The degree of aortic occlusion in the setting of trauma alters the extent of acute kidney injury associated with mitochondrial preservation.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) is used to control noncompressible hemorrhage not addressed with traditional tourniquets. However, REBOA is associated with acute kidney injury (AKI) and subsequent mortality in severely injured trauma patients. Here, we investigated how the degree of aortic occlusion altered the extent of AKI in a porcine model. Female Yorkshire-cross swine (n = 16, 68.1 ± 0.7 kg) were anesthetized and had carotid and bilateral femoral arteries accessed for REBOA insertion and distal and proximal blood pressure monitoring. Through a laparotomy, a 6-cm liver laceration was performed and balloon inflation was performed in zone 1 of the aorta for 90 min, during which animals were randomized to target distal mean arterial pressures of 25 or 45 mmHg via balloon volume adjustment. Blood draws were taken at baseline, end of occlusion, and time of death, at which point renal tissues were harvested 6 h after balloon deflation for histological and molecular analyses. Renal blood flow was lower in the 25-mmHg group (48.5 ± 18.3 mL/min) than in the 45-mmHg group (177.9 ± 27.2 mL/min) during the occlusion phase, which recovered and was not different after balloon deflation. AKI was more severe in the 25-mmHg group, as evidenced by circulating creatinine, blood urea nitrogen, and urinary neutrophil gelatinase-associated lipocalin. The 25-mmHg group had increased tubular necrosis, lower renal citrate synthase activity, increased tissue and circulating syndecan-1, and elevated systemic inflammatory cytokines. The extent of renal ischemia-induced AKI is associated with the magnitude of mitochondrial biomass and systemic inflammation, highlighting potential mechanistic targets to combine with partial REBOA strategies to prevent AKI.NEW & NOTEWORTHY Large animal models of ischemia-reperfusion acute kidney injury (IR-AKI) are lacking. This report establishes a titratable IR-AKI model in swine in which a balloon catheter can be used to alter distal pressures experienced by the kidney, thus controlling renal blood flow. Lower blood flow results in greater renal dysfunction and structural damage, as well as lower mitochondrial biomass, elevated systemic inflammation, and vascular dysfunction.

Canagliflozin Mitigates Acute Kidney Injury Secondary to Resuscitative Endovascular Balloon Occlusion of the Aorta in a Porcine Model of Hemorrhagic Shock.

OBJECTIVE: We investigated the potential of acute canagliflozin administration to mitigate acute kidney injury (AKI) and attenuate deleterious pro-inflammatory cytokine release in a clinically relevant swine model of severe renal ischemia reperfusion injury (IRI) induced by hemorrhage and aortic occlusion. BACKGROUND: Long-term canagliflozin use attenuates renal function decline and reduces AKI in diabetes mellitus and heart failure patients. Whilst several reports indicate prophylactic SGLT2 inhibition prevents AKI in IRI, the efficacy of acute administration on IRI and inflammation is not known. METHODS: Female swine (n=16) underwent controlled hemorrhage of 25% blood volume, followed by 90 min of aortic occlusion at the level of the renal ostia (via Resuscitative Endovascular Balloon Occlusion of the Aorta). A single 300 mg dose of oral canagliflozin or vehicle (saline) was delivered 5 mins into aortic occlusion. Hemodynamic monitoring, markers of renal function (serum creatinine, blood urea nitrogen, proteinuria and urinary neutrophil gelatinase-associated lipocalin) and serum cytokine concentrations (including interleukins: IL-1RA, IL-6, IL-8, IL-10, IL-18; and Tumor necrosis factor alpha) were analyzed after IRI, and during a 6h critical care phase. RESULTS: Compared to controls, animals receiving canagliflozin had less severe AKI, improved creatinine clearance, reduced proteinuria, and significantly lower tubular damage as evidenced by histopathology and urinary NGAL. Furthermore, the pro-inflammatory cytokine IL-6 was markedly attenuated without reduction in anti-inflammatory cytokines (IL-1RA and IL-10). CONCLUSIONS: A single dose of canagliflozin administered shortly into ischemic insult mitigates AKI and attenuates harmful pro-inflammatory cytokine release following trauma or surgery. These findings suggest a potential novel therapeutic role for canagliflozin in mitigating the effects of renal IRI worthy of further investigation.

Second-site suppressor mutations reveal connection between the drug-binding pocket and nucleotide-binding domain 1 of human P-glycoprotein (ABCB1).

Human P-glycoprotein (P-gp) or ABCB1 is overexpressed in many cancers and has been implicated in altering the bioavailability of chemotherapeutic drugs due to their efflux, resulting in the development of chemoresistance. To elucidate the mechanistic aspects and structure-function relationships of P-gp, we previously utilized a tyrosine (Y)-enriched P-gp mutant (15Y) and demonstrated that at least 15 conserved residues in the drug-binding pocket of P-gp are responsible for optimal substrate interaction and transport. To further understand the role of these 15 residues, two new mutants were generated, namely 6Y with the substitution of six residues (F72, F303, I306, F314, F336 and L339) with Y in transmembrane domain (TMD) 1 and 9Y with nine substitutions (F732, F759, F770, F938, F942, M949, L975, F983 and F994) in TMD2. Although both the mutants were expressed at normal levels at the cell surface, the 6Y mutant failed to transport all the tested substrates except Bodipy-verapamil, whereas the 9Y mutant effluxed all tested substrates in a manner very similar to that of the wild-type protein. Further mutational analysis revealed that two second-site mutations, one in intracellular helix (ICH) 4 (F916Y) and one in the Q loop of nucleotide-binding domain (NBD) 1 (F480Y) restored the transport function of 6Y. Additional biochemical data and comparative molecular dynamics simulations of the 6Y and 6Y+F916Y mutant indicate that the Q-loop of NBD1 of P-gp communicates with the substrate-binding sites in the transmembrane region through ICH4. This is the first evidence for the existence of second-site suppressors in human P-gp that allow recovery of the loss of transport function caused by primary mutations. Further study of such mutations could facilitate mapping of the communication pathway between the substrate-binding pocket and the NBDs of P-gp and possibly other ABC drug transporters.

Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein.

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

Evidence for the Interaction of A3 Adenosine Receptor Agonists at the Drug-Binding Site(s) of Human P-glycoprotein (ABCB1).

P-glycoprotein (P-gp) is a multidrug transporter that is expressed on the luminal surface of epithelial cells in the kidney, intestine, bile-canalicular membrane in the liver, blood-brain barrier, and adrenal gland. This transporter uses energy of ATP hydrolysis to efflux from cells a variety of structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs. In this regard, understanding the interaction with P-gp of drug entities in development is important and highly recommended in current US Food and Drug Administration guidelines. Here we tested the P-gp interaction of some A3 adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Biochemical assays of the ATPase activity of P-gp and by photolabeling P-gp with its transport substrate [125I]-iodoarylazidoprazosin led to the identification of rigidified (N)-methanocarba nucleosides (i.e., compound 3 as a stimulator and compound 8 as a partial inhibitor of P-gp ATPase activity). Compound 8 significantly inhibited boron-dipyrromethene (BODIPY)-verapamil transport mediated by human P-gp (IC50 2.4 ± 0.6 µM); however, the BODIPY-conjugated derivative of 8 (compound 24) was not transported by P-gp. In silico docking of compounds 3 and 8 was performed using the recently solved atomic structure of paclitaxel (Taxol)-bound human P-gp. Molecular modeling studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as Taxol. Thus, this study indicates that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. SIGNIFICANCE STATEMENT: Certain A3 adenosine receptor agonists are being developed for the treatment of chronic diseases. The goal of this study was to test the interaction of these agonists with the human multidrug resistance-linked transporter P-glycoprotein (P-gp). ATPase and photolabeling assays demonstrated that compounds with rigidified (N)-methanocarba nucleosides inhibit the activity of P-gp; however, a fluorescent derivative of one of the compounds was not transported by P-gp. Furthermore, molecular docking studies revealed that the binding site for these compounds overlaps with the site for paclitaxel in the drug-binding pocket. These results suggest that nucleoside derivatives, depending on structural functionalization, can modulate the function of P-gp.

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions.

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10-40 nm range. Similarly, a 30-150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment.

Real-Time Measurements of Oral Mucosal Carbon Dioxide (POMCO2) Reveals an Inverse Correlation With Blood Pressure in a Porcine Model of Coagulopathic Junctional Hemorrhage.

INTRODUCTION: Shock states that occur during, for example, profound hemorrhage can cause global tissue hypoperfusion leading to organ failure. There is an unmet need for a reliable marker of tissue perfusion during hemorrhage that can be followed longitudinally. Herein, we investigated whether longitudinal POMCO2 tracks changes in hemodynamics in a swine model of coagulopathic uncontrolled junctional hemorrhage. MATERIALS AND METHODS: Female Yorkshire-crossbreed swine (n = 7, 68.1 ± 0.7 kg) were anesthetized and instrumented for continuous measurement of mean arterial pressure (MAP). Coagulopathy was induced by the exchange of 50 to 60% of blood volume with 6% Hetastarch over 30 minutes to target a hematocrit of <15%. A 4.5-mm arteriotomy was made in the right common femoral artery with 30 seconds of free bleeding. POMCO2 was continuously measured from baseline through hemodilution, hemorrhage, and a subsequent 3-h intensive care unit period. Rotational thromboelastometry and blood gases were measured. RESULTS: POMCO2 and MAP showed no significant changes during the hemodilution phase of the experiment, which produced coagulopathy evidenced by prolonged clot formation times. However, POMCO2 increased because of the uncontrolled hemorrhage by 11.3 ± 3.1 mmHg and was inversely correlated with the drop (17.9 ± 5.9 mmHg) in MAP (Y = -0.4122*X + 2.649, P = .02, r2 = 0.686). In contrast, lactate did not significantly correlate with the changes in MAP (P = .35) or POMCO2 (P = .37). CONCLUSIONS: Despite the logical appeal of measuring noninvasive tissue CO2 measurement as a surrogate for gastrointestinal perfusion, prior studies have only reported snapshots of this readout. The present investigation shows real-time longitudinal measurement of POMCO2 to confirm that MAP inversely correlates to POMCO2 in the face of coagulopathy. The simplicity of measuring POMCO2 in real time can provide an additional practical option for military or civilian medics to monitor trends in hypoperfusion during hemorrhagic shock.

Interaction of a Homologous Series of Amphiphiles with P-glycoprotein in a Membrane Environment-Contributions of Polar and Non-Polar Interactions.

The transport of drugs by efflux transporters in biomembranes limits their bioavailability and is a major determinant of drug resistance development by cancer cells and pathogens. A large number of chemically dissimilar drugs are transported, and despite extensive studies, the molecular determinants of substrate specificity are still not well understood. In this work, we explore the role of polar and non-polar interactions on the interaction of a homologous series of fluorescent amphiphiles with the efflux transporter P-glycoprotein. The interaction of the amphiphiles with P-glycoprotein is evaluated through effects on ATPase activity, efficiency in inhibition of [125I]-IAAP binding, and partition to the whole native membranes containing the transporter. The results were complemented with partition to model membranes with a representative lipid composition, and details on the interactions established were obtained from MD simulations. We show that when the total concentration of amphiphile is considered, the binding parameters obtained are apparent and do not reflect the affinity for P-gp. A new formalism is proposed that includes sequestration of the amphiphiles in the lipid bilayer and the possible binding of several molecules in P-gp's substrate-binding pocket. The intrinsic binding affinity thus obtained is essentially independent of amphiphile hydrophobicity, highlighting the importance of polar interactions. An increase in the lipophilicity and amphiphilicity led to a more efficient association with the lipid bilayer, which maintains the non-polar groups of the amphiphiles in the bilayer, while the polar groups interact with P-gp's binding pocket. The presence of several amphiphiles in this orientation is proposed as a mechanism for inhibition of P-pg function.

Evaluation of novel hemostatic agents in a coagulopathic swine model of junctional hemorrhage.

BACKGROUND: Hemostatic dressings are used extensively in both military and civilian trauma to control lethal noncompressible hemorrhage. The ideal topical hemostatic agent would provide reliable hemostasis in patients with profound acidosis, coagulopathy, and shock. This study aimed to compare next-generation hemostatic agents against the current military standard in a translational swine model of vascular injury and coagulopathy. METHODS: Female Yorkshire swine were randomized to eight groups (total n = 63; control n = 14, per group n = 7) of hemostatic agents and included: QuikClot Combat Gauze (Teleflex, Morrisville, NC), which served as the control; BloodSTOP IX (LifeScience Plus, Mountain View, CA); Celox Rapid (Medtrade Product, Crewe, United Kingdom); ChitoSAM 100 (Sam Medical, Tualatin, OR); EVARREST Fibrin Sealant Patch (Ethicon, Raritan, NJ); TAC Wrapping Gauze (H&H Medical, Williamsburg, VA); ChitoGauze XR Pro (Tricol Biomedical, Portland, OR); and X-Stat 30 (RevMedX, Wilsonville, OR). Hemodilution via exchange transfusion of 6% hetastarch was performed to induce acidosis and coagulopathy. An arteriotomy was created, allowing 30 seconds of free bleeding followed by application of the hemostatic agent and compression via an external compression device. A total of three applications were allowed for continued/recurrent bleeding. All blood loss was collected, and hemostatic agents were weighed to calculate blood volume loss. Following a 180-minute observation period, angiography was completed to evaluate for technical complication and distal perfusion of the limb. Finally, the limb was ranged five times to assess for rebleeding and clot stability. RESULTS: All swine were confirmed coagulopathic with rotational thromboelastography and acidotic (pH 7.2 ± 0.02). BloodSTOP IX allowed a significant increase in blood loss and number of applications required to obtain hemostasis compared with all other groups. BloodSTOP IX demonstrated a decreased survival rate (29%, p = 0.02). All mortalities were directly attributed to exsanguination as a result of device failure. In surviving animals, there was no difference in extravasation. BloodSTOP IX had an increased rebleeding rate after ranging compared with QuikClot Combat Gauze ( p = 0.007). CONCLUSION: Most novel hemostatic agents demonstrated comparable efficacy compared with the currently military standard hemostatic dressing, CG.

Interaction of A3 adenosine receptor ligands with the human multidrug transporter ABCG2.

Various adenosine receptor nucleoside-like ligands were found to modulate ATP hydrolysis by the multidrug transporter ABCG2. Both ribose-containing and rigidified (N)-methanocarba nucleosides (C2-, N6- and 5'-modified), as well as adenines (C2-, N6-, and deaza modified), were included. 57 compounds out of 63 tested either stimulated (50) or inhibited (7) basal ATPase activity. Structure-activity analysis showed a separation of adenosine receptor and ABCG2 activities. The 7-deaza modification had favorable effects in both (N)-methanocarba nucleosides and adenines. Adenine 37c (MRS7608) and (N)-methanocarba 7-deaza-5'-ethyl ester 60 (MRS7343) were found to be potent stimulators of ABCG2 ATPase activity with EC50 values of 13.2 ± 1.7 and 13.2 ± 2.2 nM, respectively. Both had affinity in the micromolar range for A3 adenosine receptor and lacked the 5'-amide agonist-enabling group (37c was reported as a weak A3 antagonist, Ki 6.82 µM). Compound 60 significantly inhibited ABCG2 substrate transport (IC50 0.44 µM). Docking simulations predicted the interaction of 60 with 21 residues in the drug-binding pocket of ABCG2.

Characterization of the Lipidome and Biophysical Properties of Membranes from High Five Insect Cells Expressing Mouse P-Glycoprotein.

The lipid composition of biomembranes influences the properties of the lipid bilayer and that of the proteins. In this study, the lipidome and the lipid/protein ratio of membranes from High Five™ insect cells overexpressing mouse P-glycoprotein was characterized. This provides a better understanding of the lipid environment in which P-glycoprotein is embedded, and thus of its functional and structural properties. The relative abundance of the distinct phospholipid classes and their acyl chain composition was characterized. A mass ratio of 0.57 ± 0.11 phospholipids to protein was obtained. Phosphatidylethanolamines are the most abundant phospholipids, followed by phosphatidylcholines. Membranes are also enriched in negatively charged lipids (phosphatidylserines, phosphatidylinositols and phosphatidylglycerols), and contain small amounts of sphingomyelins, ceramides and monoglycosilatedceramides. The most abundant acyl chains are monounsaturated, with significant amounts of saturated chains. The characterization of the phospholipids by HPLC-MS allowed identification of the combination of acyl chains, with palmitoyl-oleoyl being the most representative for all major phospholipid classes except for phosphatidylserines, which are mostly saturated. A mixture of POPE:POPC:POPS in the ratio 45:35:20 is proposed for the preparation of simple representative model membranes. The adequacy of the model membranes was further evaluated by characterizing their surface potential and fluidity.

Comprehensive Synthesis of Amino Acid-Derived Thiazole Peptidomimetic Analogues to Understand the Enigmatic Drug/Substrate-Binding Site of P-Glycoprotein.

A novel set of 64 analogues based on our lead compound 1 was designed and synthesized with an initial objective of understanding the structural requirements of ligands binding to a highly perplexing substrate-binding site of P-glycoprotein (P-gp) and their effect on modulating the ATPase function of the efflux pump. Compound 1, a stimulator of P-gp ATPase activity, was transformed to ATPase inhibitory compounds 39, 53, and 109. The ATPase inhibition by these compounds was predominantly contributed by the presence of a cyclohexyl group in lieu of the 2-aminobenzophenone moiety of 1. The 4,4-difluorocyclohexyl analogues, 53 and 109, inhibited the photolabeling by [125I]-IAAP, with IC50 values of 0.1 and 0.76 µM, respectively. Selected compounds were shown to reverse paclitaxel resistance in HEK293 cells overexpressing P-gp and were selective toward P-gp over CYP3A4. Induced-fit docking highlighted a plausible binding pattern of inhibitory compounds in the putative-binding pocket of P-gp. The current study underscores the stringent requirement by P-gp to bind to chemically similar molecules.

Enhancement of the Chemiluminescence Response of Enzymatic Reactions by Plasmonic Surfaces for Biosensing Applications.

We report the enhancement of chemiluminescence response of horseradish peroxidase (HRP) in bioassays by plasmonic surfaces, which are comprised of (i) silver island films (SIFs) and (ii) metal thin films (silver, gold, copper, and nickel, 1 nm thick) deposited onto glass slides. A model bioassay, based on the interactions of avidin-modified HRP with a monolayer of biotinylated poly(ethylene-glycol)-amine, was employed to evaluate the ability of plasmonic surfaces to enhance chemiluminescence response of HRP. Chemiluminescence response of HRP in model bioassays were increased up to ~3.7-fold as compared to the control samples (i.e. glass slides without plasmonic nanoparticles), where the largest enhancement of the chemiluminescence response was observed on SIFs with high loading. These findings allowed us to demonstrate the use of SIFs (high loading) for the detection of a biologically relevant target protein (glial fibrillary acidic protein or GFAP), where the chemiluminescence response of the standard bioassay for GFAP was enhanced up to ~50% as compared to bioassay on glass slides.

Enhancement of Colorimetric Response of Enzymatic Reactions by Thermally Evaporated Plasmonic Thin Films: Application to Glial Fibrillary Acidic Protein.

We report the enhancement of the colorimetric response of horseradish peroxidase (HRP) and alkaline phosphatase (AP) in bioassays by thermally evaporated silver, gold, copper and nickel thin films. In this regard, a model bioassay based on biotin-avidin interactions was employed. Biotin groups and enzymes were introduced to all surfaces using a biotinylated linker molecule and avidin, respectively. The colorimetric response of HRP in the model bioassay carried out on the plasmonic thin films were up to 4.4-fold larger as compared to control samples (i.e., no plasmonic thin films), where the largest enhancement of colorimetric response was observed on silver thin films. The colorimetric response of AP on plasmonic thin films was found to be similar to those observed on control samples, which was attributed to the loss of enzymes from the surface during the bioassay steps. The extent of enzymes immobilized on to plasmonic thin films was found to affect the colorimetric response of the model bioassay. These findings allowed us to demonstrate the use of silver thin films for the detection of glial fibrillary acidic protein (GFAP), where the colorimetric response of the standard bioassays for GFAP was enhanced up to 67% as compared to bioassays on glass slides.

Metal-Enhanced Fluorescence from Silver Nanowires with High Aspect Ratio on Glass Slides for Biosensing Applications.

High enhancement of fluorescence emission, improved fluorophore photostability, and significant reduction of fluorescence lifetimes have been obtained from high aspect ratio (>100) silver (Ag) nanowires. These quantities are found to depend on the surface loading of Ag nanowires on glass slides, where the enhancement of fluorescence emission increases with the density of nanowires. The surface loading dependence was attributed to the creation of intense electric fields around the network of Ag nanowires and to the coupling of fluorophore excited states that takes place efficiently at a distance of 10 nm from the surface of nanowires, which was confirmed by theoretical calculations. The enhancement of fluorescence emission of fluorescein isothiocyanate (FITC) was assessed by fluorescence spectroscopy and fluorescence-lifetime imaging microscopy (FLIM) to demonstrate the potential of high aspect ratio Ag nanowires. Fluorescence enhancement factors exceeding 14 were observed on Ag nanowires with high loading by FLIM. The photostability of FITC was the highest on nanowires with medium loading under continuous laser excitation for 10 min because of the significant reduction in the fluorescence lifetime of FITC on these surfaces. These results clearly demonstrate the potential of Ag nanowires in metal-enhanced fluorescence-based applications of biosensing on planar surfaces and cellular imaging.

Immobilization of enzymes to silver island films for enhanced enzymatic activity.

HYPOTHESIS: The performance of the enzyme-based biosensors depends on the enzymatic activity and the use of an appropriate technique for immobilization of enzymes. The incorporation of silver island films (SIFs) into the enzyme-based biosensors is expected to enhance the enzymatic activity and to increase the detectability of analytes of interest. EXPERIMENTS: Two enzymes, ß-galactosidase (ß-Gal) and alkaline phosphatase (AP) were immobilized onto SIFs using the interactions of avidin-modified enzymes with (i) a monolayer of biotinylated bovine serum albumin (b-BSA) and/or (ii) a monolayer of biotinylated poly(ethylene-glycol)-amine (BEA molecular weight: 550-10,000Da). To confirm the effect of SIFs on enzymatic activity, two control surfaces (no silver) were also employed. FINDINGS: No enhancement in enzymatic activity for ß-Gal on all SIFs was observed, which was attributed to the inhibition of ß-Gal activity due to direct interactions of ß-Gal with SIFs. The AP activity on SIFs with BEA was significantly larger than that observed on SIFs with b-BSA, where a 300% increase in AP activity was observed as compared to control surfaces. These observations suggest that SIFs can significantly enhance AP activity, which could help improve the detection limits of ELISAs and immunoassays that employ AP.

Enhancement of enzymatic colorimetric response by silver island films on high throughput screening microplates.

In this study, we report the use of an enzyme-based hybrid platform, which is comprised of silver island films, enzymes (HRP and AP) and high-throughput screening (HTS) microplates, to enhance the colorimetric response of enzymatic reactions. The hybrid platform was designed in a two-step process: (i) deposition of SIFs onto HTS microplates with low, medium, and high loading (refers to the extent of the surface plasmon resonance peak of SIFs at 460 nm) using Tollen's reaction scheme; and (ii) attachment of b-BSA or BEA as linkers for the immobilization of enzymes. The presence of SIFs within the wells of the HTS microplates was confirmed using an optical spectrophotometer and real-color photography. Control experiments, where SIFs were omitted from the surfaces were carried out to confirm the effect of SIFs on the enzymatic colorimetric response. Significant colorimetric signal enhancement was observed for HRP or AP on SIFs (high loading) deposited HTS microplates using b-BSA (up to ~3-fold for AP and ~6-fold HRP) or BEA (up to ~7-fold for both HRP and AP), as compared to our control samples. The observed increase in colorimetric response can be attributed to the nature of BEA, which exposes surface-bound enzymes to the substrate present in bulk more efficiently than b-BSA. This study proves that SIFs can serve as a valuable tool to improve the signal output of existing bioassays carried out in HTS microplates, which can be applicable to the field biosensors and plasmonics.

De-crystallization of Uric Acid Crystals in Synovial Fluid Using Gold Colloids and Microwave Heating.

In this study, we demonstrated a unique application of our Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC) technique for the de-crystallization of uric acid crystals, which causes gout in humans when monosodium urate crystals accumulate in the synovial fluid found in the joints of bones. Given the shortcomings of the existing treatments for gout, we investigated whether the MA-MAEC technique can offer an alternative solution to the treatment of gout. Our technique is based on the use of metal nanoparticles (i.e., gold colloids) with low microwave heating to accelerate the de-crystallization process. In this regard, we employed a two-step process; (i) crystallization of uric acid on glass slides, which act as a solid platform to mimic a bone, (ii) de-crystallization of uric acid crystals on glass slides with the addition of gold colloids and low power microwave heating, which act as "nano-bullets" when microwave heated in a solution. We observed that the size and number of the uric acid crystals were reduced by >60% within 10 minutes of low power microwave heating. In addition, the use of gold colloids without microwave heating (i.e. control experiment) did not result in the de-crystallization of the uric acid crystals, which proves the utility of our MA-MAEC technique in the de-crystallization of uric acid.

Plasmon-Enhanced Enzymatic Reactions 2:Optimization of Enzyme Activity by Surface Modification of Silver Island Films with Biotin-Poly (Ethylene-glycol)-Amine.

Surface modification of silver island films (SIFs) was carried out with Biotin-Poly (Ethylene-glycol)-Amine (BEA), which acts as a cross-linker between the silver surface and horse radish peroxidase (HRP) enzyme for optimum plasmon-enhanced enzymatic activity. SIFs-deposited blank glass slides and SIFs-deposited 3-Aminopropyltriethoxysilane(APTES)-coated glass slides were used as our plasmonic surfaces.In this regard, three different extent of loading of SIFs were also prepared (low, medium and high) on APTES-coated glass slides. Streptavidin-linked HRP enzyme was attached to SIFs-deposited blank glass slides and SIFs-deposited APTES-coated glass slides through the well-known biotin-streptavidin interactions. The characterization of these surfaces was done using optical absorption spectroscopy. The loading of SIFs on glass slides was observed to have significant effect on the efficiency of plasmon-enhanced enzymatic activity, where an enhancement of 200% in the enzymatic activity was observed when compared to our previously used strategies for enzyme immobilization in our preceding work[1]. In addition, SIFs-deposited on APTES-coated glass slides were found to be re-usable for plasmon-enhanced enzymatic reactions unlike SIFs deposited on to blank glass slides.

Surface modification of plasmonic nanostructured materials with thiolated oligonucleotides in 10 seconds using selective microwave heating.

This study demonstrates the proof-of-principle of rapid surface modification of plasmonic nanostructured materials with oligonucleotides using low power microwave heating. Due to their interesting optical and electronic properties, silver nanoparticle films (SNFs, 2 nm thick) deposited onto glass slides were used as the model plasmonic nanostructured materials. Rapid surface modification of SNFs with oligonucleotides was carried out using two strategies (1) Strategy 1: for ss-oligonucleotides, surface hybridization and (2) Strategy 2: for ds-oligonucleotides, solution hybridization), where the samples were exposed to 10, 15, 30 and 60 seconds microwave heating. To assess the efficacy of our new rapid surface modification technique, identical experiments carried out without the microwave heating (i.e., conventional method), which requires 24 hours for the completion of the identical steps. It was found that SNFs can be modified with ss- and ds-oligonucleotides in 10 seconds, which typically requires several hours of incubation time for the chemisorption of thiol groups on to the planar metal surface using conventional techniques.