Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.

Real-time shape approximation and fingerprinting of single proteins using a nanopore.

Sericin, a major component of silk, has a long history of being discarded as a waste during silk processing. The value of sericin for tissue engineering is underestimated and its potential application in regenerative medicine has just begun to be explored. Here we report the successful fabrication and characterization of a covalently-crosslinked 3D pure sericin hydrogel for delivery of cells and drugs. This hydrogel is injectable, permitting its implantation through minimally invasive approaches. Notably, this hydrogel is found to exhibit photoluminescence, enabling bioimaging and in vivo tracking. Moreover, this hydrogel system possesses excellent cell-adhesive capability, effectively promoting cell attachment, proliferation and long-term survival of various types of cells. Further, the sericin hydrogel releases bioactive reagents in a sustained manner. Additionally, this hydrogel demonstrates good elasticity, high porosity, and pH-dependent degradation dynamics, which are advantageous for this sericin hydrogel to serve as a delivery vehicle for cells and therapeutic drugs. With all these unique features, it is expected that this sericin hydrogel will have wide utility in the areas of tissue engineering and regenerative medicine.

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Exploring natural silk protein sericin for regenerative medicine: an injectable, photoluminescent, cell-adhesive 3D hydrogel

With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (kD) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25°C and 40°C) and long-term storage conditions (2-8°C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2-8°C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the CH2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability.

In this study, we have reported the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and structural stability of Rhizopus niveus lipase Secondary structural changes were monitored by Far-UV CD which shows that surfactant induces helicity in the Rhizopus niveus lipase protein which was maximum in case of CTAB followed by SDS, CHAPS, and Brij-35 Similarly, tertiary structural changes were monitored by tryptophan fluorescence We also carried out enzyme kinetics assays which showed that activity was enhanced by 15- and 11-fold in the presence of CHAPS and Brij-35, respectively Furthermore, there was a decline in activity by 20 and 30 % in case of SDS and CTAB, respectively These studies may be helpful in understanding detergent–lipase interaction in greater detail as lipases are used in many industrial processes

The surfactant-induced conformational and activity alterations in Rhizopus niveus lipase.

Carbazole-substituted dicyanobenzenes and anthraquinone based intramolecular charge-transfer dyes, two classes of organic thermally activated delayed fluorescence (TADF) emitters developed for organic light-emitting diode (OLED) technology, are prepared and investigated for their suitability for optical oxygen and temperature sensing. The new materials do not offer any improvement compared to the state-of-the-art probes in respect to oxygen sensing. In contrast, due to very high temperature dependency of the TADF decay times, the new dyes are particularly advantageous for optical thermometry. Contrary to many state-of-the-art optical thermometers, the dyes show only moderate decrease of TADF intensity at increased temperatures. The dyes immobilized into a nearly oxygen impermeable poly(vinylidene chloride-co-acrylonitrile) matrix feature excellent temperature sensitivity in the investigated temperature range (278–323 K) with 1.4–3.7% K−1 change of TADF lifetime at 298 K. Indicator-loaded cell penetrating cationic nanoparticles are prepared for biological applications and feature sensitivities from 2.2 to 2.8% K−1 change of TADF lifetime at 298 K. Moreover, fiber-optic minisensors enable fast monitoring of temperature fluctuations in small sample volumes. The outcome of our work demonstrates the high potential of organic TADF emitters for optical temperature sensing and imaging.

Purely Organic Dyes with Thermally Activated Delayed Fluorescence—A Versatile Class of Indicators for Optical Temperature Sensing

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Topics In Fluorescence Spectroscopy