The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself —and motor organization on the cargo—affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s), significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their ‘on’ rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.

This work reports on a new approach to rapidly and selectively detect and discriminate enzymes of pathogenic from those of nonpathogenic bacteria using a patterned autonomously reporting hydrogel on a transparent support, in which the selectivity has been encoded by the pattern shape to enable facile detection by a color change at one single wavelength. In particular, enzyme-responsive chitosan hydrogel layers that report the presence of the enzymes β-glucuronidase (β-Gus) and β-galactosidase (β-Gal), produced by the nonvirulent Escherichia coli K12 and the food-borne biosafety level 3 pathogen enterohemorrhagic E. coli, respectively, via the blue color of an indigo dye were patterned by two complementary strategies. The comparison of the functionalization of patterned chitosan patches on a solid support with two chromogenic substrates on one hand and the area-selective conjugation of the substrates on the other hand showed that the two characteristic enzymes could indeed be rapidly and selectively discriminated. The limits of detection of the highly stable sensing layers for an observation time of 60 min using a spectrophotometer correspond to enzyme concentrations of β-Gus and β-Gal of ≤5 and ≤3 nM, respectively, and to ≤62 and ≤33 nM for bare eye detection in nonoptimized sensor patches. These results confirm the applicability of this approach, which is compatible with the simple measurement of optical density at one single wavelength only as well as with parallel, multiplexed detection, to differentiate the enzymes secreted by a highly pathogenic E. coli from a nonpathogenic E. coli on the basis of specifically secreted enzymes. Hence, a general approach for the rapid and selective detection of enzymes of different bacterial species for potential applications in food safety as well as point-of-care microbiological diagnostics is described.

In this work, patterned β‐GUS sensing chitosan hydrogels functionalized with three different colorimetric substrates were fabricated for the multiplexed detection of the enzyme β‐glucuronidase (β‐GUS), which is secreted by >98% of all known Escherichia coli (E. coli) strains. The immobilization of fluorogenic and chromogenic substrates in specified areas allows a spatially resolved readout of the corresponding colorimetric signal. The apparent initial rate of the β‐GUS induced cleavage of the reporter moieties of chitosan films functionalized with the chromogenic substrate 4‐nitrophenyl‐β‐D‐glucuronide (PNPG), the chromogenic substrate 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronide (X‐Gluc) and the fluorogenic substrate 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) were analyzed spectroscopically and confirmed a detectable reaction within less than 60 min. Likewise the released dyes were observed in the patterns owing to different colors by naked eye detection under appropriate illumination in less than 80 min. Hence the presence of a characteristic enzyme secreted by E. coli bacteria was successfully detected by three independent sensing moieties, which is important to reduce false positives by introducing redundancy. Patterned enzyme sensing chitosan hydrogels, which are functionalized with different substrates, open the possibility for multiplexed bacteria detection and in the long run also the identification of different bacteria strains.

We report on the fabrication and characterization of color-encoded chitosan hydrogels for the rapid, sensitive and specific detection of bacterial enzymes as well as the selective detection of a set of tested bacteria through characteristic enzyme reactions. These patterned sensor hydrogels are functionalized with three different colorimetric enzyme substrates affording the multiplexed detection and differentiation of α-glucosidase, β-galactosidase and β-glucuronidase. The limits of detection of the hydrogels for an observation time of 60 min using a conventional microplate reader correspond to concentrations of 0.2, 3.4 and 4.5 nM of these enzymes, respectively. Based on their different enzyme expression patterns, Staphylococcus aureus strain RN4220, methicillin-resistant S . aureus (MRSA) strain N315, both producing α-glucosidase, but not β-glucuronidase and β-galactosidase, Escherichia coli strain DH5α, producing β-glucuronidase and α-glucosidase, but not β-galactosidase, and the enterohemorrhagic E . coli (EHEC) strain E32511, producing β-galactosidase, but none of the other two enzymes, can be reliably and rapidly distinguished from each other. These results confirm the applicability of enzyme sensing hydrogels for the detection and discrimination of specific enzymes to facilitate differentiation of bacterial strains. Patterned hydrogels thus possess the potential to be further refined as detection units of a multiplexed format to identify certain bacteria for future application in point-of-care microbiological diagnostics in food safety and medical settings.

Here, we report a new pentafluoropropanamido rhodamine fluorescent probe (ACS-HNE) that allows for the selective detection of neutrophil elastase (NE). ACS-HNE displayed high sensitivity, with a low limit of detection (<5.3 nM), and excellent selectivity toward elastase over other relevant biological analytes and enzymes. The comparatively poor solubility and cell permeability of neat ACS-HNE was improved by creating an ACS-HNE-albumin complex; this approach allowed for improvements in the in situ visualization of elastase activity in RAW 264.7 cells relative to ACS-HNE alone. The present study thus serves to demonstrate a simple universal strategy that may be used to overcome cell impermeability and solubility limitations, and to prepare probes suitable for the cellular imaging of enzymatic activity in vitro.