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The permeation and translational diffusion of antibodies through the porous matrix of hydrogel materials is of fundamental relevance for many biological systems in living nature, but equally important in medical and technological applications, such as implanted drug release systems and biosensors. In this respect the diffusion of fluorophore-labeled protein immunoglobulin G (IgG) in micrometer thick, grafted hydrogel layers based on thermoresponsive poly­(N-isopropyl­acrylamide) (pNiPAAm) is studied here by fluorescence correlation spectroscopy (FCS). The pore size of the gel gradually changes with its swelling state, which is controlled by the cross-link density of the network, temperature, and pH value of the surrounding medium. Notably, IgG permeation in these hydrogel layers exhibits a much more complex dependence on these factors. This rich variability of IgG permeation is attributed to the varying balance of protein interactions with the polymer network through electrostatics, controlled pH-dependent protein ionization, excluded volume repulsion, and hydrophobic attraction. A combined analysis of the fluorescence intensity profiles and the dynamics monitored by FCS allows us to quantify the thermodynamically controlled partitioning of IgG as well as the slowdown of its diffusion. Contrary to the complex behavior of the permeation, the diffusion slowdown seems to be a universal function of polymer volume fraction, which is rather robust with respect to temperature or pH changes. The presented findings suggest a model approach to explore the synergy between crowding and thermodynamics with respect to the controlled protein transport in pNiPAAm-based hydrogels.

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 “CRC genes.” These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology.

The preparation and investigation of a free-standing membrane made from a composite of thermoresponsive poly(N-isopropylacrylamide) (pNIPAAm) and polystyrene nanoparticles (PS NP) with temperature-controlled permeability is reported. The method exploits the light-induced crosslinking of the photo-reactive pNIPAAm-based polymer and mechanical reinforcement of the membrane structure by the polystyrene nanoparticles. About micrometer thick layers were either directly attached to a gold surface or prepared as free-standing layers spanning over arrays of microfluidic channels with a width of about hundred microns by using template stripping. Diffusion of liquid medium, low molecular weight molecules, and large molecular weight proteins contained in blood through the composite membrane was observed with combined surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The swelling ratio, permeability, and nonspecific sorption to these composite membranes were investigated by SPR and OWS as a function of molecular weight of analyte, loading of PS NP in the composite film, and temperature. The authors show successful preparation of a defect-free membrane structure that acts as a thermoresponsive filter with nanoscale pores spanning over an area of several square millimeters. This membrane can be reversibly switched to block or allow the diffusion of low mass molecules to the sensor surface by temperature-triggered swelling and collapsing of the hydrogel component. Blocking of diffusion and low unspecific sorption of proteins contained in blood serum is observed. These features make this platform interesting for potential future applications in continuous monitoring biosensors for the analysis of low molecular weight drug analytes or for advanced cell-on-chip microfluidic studies.

Surface plasmon field-enhanced fluorescence is reported for the readout of a heterogeneous assay that utilizes low affinity split aptamer ligands. Weak affinity ligands that reversibly interact with target analytes hold potential for facile implementation in continuous monitoring biosensor systems. This functionality is not possible without the regeneration of more commonly used assays relying on high affinity ligands and end-point measurement. In fluorescence-based sensors, the use of low affinity ligands allows avoiding this step but it imposes a challenge associated with the weak optical response to the specific capture of the target analyte which is also often masked by a strong background. The coupling of fluorophore labels with a confined field of surface plasmons is reported for strong amplification of the fluorescence signal emitted from the sensor surface and its efficient discrimination from the background. This optical scheme is demonstrated for time-resolved analysis of chosen model analytes - adenoside and adenosine triphosphate - with a split aptamer that exhibits an equilibrium affinity binding constant between 0.73 and 1.35 mM. The developed biosensor enables rapid and specific discrimination of target analyte concentration changes from low μM to mM in buffer as well as in 10% serum.

Surface plasmon field-enhanced fluorescence energy transfer is employed for sensitive optical readout of a reversible hairpin aptamer assay that is suitable for continuous monitoring of low-molecular-weight chemical analytes. A hairpin aptamer specific to adenosine and adenosine triphosphate with Alexa Fluor 647 fluorophore attached to its 5′ end was anchored via 3′ end thiol to a gold thin film. Molecular spacers were used to control the distance of the fluorophore from the surface in the aptamer “off” and “on” states. The specific binding of the target analyte changes the aptamer conformation, which alters the distance of the fluorophore from the gold surface and translates to variations in the detected fluorescence intensity. The plasmonically mediated fluorescence signal increases the measured signal-to-noise ratio and allows for real-time observation of the analyte binding. Theoretical as well as experimental study of the optical signal dependence on fluorophore orientation, design of spacers, and angular distribution of collected light is presented for rational design of the assay. The detected sensor signal increased by a factor as large as 23 upon switching the aptamer from the “off” to “on” state due to the hairpin opening associated with the specific capture of target analyte.

Free-standing hydrogel membrane with dynamically controlled permeability for lab-on-chip applications, Biointerphases, submitted.