u-track3D: Measuring, navigating, and validating dense particle trajectories in three dimensions.

We describe u-track3D, a software package that extends the versatile u-track framework established in 2D to address the specific challenges of 3D particle tracking. First, we present the performance of the new package in quantifying a variety of intracellular dynamics imaged by multiple 3D microcopy platforms and on the standard 3D test dataset of the particle tracking challenge. These analyses indicate that u-track3D presents a tracking solution that is competitive to both conventional and deep-learning-based approaches. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability that automatically defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track3D provides a complete framework for unbiased studies of molecular processes in complex volumetric sequences.

Cellular harmonics for the morphology-invariant analysis of molecular organization at the cell surface.

The spatiotemporal organization of membrane-associated molecules is central to the regulation of cellular signals. Powerful new microscopy techniques enable the three-dimensional visualization of localization and activation of these molecules; however, the quantitative interpretation and comparison of molecular organization on the three-dimensional cell surface remains challenging because cells themselves vary greatly in morphology. Here we introduce u-signal3D, a framework to assess the spatial scales of molecular organization at the cell surface in a cell-morphology-invariant manner. We validated the framework by analyzing synthetic signaling patterns painted onto observed cell morphologies, as well as measured distributions of cytoskeletal and signaling molecules. To demonstrate the framework's versatility, we further compared the spatial organization of cell surface signals both within, and between, cell populations, and powered an upstream machine-learning-based analysis of signaling motifs.

Identification of peptide inhibitors of penicillinase using a phage display library.

There is a constant need to identify novel inhibitors to combat ß-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM(-1)) and binding affinity (KD = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV-vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a Ki of 9.22 µM and a Ki' of 33.12 µM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of ß-lactamases and development of novel inhibitors of ß-lactamases.

Electrografted diazonium salt layers for antifouling on the surface of surface plasmon resonance biosensors.

Electrografted diazonium salt layers on the surface of surface plasmon resonance (SPR) sensors present potential for a significant improvement in antifouling coatings. A pulsed potential deposition profile was used in order to circumvent mass-transport limitations for layer deposition rate. The influence of number of pulses with respect to antifouling efficacy was evaluated by nonspecific adsorption surface coverage of crude bovine serum proteins. Instead of using empirical and rough estimated values, the penetration depth and sensitivity of the SPR instrument were experimentally determined for the calculation of nonspecific adsorption surface coverage. This provides a method to better examine antifouling surface coatings and compare crossing different coatings and experimental systems. Direct comparison of antifouling performance of different diazonium salts was facilitated by a tripad SPR sensor design. The electrografted 4-phenylalanine diazonium chloride (4-APhe) layers with zwitterionic characteristic demonstrate ultralow fouling.

Development and investigation of a dual-pad in-channel referencing surface plasmon resonance sensor.

Herein, we describe the construction of a novel dual-pad referencing surface plasmon resonance (SPR) sensor utilizing electrolytic grafting of diazonium salts to individually functionalize two gold pads positioned in a single fluidic channel. Using a dove prism, a simple single axis optical train may be employed without compromising the analytical performance. Once functionalized, one pad is used as the analytical sensing pad for detection of molecular interactions while the other serves as the reference pad, compensating for background refractive index fluctuations. The reference pad effectively compensates bulk refractive index changes and temperature variations as well as other nonspecific effects. The sensor was applied to calibration of a pH-responsive polymer layer in the presence of bulk refractive index and temperature variations. Monitoring selective attachment of a protein is also demonstrated. To our knowledge, this is the first implementation of in-channel referencing SPR sensor utilizing diazonium salt-based surface chemistry.

Glucose detection with surface plasmon resonance spectroscopy and molecularly imprinted hydrogel coatings.

Molecularly imprinted hydrogel membranes were developed and evaluated for detection of small analytes via surface plasmon resonance spectroscopy. Imprinting of glucose phosphate barium salt into a poly(allylamine hydrochloride) network covalently bound to gold surfaces yielded a selective sensor for glucose. Optimization of relative amounts of chemicals used for preparation of the hydrogel was performed to obtain highest sensitivity. Addition of gold nanoparticles into the hydrogel matrix significantly amplified its response and sensitivity due to the impact of gold nanoparticles on the refractive index of the sensing layer. Evaluation of its selectivity showed that the sensor displayed preferential recognition to glucose compared to structurally related sugars in addition to being unaffected by phosphate as well as compounds containing amine groups, like creatinine. The detection limit of glucose in deionized water was calculated to be 0.02 mg/mL. The developed sensor was finally exposed to human urine spiked with glucose illustrating the coating's ability to re-bind the analyte in complex matrices. While the working concentration range in water was determined to be suitable for glucose monitoring in diabetic individuals at physiological levels, the detection in urine was determined to be 0.12 mg/mL. The decreased performance in urine provided an initial perspective on the difficulties associated with measurements in complex media.