistently monitoring their condition and performance.

Tips to Streamline Your Cooling Tower Maintenance

Cooling towers are widely used to keep large facilities comfortable and cool. Cooling towers are the size of Large Dinosaurs and keeping them working in top condition can take some work, especially if you fall behind on your maintenance schedule. It is not difficult to keep your towers humming; it just takes some dedication to the process and the time to do so. This blog entry is going to suggest some ways you can stay ahead on maintenance and keep your cooling towers working efficiently without having to break your budget.

Make needed updates

Cooling towers are a large investment to have installed as a part of your facilities operations. That kind of investment is meant to last and that means there are plenty of cooling towers out there that have already seen decades of service. These towers can stay efficient because their owners have made necessary upgrades as time when on and innovations were made. Keeping up with the times in regard to your cooling tower will maintain and even increase the efficiency of your tower’s performance.

Make and execute a plan

Developing a methodology for the upkeep of your towers is both extremely important and massively beneficial.  It takes time to learn your towers and their idiosyncrasies, but once you have a good idea on their behavior you can draw up a standard procedure for all to follow in regard to maintaining your cooling towers.

Collect as much data as possible

As stated above, it takes time to learn the behavior patterns of your cooling tower.  In the initial stages and beyond, collecting as much data about the various aspects of your cooling towers will arm you to be able to keep them running well with the least amount of effort put into their upkeep. Streamlining the maintenance process is only possible with enough data available to do so.

Cooling Tower Fill Types

At first assumption one would probably believe water to be the core of the cooling towers operations, however the real star of the show are the fills or wet decks.  Fills provide the cooling tower operation with extra surface area for the water in the tower to be exposed to air and absolutely maximize the evaporation process. There are several types of fills out there and different types of cooling towers will utilize different fill types depending on their methodology when it comes to creating evaporation. We will outline a few different fill types in brief in order to set you on the path to furthering your understanding of how cooling towers actually work!

Splash Fills:

Splash fills work by breaking up the flow of water crating droplets by ‘splashing’ the water. The droplets evaporate quickly and increase the rate of evaporation in the right conditions and tower. As the water descends  various splash fills increasingly break down the water into smaller and smaller droplets that have air flowing against the descending water.

Film Fills:

Film Fills operate by spreading water into a thin flowing layer over a large surface area exposed to air flowing across the water in order to extract heat and elevate it out of the tower.  There are patterns on the fill called flutes to help control how the water flows and to keep it at the desirable level for the evaporation to happen at the rate the tower operator is looking for. The goal of the flutes is to have the water take the longest possible route across the fill so air has the most time possible to do it’s part of the evaporative process that the tower is designed to perform.

The fills listed above present different advantages and disadvantages and there really is no right choice. The main factor in deciding which one to choose is the process they will be deployed to be a part of. This is a very brief description and there is definitely plenty more to learn. All Kote Lining inc is happy to answer any questions you may have and to assist you with any of your cooling tower needs.

How Do Cooling Towers Work?

Cooling towers have a very important commonality with the human body, sweating. At least in principle. We sweat when our body needs to cool itself down by emitting water over the skin and allowing the evaporation to cause a cooling effect which lowers the core temperature of the body. That effect isn’t exclusive to the human body, spreading water over any surface and allowing it to evaporate will leave the area or item much cooler than it was prior.

The environment you are in dictates how well this process works to cool via moisture. Air has a saturation threshold and one it’s reached it’s limit it cannot hold any more moisture. In areas with extremely humid conditions this process doesn’t work very well because the sweat and moisture remain on the skin instead of evaporating and can actually cause an increase in heat. Sweating in Arizona is much different than sweating in Georgia because the air in Arizona is very dry and has plenty of room for saturation whereas Georgia is very, very humid and the air becomes fully saturated very quickly even though it is much hotter temperature-wise in Arizona’s desert. This means that towers will work differently and utilize different measures to achieve optimal efficiency. There are variations to almost every component of cooling towers on top of the various styles that the towers themselves are designed within in order to accommodate differing needs and climates. Knowing which type of tower works best in your region is paramount to getting the most out of your cooling tower investment. The type of maintenance as well as the schedule you keep in regard to maintenance will differ by region as well.

Cooling Towers work in much the same way although the amount of water involved that needs to be evaporated is much greater than what the human body produces. This means that extra components will be needed in order for the process to be carried out efficiently. Fans are used to cycle saturated air out of the tower so unsaturated air can be brought in to promote further evaporation. Water is constantly circulated via various devices in order to replace evaporated water and keep the thermodynamic process going. Heat sources are also involved to ensure the water is always being heated to levels it can vaporize and evaporate. Cooling towers and their maintenance are very complex and their components can vary in type and number, but at the heart of the whole operation, the fundamentals are as simple as sweat.

Your Damaged Concrete Floor Could Be Damaging Your Forklifts

If you have damaged concrete floors in your facility you not only run the risk of lost efficiency and unsightly patches of your flooring, you may also be damaging and accelerating the wear and tear on your forklifts. It may seem easier and cheaper to repair your forklifts instead of repairing your concrete flooring, but it is not the case over time and you may push your forklifts to the point that replacements could be necessary.  The prospect of having a floor repaired in uncomfortable because it brings a lot of inconvenient possibilities into the fold such as temporary closures and interruptions to everyday functions. Those inconvenient possibilities pale in scale to having to potentially replace your fleet of forklifts or constantly have them repaired throughout their serviceable lifetimes.  Once your floor is repaired the right way, the temporary inconveniences listed above will prove to be worth dealing with as your floor and facility return to optimum levels of efficiency and performance. Those forklifts will enjoy the benefit of increased longevity and you will enjoy the benefit of not footing all of those repair bills constantly.

Both your flooring and your forklift fleet are major investments that are integral to the overall success of your operation. If you floor in such disrepair that it damages your forklifts you will actually see your investments working against each other and harming the overall durability and serviceable lifetime of the forklifts. Keeping both your flooring and your forklift fleet in good working condition is integral to the success level that will be possible for your operation.

Making sure you find the right company to come to your facility in order to assess and ultimately repair your floor is very important and can save you a lot of headaches.   All Kote Lining Inc in the Phoenix, AZ metro area will provide the results you desire with our valley leading concrete flooring restoration services. A member of our team will come out to your facility, inspect the situation and make recommendations to you while explain your options and pricing quote. Once you have made your decision our team will quickly attend to the damaged portions of your floor and be out of your facility as soon as possible. Give us a call today at (480)966-4446 and let us get your concrete flooring back in top shape!


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ASK ROTOMAN: Holliday for Machado? Blame It On Cain.

Hey Rotoman:

I have Longoria as my 3B, and have Manny Machado. I want to trade Machado to improve my outfield.

What do you think of this trade: I get Matt Holliday for Machado and Matt Cain?

“Machado Man”

Dear Macho:

Some music while we think about this.

First off, I don’t have enough information to judge. It sounds like you won’t miss Machado much, playing Longoria instead, but whose working week does Holliday end? And who is filling Cain’s shoes?

The problem here is that Cain has not been a Top-20 starter so far this year, and that makes it easy to undervalue him after a few bad outings. I recently l0oked at last year’s slow starting pitchersÂ and found that pitchers who were expected to be good usually did okay after a bad start. Matt Cain was one of those guys last year, and he recovered, at least part way. A bad start doesn’t mean the rest of the season is a disaster.

Still, over the five months after his bad start Cain wasn’t the Cain anyone who bought him last year hoped for him to be. And it has to be disturbing that he’s starting this season in similar fashion. So I can understand disenchantment.

Okay, another musical interlude.

But I can’t really judge your trade because I’m not seeing all the pieces. The two parts that stand out are: It’s a great idea to upgrade your outfield by dealing a reserve corner. Not that it isn’t valuable to have a solid backup like Machado, but to win you probably have to maximize even if that’s a little risky. So that makes sense if Holliday is a nice bump on the guy you’re replacing.

But Matt Cain shouldn’t be a throw in. If you have a great staff without him, making him expendable because of his poor start, I don’t have a problem with that. But if you’re dealing him because you’re expecting him to be replacement level the rest of the way, I think there’s a fair chance you’re selling him a little short.