Concentrated protein solutions investigated using acoustic levitation and small-angle X-ray scattering.

An acoustically levitated droplet has been used to collect synchrotron SAXS data on human serum albumin protein solutions up to a protein concentration of 400 mg ml-1. A careful selection of experiments allows for fast data collection of a large amount of data, spanning a protein concentration/solvent concentration space with limited sample consumption (down to 3 µL per experiment) and few measurements. The data analysis shows data of high quality that are reproducible and comparable with data from standard flow-through capillary-based experiments. Furthermore, using this methodology, it is possible to achieve concentrations that would not be accessible by conventional cells. The protein concentration and ionic strength parameter space diagram may be covered easily and the amount of protein sample is significantly reduced (by a factor of 100 in this work). Used in routine measurements, the benefits in terms of protein cost and time spent are very significant.

Investigating the Time Response of an Optical pH Sensor Based on a Polysiloxane-Polyethylene Glycol Composite Material Impregnated with a pH-Responsive Triangulenium Dye.

Determining the time it takes a sensor to report a change in the concentration of its target analyte may appear to be an easy task, but it is not. The dynamic characteristic of a sensor is determined by all components in the sensor system and the hydrodynamics of the sample. Here, the dynamic properties of an optical pH sensor were determined using the IUPAC-recommended activity step method in experimental setups that can determine sensor-limited response times longer than 5 s. In order to do so, experimental setups for the injection and for the dipping method of determining the sensor time response were developed, tested, and shown to be able to determine time-response curves with 1 s time resolution. This time resolution is shown to be sufficient for determining dynamic characterization of this optical pH sensor. The sensor chemistry-limited time-response curves were analyzed using curve fitting. It was found that the optode response time is limited by diffusion of protons within the sensor material when the proton concentration is reduced and limited by diffusion from the bulk to the boundary layer at the optode surface when proton concentration is increased. The latter is dependent on the magnitude of the change in analyte concentration and cannot be reported as a single response time. The investigation of the time response of the optical pH sensor reveals detailed information of the sensor chemistry, but does not yield a single response time of the sensor capable of describing the dynamic sensor characteristics of the optical pH sensor system.

A unified approach for investigating chemosensor properties - dynamic characteristics.

Chemosensors are a group of sensors-responsive sensor chemistry, sensor hardware, and software-that report on the composition of solutions and gaseous samples. Dynamic properties are fundamental for all sensor characterization. While electrochemical chemosensors have seen a century of research and are well-described, research on chemosensors using other modes of transductions are still at an early stage. The dynamic properties of chemosensors-independent of their mode of transduction-are not reported consistently in the literature. This makes it impossible to compare sensor performance of chemosensors from different manufacturers and laboratories. To remedy this, standardized experimental methods that exclude the influence of drift and any dependence on activity step change must be used. Subsequently, the resulting data must be treated using a unifying analysis formalism, and robust values must be used to describe chemosensor characteristics. Characterizing the sensor properties in turn enables rationalizing the link between sensor performance and sensor chemistry. Following a review of sensor theory, a thorough discussion of experimental methods and data analysis models for determining dynamic sensor properties, we arrive at evidence-based recommendations for good practice when describing new chemosensors. Adhering to these recommendations, sensor performance can be compared between laboratories, and information on the sensor chemistry may be revealed in the data analysis. This topic is particularly relevant in the rapidly maturing field of optical chemosensors.

Tuning the p Ka of a pH Responsive Fluorophore and the Consequences for Calibration of Optical Sensors Based on a Single Fluorophore but Multiple Receptors.

Since Sørensen and Bjerrum defined the pH scale, we have relied on two methods for determining pH, the colorimetric or the electrochemical. For pH electrodes, calibration is easy as a linear response is observed in the interesting pH range from 1 to ∼12. For colorimetric sensors, the response follows the sigmoidal Bjerrum diagram of an acid-base equilibrium. Thus, calibration of colorimetric sensors is more complex. Here, seven pH responsive fluorescent dyes based on the same diazaoxatriangulenium (DAOTA) fluorophore linked to varying receptor groups were prepared. Photoinduced electron transfer (PeT) quenching from appended aniline or phenol receptors generated the pH response of the DAOTA dyes, and the position of the p Ka value of the dye was tuned using the Hammett relationship as a guideline. The fluorescence intensity of the dyes in a sol-gel matrix environment was measured as a function of pH in universal buffer, and it was found that the dyes behave as perfect pH responsive probes under these conditions. The response of optical pH sensors is nonlinear and was found to be limited to 2-3 pH units for a precision of 0.01 pH unit. As sensors with a broader sensitivity range can be achieved by mixing multiple dyes with different p Ka values, mixtures of dyes in solution were investigated, and a broad range pH sensor with a precision of 0.006 pH units over a range of 3.6 pH units was demonstrated. Further, approximating the sensor response as linear was considered, and a limiting precision for this approach was determined. As the responses of the pH responsive DAOTA dyes were found to be ideally sigmoidal and as the six dyes were shown to have p Ka values scattered over a range from ∼2 to ∼9, this allows for design of a broad range optical pH sensor in the pH range from 1 to 10. This hypothesis was tested using quaternary mixtures of the different DAOTA dyes, and these were found to behave as a direct sum of the individual components. Thus, while linear calibration is limited to a precision of 0.02 in a range of 2-3 pH units, calibration using ideal sigmoidal functions is possible in the range of 1-10 with a precision better than 0.01, and as good as 0.002 pH units.

Optical Chemical Sensor Using Intensity Ratiometric Fluorescence Signals for Fast and Reliable pH Determination.

Optical pH sensors enable noninvasive monitoring of pH, yet in pure sensing terms, the potentiometric method of measuring pH is still vastly superior. Here, we report a full spectrometer-based optical pH sensor system consisting of sensor chemistry, hardware, and software that for the first time is capable of challenging the performance of an electrode-based pH meter in specific applications such as biopharmaceutical process monitoring and in single-use bioproduction. A highly photostable triangulenium fluorophore emitting at 590 nm was immobilized in an organically modified silicon matrix that allows for fast time-response by rapid diffusion of water in and out of the resulting composite polymer deposited on a polycarbonate substrate. Fluctuations from the fiber optical sensor hardware have been reduced by including a highly photostable terrylene-based reference dye emitting at 660 nm, thus enabling intensity-based ratiometric readouts. The dyes were excited by 505 nm light from a light emitting diode. The sensor was operational within a pH range of 4.6-7.6, and was characterized and demonstrated to have properties that are comparable to those of commercial pH electrodes considering time-response ( t90 < 90 s), precision (0.03 pH-units), and drift.

Remote Loading of (64)Cu(2+) into Liposomes without the Use of Ion Transport Enhancers.

Due to low ion permeability of lipid bilayers, it has been and still is common practice to use transporter molecules such as ionophores or lipophilic chelators to increase transmembrane diffusion rates and loading efficiencies of radionuclides into liposomes. Here, we report a novel and very simple method for loading the positron emitter (64)Cu(2+) into liposomes, which is important for in vivo positron emission tomography (PET) imaging. By this approach, copper is added to liposomes entrapping a chelator, which causes spontaneous diffusion of copper across the lipid bilayer where it is trapped. Using this method, we achieve highly efficient (64)Cu(2+) loading (>95%), high radionuclide retention (>95%), and favorable loading kinetics, excluding the use of transporter molecule additives. Therefore, clinically relevant activities of 200-400 MBq/patient can be loaded fast (60-75 min) and efficiently into preformed stealth liposomes avoiding subsequent purification steps. We investigate the molecular coordination of entrapped copper using X-ray absorption spectroscopy and demonstrate high adaptability of the loading method to pegylated, nonpegylated, gel- or fluid-like, cholesterol rich or cholesterol depleted, cationic, anionic, and zwitterionic lipid compositions. We demonstrate high in vivo stability of (64)Cu-liposomes in a large canine model observing a blood circulation half-life of 24 h and show a tumor accumulation of 6% ID/g in FaDu xenograft mice using PET imaging. With this work, it is demonstrated that copper ions are capable of crossing a lipid membrane unassisted. This method is highly valuable for characterizing the in vivo performance of liposome-based nanomedicine with great potential in diagnostic imaging applications.

Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: a powerful quality-control tool.

A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Šand a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Šwith an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.

Polymeric strontium ranelate nona-hydrate.

The title compound, poly[[µ-aqua-tetra-aqua{µ-5-[bis-(carboxyl-atometh-yl)amino]-3-carboxyl-atomethyl-4-cyano-thio-phene-2-carboxyl-ato}distrontium(II)] tetra-hydrate], [Sr(2)(C(12)H(6)N(2)O(8)S)(H(2)O)(5)]·3.79H(2)O, crystallizes with nine- and eight-coordinated Sr(2+) cations. They are bound to seven of the eight ranelate O atoms and five of the water mol-ecules. The SrO(8) and SrO(9) polyhedra are inter-connected by edge-sharing, forming hollow layers parallel to (011). The layers are, in turn, inter-connected by ranelate anions, forming a metal-organic framework (MOF) structure with channels along the a axis. The four water mol-ecules not coordinated to strontium are located in these channels and hydrogen bonded to each other and to the ranelates. Part of the water H atoms are disordered. The compound dehydrates very easily and 0.210 (4) water mol-ecules out of nine were lost during crystal mounting causing additional disorder in the water structure.