Nonperturbative Fluorogenic Labeling of Immunophilins Enables the Wash-free Detection of Immunosuppressants.

Immunosuppressants are clinically approved drugs to treat the potential rejection of transplanted organs and require frequent monitoring due to their narrow therapeutic window. Immunophilins are small proteins that bind immunosuppressants with high affinity, yet there are no examples of fluorogenic immunophilins and their potential application as optical biosensors for immunosuppressive drugs in clinical biosamples. In the present work, we designed novel diazonium BODIPY salts for the site-specific labeling of tyrosine residues in peptides via solid-phase synthesis as well as for late-stage functionalization of whole recombinant proteins. After the optimization of a straightforward one-step labeling procedure for immunophilins PPIA and FKBP12, we demonstrated the application of a fluorogenic analogue of FKBP12 for the selective detection of the immunosuppressant drug tacrolimus, including experiments in urine samples from patients with functioning renal transplants. This chemical methodology opens new avenues to rationally design wash-free immunophilin-based biosensors for rapid therapeutic drug monitoring.

Energy Landscape of Negatively Charged BSA Adsorbed on a Negatively Charged Silica Surface.

We study the energy landscape of the negatively charged protein bovine serum albumin adsorbed on a negatively charged silica surface at pH 7. We use fully atomistic molecular dynamics (MD) and steered MD (SMD) to probe the energy of adsorption and the pathway for the surface diffusion of the protein and its associated activation energy. We find an adsorption energy ∼1.2 eV, which implies that adsorption is irreversible even on experimental time scales of hours. In contrast, the activation energy for surface diffusion is ∼0.4 eV so that it is observable on the MD simulation time scale of 100 ns. This analysis paves the way for a more detailed understanding of how a protein layer forms on biomaterial surfaces, even when the protein and surface share the same electrical polarity.

Quantitative interpretation of PAMAM dendrimers adsorption on silica surface.

Understanding the dendrimer-solid support interaction is of great importance for dendrimer-based drug delivery system design. The maximum surface coverage on a hydrophilic silica surface was determined using the quartz crystal microbalance with dissipation monitoring (QCM-D) and multi-parametric surface plasmon resonance (MP-SPR) methods: the adsorption process depends on ionic strength and pH of solutions. The effectiveness of G6 adsorption is mainly determined by the range of electrostatic inter-dendrimer interactions and dendrimer-silica surface interactions. Changes in ionic strength have a strong effect on the binding affinity of dendrimers to the surface. The trends in the binding affinity and the surface saturation amount correspond well with the degree of change of protonation of the adsorbed molecules. The development of new research techniques makes it possible to attain a more profound understanding of the self-assembling behaviour of dendrimers. The comparison of QCM-D and MP-SPR allowed the estimation that the dendrimer film contains approximately 70% water. These results indicate that 6th generation PAMAM dendrimers form very hydrated films on silica surfaces. In this case the number of water molecules associated per dendrimer molecule varied from 10,450 to 9,200. The hydration of dendrimer films seems to be a crucial aspect of their implementation. This data confirmed that dendrimers are very promising candidates for many biological applications.

Bovine Serum Albumin Adsorption at a Silica Surface Explored by Simulation and Experiment.

Molecular details of BSA adsorption on a silica surface are revealed by fully atomistic molecular dynamics (MD) simulations (with a 0.5 µs trajectory), supported by dynamic light scattering (DLS), zeta potential, multiparametric surface plasmon resonance (MP-SPR), and contact angle experiments. The experimental and theoretical methods complement one another and lead to a wider understanding of the mechanism of BSA adsorption across a range of pH 3-9. The MD results show how the negatively charged BSA at pH7 adsorbs to the negatively charged silica surface, and reveal a unique orientation with preserved secondary and tertiary structure. The experiments then show that the protein forms complete monolayers at ∼ pH6, just above the protein's isoelectric point (pH5.1). The surface contact angle is maximum when it is completely coated with protein, and the hydrophobicity of the surface is understood in terms of the simulated protein conformation. The adsorption behavior at higher pH > 6 is also consistently interpreted using the MD picture; both the contact angle and the adsorbed protein mass density decrease with increasing pH, in line with the increasing magnitude of negative charge on both the protein and the surface. At lower pH < 5 the protein starts to unfold, and the adsorbed mass dramatically decreases. The comprehensive picture that emerges for the formation of oriented protein films with preserved native conformation will help guide efforts to create functional films for new technologies.