Aqueous photolysis of naproxen exposed to UV and natural sunlight: Formation of excited triplet and photosensitizing product.

Photochemical transformation is an important attenuation process for the non-steroidal anti-inflammatory drug naproxen (NPX) in both engineered and natural waters. Herein, we investigated the photolysis of NPX in aqueous solution exposed to both ultraviolet (UV, 254 nm) and natural sunlight irradiation. Results show that N2 purging significantly promoted NPX photolysis under UV irradiation, suggesting the formation of excited triplet state (3NPX*) as a critical transient. This inference was supported by benzophenone photosensitization and transient absorption spectra. Sunlight quantum yield of NPX was only one fourteenth of that under UV irradiation, suggesting the wavelength-dependence of NPX photochemistry. 3NPX* formed upon irradiation of NPX underwent photodecarboxylation leading to the formation of 2-(1-hydroxyethyl)-6-methoxynaphthalene (2HE6MN), 2-(1-hydroperoxyethyl)-6-methoxynaphthalene (2HPE6MN), and 2-acetyl-6-methoxynaphthalene (2A6MN). Notably, the conjugation and spin-orbit coupling effects of carbonyl make 2A6MN a potent triplet sensitizer, therefore promoting the photodegradation of the parent NPX. In hospital wastewater, the photolysis of NPX was influenced because the photoproduct 2A6MN and wastewater components could competitively absorb photons. Bioluminescence inhibition assay demonstrated that photoproducts of NPX exhibited higher toxicity than the parent compound. Results of this study provide new insights into the photochemical behaviors of NPX during UV treatment and in sunlit surface waters.

Synergistic bactericidal effects of pairs of photosensitizer molecules activated by ultraviolet A light against bacteria in plasma.

BACKGROUND: The current approach to reducing bacterial contamination in blood transfusion products is through detection or pathogen reduction methods, some of which utilize ultraviolet (UV) light photosensitizers. A small number of photosensitizers are being used as single agents in combination with UV light, but their efficacy can be limited against some pathogens. Benzophenone (BP) and vitamins B1, B6, and K3 have been identified as effective UVA photosensitizers for inactivation of bacteria. We evaluated whether combining pairs of photosensitizers in this group would have synergistic bactericidal effects on Gram-negative and Gram-positive bacteria. STUDY DESIGN AND METHODS: Bacteria species of Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Klebsiella pneumoniae were mixed with 0 to 100 mM concentrations of photosensitizers and exposed to UVA irradiation at 18 J/cm2 to assess their bactericidal effects. RESULTS: Single photosensitizers irradiated with UVA produced a range of bactericidal activity. When combined in pairs, all demonstrated some synergistic bactericidal effects with up to 4-log reduction above the sum of activities of individual molecules in the pair against bacteria in plasma. Photosensitizer pairs with BP had the highest synergism across all bacteria. With vitamin K3 in the pair, synergism was evident for Gram-positive but not for Gram-negative bacteria. Vitamin B1 and vitamin B6 had the least synergism. These results indicate that a combination approach with multiple photosensitizers may extend effectiveness of pathogen reduction in plasma. CONCLUSIONS: Combining photosensitizers in pathogen reduction methods could improve bactericidal efficacy and lead to use of lower concentrations of photosensitizers to reduce toxicities and unwanted side effects.

Sodium cationization can disrupt the intramolecular hydrogen bond that mediates the sunscreen activity of oxybenzone.

A key decay pathway by which organic sunscreen molecules dissipate harmful UV energy involves excited-state hydrogen atom transfer between proximal enol and keto functional groups. Structural modifications of this molecular architecture have the potential to block ultrafast decay processes, and hence promote direct excited-state molecular dissociation, profoundly affecting the efficiency of an organic sunscreen. Herein, we investigate the binding of alkali metal cations to a prototype organic sunscreen molecule, oxybenzone, using IR characterization. Mass-selective IR action spectroscopy was conducted at the free electron laser for infrared experiments, FELIX (600-1800 cm-1), on complexes of Na+, K+ and Rb+ bound to oxybenzone. The IR spectra reveal that K+ and Rb+ adopt binding positions away from the key OH intermolecular hydrogen bond, while the smaller Na+ cation binds directly between the keto and enol oxygens, thus breaking the intramolecular hydrogen bond. UV laser photodissociation spectroscopy was also performed on the series of complexes, with the Na+ complex displaying a distinctive electronic spectrum compared to those of K+ and Rb+, in line with the IR spectroscopy results. TD-DFT calculations reveal that the origin of the changes in the electronic spectra can be linked to rupture of the intramolecular bond in the sodium cationized complex. The implications of our results for the performance of sunscreens in mixtures and environments with high concentrations of metal cations are discussed.

Identification of SUMO Binding Proteins Enriched after Covalent Photo-Cross-Linking.

Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a rewiring of protein-protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO; however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-cross-linker positioned next to the SIM binding groove. Following photoinduced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes was corroborated by the presence of many established SUMO interacting proteins and the high percentage (>90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional noncovalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent cross-links for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.

Benzophenone Photoreactivity in a Lipid Bilayer To Probe Peptide/Membrane Interactions: Simple System, Complex Information.

Affinity photo-cross-linking coupled to mass spectrometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ9), 18:1 (trans Δ9) PG, and DLoPG (18:2 cis Δ9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: C═C double bond formation on saturated fatty acids, covalent adducts formation via classical C-C bond, and Paternò-Büchi oxetane formation followed or not by fragmentation (retro-Paternò-Büchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction.

Degradation of benzophenone-4 in a UV/chlorine disinfection process: Mechanism and toxicity evaluation.

This study investigated the degradation of benzophenone-4 (BP-4) in a UV/chlorine disinfection process, with chlorination and UV disinfection as comparisons. With a degradation efficiency of 80% after 10 s, the UV/chlorine process significantly enhanced the degradation of BP-4. However, a rebound of 36% of the initial concentration was observed in the UV/chlorine process ([free active chlorine (FAC)]0:[BP-4]0 = 1:1, pH = 7). The same tendency appeared under the addition of alkalinity, Cl-, and humic acid (HA). This work interpreted this interesting kinetic tendency from the perspective of mechanism. In fact, the transformation between the chlorinated product P1 and BP-4 was reversible under certain conditions. The inhomogeneous charge distribution of the CCl bond in P1 led to the photolytic dechlorination of P1. This transformation caused an increase in BP-4 concentration. In addition, the increase in the UV light power promoted the photodecomposition of P1 under the experimental condition. In addition, this study evaluated the change in absorbable organic halogens (AOX) and three kinds of toxicity changes in the BP-4 solution after chlorination and the UV/chlorine process, including the acute toxicity of luminescent bacteria, endocrine disrupting effect and cytotoxicity. The UV/chlorine process exhibited lower ecotoxicity than chlorination in water treatment.

Competition of singlet and triplet recombination of radical pairs in photoreactions of carboxy benzophenones and aromatic amino acids.

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) and transient absorption (TA) were applied to reveal the branching ratio of the singlet and triplet recombination channels in the reaction of short-lived radicals of carboxy benzophenones and the aromatic amino acids histidine, tryptophan, and tyrosine in neutral aqueous solution. It was established that the share of triplet recombination increases with increasing number of carboxylic groups: no triplet recombination was found for 4-carboxy benzophenone, whereas ∼13% of radicals of 4,4'-dicarboxy benzophenone (DCBP) and ∼27% of radicals of 3,3',4,4'-tetracarboxy benzophenone (TCBP) react with histidine radicals from the triplet state of radical pairs. The main idea is that the protonated (π,π*) triplet state of TCBP or DCBP is populated via back electron transfer from the ketyl radical of TCBP or DCBP to the radical of the amino acid. The protonated triplet state of the ketone decays with the formation of a metastable hydroxylated product, which is detected by TA. Taking into account triplet recombination provides excellent coincidence between experimental data and the simulated CIDNP kinetics.

Antifouling Photograftable Zwitterionic Coatings on PDMS Substrates.

The foreign body response (FBR) to implantable materials can negatively impact performance of medical devices such as the cochlear implant. Engineering surfaces that resist the FBR could lead to enhanced functionality including potentially improving outcomes for cochlear implant recipients through reduction in fibrosis. In this work, we coat poly(dimethylsiloxane) (PDMS) surfaces with two zwitterionic polymers, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA), using a simultaneous photografting/photo-cross-linking process to produce a robust grafted zwitterionic hydrogel. reduce nonspecific protein adsorption, the first step of the FBR. The coating process uses benzophenone, a photografting agent and type II photoinitiator, to covalently link the cross-linked zwitterionic thin film to the PDMS surface. As the concentration of benzophenone on the surface increases, the adhesive strength of the zwitterionic thin films to PDMS surfaces increases as determined by shear adhesion. Additionally, with increased concentration of the adsorbed benzophenone, failure of the system changes from adhesive delamination to cohesive failure within the hydrogel, demonstrating that durable adhesive bonds are formed from the photografting process. Interestingly, antifouling properties of the zwitterionic polymers are preserved with significantly lower levels of nonspecific protein adsorption on zwitterion hydrogel-coated samples compared to uncoated controls. Fibroblast adhesion is also dramatically reduced on coated substrates. These results show that cross-linked pSBMA and pCBMA hydrogels can be readily photografted to PDMS substrates and show promise in potentially changing the fibrotic response to implanted biomaterials.

Clickable photoaffinity ligands for the human serotonin transporter based on the selective serotonin reuptake inhibitor (S)-citalopram.

To date, the development of photoaffinity ligands targeting the human serotonin transporter (hSERT), a key protein involved in disease states such as depression and anxiety, have been radioisotope-based (i.e., 3H or 125I). This letter instead highlights three derivatives of the selective serotonin reuptake inhibitor (SSRI) (S)-citalopram that were rationally designed and synthesized to contain a photoreactive benzophenone or an aryl azide for protein target capture via photoaffinity labeling and a terminal alkyne or an aliphatic azide for click chemistry-based proteomics. Specifically, clickable benzophenone-based (S)-citalopram photoprobe 6 (hSERT Ki = 0.16 nM) displayed 11-fold higher binding affinity at hSERT when compared to (S)-citalopram (hSERT Ki = 1.77 nM), and was subsequently shown to successfully undergo tandem photoaffinity labeling-biorthogonal conjugation using purified hSERT. Given clickable photoprobes can be used for various applications depending on which reporter is attached by click chemistry subsequent to photoaffinity labeling, photoprobe 6 is expected to find value in structure-function studies and other research applications involving hSERT (e.g., imaging).

Modelling the isothermal degradation kinetics of metrafenone and mepanipyrim in a grape juice analog.

Five photodegradation products of metrafenone (MTF) and six of mepanipyrim (MEP) were identified in synthetic grape juice at 25 °C and the structures of the main reaction products established. The degradation of MTF and MEP was modelled by using three different strategies involving monitoring (a) the disappearance of the parent compound, (b) the conversion of the parent compound into its main structurally related reaction products and (c) the degradation of the parent compound to all intermediates and degradation end-products. The kinetic coefficients of degradation for these fungicides were determined and the corresponding half-lives found to be 20.8 h for MFT and 10.1 h for MEP. The proposed models afford reasonably accurate interpretation of the experimental data. Based on the results, modelling the kinetics of disappearance of the parent compound by itself does not ensure the best fit of the degradation behaviour of the fungicides.

Photo-Cross-Linking of IKs Demonstrates State-Dependent Interactions between KCNE1 and KCNQ1.

The slow delayed rectifier potassium current (IKs) is a key repolarizing current during the cardiac action potential. It consists of four KCNQ1 α-subunits and up to four KCNE1 ß-subunits, which are thought to reside within external clefts of the channel. The interaction of KCNE1 with KCNQ1 dramatically delays opening of the channel but the mechanisms by which this occur are not yet fully understood. Here, we have used unnatural amino acid photo-cross-linking to investigate the dynamic interactions that occur between KCNQ1 and KCNE1 during activation gating. The unnatural amino acid p-Benzoylphenylalanine was successfully incorporated into two residues within the transmembrane domain of KCNE1: F56 and F57. UV-induced cross-linking suggested that F56Bpa interacts with KCNQ1 in the open state, whereas F57Bpa interacts predominantly in resting channel conformations. When UV was applied at progressively more depolarized preopen holding potentials, cross-linking of F57Bpa with KCNQ1 was slowed, which indicates that KCNE1 is displaced within the channel's cleft early during activation, or that conformational changes in KCNQ1 alter its interaction with KCNE1. In E1R/R4E KCNQ1, a mutant with constitutively activated voltage sensors, F56Bpa and F57Bpa KCNE1 were cross-linked in open and closed states, respectively, which suggests that their actions are mediated mainly by modulation of KCNQ1 pore function.

The Life of Pi Star: Exploring the Exciting and Forbidden Worlds of the Benzophenone Photophore.

The widespread applications of benzophenone (BP) photochemistry in biological chemistry, bioorganic chemistry, and material science have been prominent in both academic and industrial research. BP photophores have unique photochemical properties: upon n-π* excitation at 365 nm, a biradicaloid triplet state is formed reversibly, which can abstract a hydrogen atom from accessible C-H bonds; the radicals subsequently recombine, creating a stable covalent C-C bond. This light-directed covalent attachment process is exploited in many different ways: (i) binding/contact site mapping of ligand (or protein)-protein interactions; (ii) identification of molecular targets and interactome mapping; (iii) proteome profiling; (iv) bioconjugation and site-directed modification of biopolymers; (v) surface grafting and immobilization. BP photochemistry also has many practical advantages, including low reactivity toward water, stability in ambient light, and the convenient excitation at 365 nm. In addition, several BP-containing building blocks and reagents are commercially available. In this review, we explore the "forbidden" (transitions) and excitation-activated world of photoinduced covalent attachment of BP photophores by touring a colorful palette of recent examples. In this exploration, we will see the pros and cons of using BP photophores, and we hope that both novice and expert photolabelers will enjoy and be inspired by the breadth and depth of possibilities.

A model assessment of the ability of lake water in Terra Nova Bay, Antarctica, to induce the photochemical degradation of emerging contaminants.

The shallow lakes located in Terra Nova Bay, Antarctica, are free from ice for only up to a couple of months (mid December to early/mid February) during the austral summer. In the rest of the year, the ice cover shields the light and inhibits the photochemical processes in the water columns. Previous work has shown that chromophoric dissolved organic matter (CDOM) in these lakes is very reactive photochemically. A model assessment is here provided of lake-water photoreactivity in field conditions, based on experimental data of lake water absorption spectra, chemistry and photochemistry obtained previously, taking into account the water depth and the irradiation conditions of the Antarctic summer. The chosen sample contaminants were the solar filter benzophenone-3 and the antimicrobial agent triclosan, which have very well known photoreactivity and have been found in a variety of environmental matrices in the Antarctic continent. The two compounds would have a half-life time of just a few days or less in the lake water during the Antarctic summertime, largely due to reaction with CDOM triplet states ((3)CDOM*). In general, pollutants that occur in the ice and could be released to lake water upon ice melting (around or soon after the December solstice) would be quickly photodegraded if they undergo fast reaction with (3)CDOM*. With some compounds, the important (3)CDOM* reactions might favour the production of harmful secondary pollutants, such as 2,8-dichlorodibenzodioxin from the basic (anionic) form of triclosan.

Preparation of Polypropylene Spin Tips Filled with Immobilized Titanium(IV) Ion Monolithic Adsorbent for Robust Phosphoproteome Analysis.

In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol ß-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 µg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells.

Photolytic fate and genotoxicity of benzophenone-derived compounds and their photodegradation mixtures in the aqueous environment.

This study investigates the environmental fate of eight benzophenone derivatives (the pharmaceutical ketoprofen, its phototransformation products 3-ethylbenzophenone and 3-acetylbenzophenone, and five benzophenone-type UV filters) by evaluating their photolytic behaviour. In addition, the genotoxicity of these compounds and the produced photodegradation mixtures was studied. Laboratory-scale irradiation experiments using a medium pressure UV lamp revealed that photodegradation of benzophenones follows pseudo-first-order kinetics. Ketoprofen was the most photolabile (t1/2 = 0.8 min), while UV filters were more resistant to UV light with t1/2 between 17 and 99 h. The compounds were also exposed to irradiation by natural sunlight and showed similar photostability as predicted under laboratory conditions. Solar photodegradation experiments were performed in distilled water, lake and seawater, and revealed that photosensitizers present in natural waters significantly affect the photolytic behaviour of the investigated compounds. In this case, the presence of lake water resulted in accelerated photodecomposition, while seawater showed different effects on photodegradation, depending on a compound. Further, it was shown that the transformation products of ketoprofen 3-ethylbenzophenone and 3-acetylbenzophenone were formed under environmental conditions when ketoprofen was exposed to natural sunlight. Genotoxicity testing of parent benzophenone compounds using the SOS/umuC assay revealed that UV filters exhibited weak genotoxic activity in the presence of a metabolic activation system, however the concentrations tested were much higher than found in the environment (≥125 µg mL(-1)). After irradiation of benzophenones, the produced photodegradation mixtures showed that, with the exception of benzophenone that exhibited weak genotoxic activity, all the other compounds tested did not elicit any activity when exposed to UV light.

Role of type I & type II reactions in DNA damage and activation of caspase 3 via mitochondrial pathway induced by photosensitized benzophenone.

Sunscreen users have been increased, since excessive sun exposure increased the risk of skin diseases. Benzophenone (BP) and its derivatives are commonly used in sunscreens as UV blocker. Its photosafety is concern for human health. Our study showed the role of type-I and type-II radicals in activation of caspase 3 and phototoxicity of BP under sunlight/UV radiation. BP photodegraded and formed two photoproducts. BP generates reactive oxygen species (ROS) singlet oxygen ((1)O2), superoxide anion (O2˙(-)) and hydroxyl radical (˙OH) through type-I and type-II photodynamic mechanisms. Photocytotoxicity significantly reduced cell viability under sunlight, UVB and UVA. DCF fluorescence confirmed intracellular ROS generation. BP showed single strand DNA breakage, further proved by cyclobutane pyrimidine dimmers (CPDs) formation. Lipid peroxidation and LDH leakage were enhanced by BP. P21 dependent cell cycle study showed sub G1 population which advocates apoptotic cell death, confirmed through AO/EB and annexin V/PI staining. BP decreased mitochondrial membrane potential, death protein released and activated caspase. We proposed cytochrome c regulated caspase 3 dependent apoptosis in HaCaT cell line through down regulation of Bcl2/Bax ratio. Phototoxicity potential of its photoproducts is essential to understand its total environmental fate. Hence, we conclude that BP may replace from cosmetics preparation of topical application.

Combination of retinyl palmitate and UV-filters: phototoxic risk assessment based on photostability and in vitro and in vivo phototoxicity assays.

This study aimed to assess the phototoxic potential of combined UV-filters and retinyl palmitate (RP) in the presence or not of bemotrizinol (BMTZ), employing photostability and in vitro and in vivo phototoxicity assays. The formulations tested contained octocrylene (OCT), octyl methoxycinnamate (OMC), benzophenone-3 (BZP-3) and RP (photostable) or octocrylene (OCT), octyl methoxycinnamate (OMC), avobenzone (AVO) and RP (less photostable). Both formulations were supplemented with bemotrizinol. Photostability was evaluated by exposing, or not, formulations spread on a glass plate to UVA/UVB irradiation. The resulting products were quantified by HPLC analysis. In vitro phototoxicity of UV-filters and combinations were evaluated using 3T3 viable monolayer fibroblast cultures submitted, or not, to irradiation according to OECD TG 432. In vivo photoallergy and photoxicity were assessed by clinical studies (photopatch test). Photostability assays showed that UV-filter bemotrizinol was a better photostabilizer for RP/benzophenone-3 than for RP/avobenzone. The in vitro phototoxicity of the combination RP/avobenzone was reduced by bemotrizinol. Clinical studies did not indicate phototoxic or photoallergenic potentials in all formulations tested. It is concluded that the 3T3 NRU phototoxicity test may be considered a supplementary assay in formulation developments, since it can detect chemically unstable and potentially phototoxic combinations. However, extrapolation of in vitro positive results to human photopatch tests may be performed only to a limited extent.

Investigation of the stabilizing effects of antioxidants and benzophenone-3 on desonide photostability.

Desonide is a topical corticoid used in the treatment of skin diseases and is marketed in different pharmaceutical dosage forms. Recently, the poor photostability of a commercially available hair solution after direct exposure to UVA light was verified. In this study, we investigated the ability of the antioxidants ascorbic acid, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), α-tocopherol, and the UV filter benzophenone-3 (BP-3) to prevent the photodegradation of desonide in hair solution (desonide 0.1%) and the stability of the proposed formulation under environmental conditions. The tested antioxidants were not able to prevent the photolysis of desonide, whereas the addition of 0.3% BP-3 enhanced the photostability of the drug. After 15 h of direct exposure to UVA radiation, the desonide remaining content in the hair solution with BP-3 was approximately 98%. Higher photostability was also verified under UVC radiation. Additionally, the results indicated that the formulation was stable under accelerated and room temperature conditions for 70 days, corresponding to the total period of the study.

A simple strategy for in situ fabrication of a smart hydrogel microvalve within microchannels for thermostatic control.

Self-regulation of temperature in microchip systems is crucial for their applications in biomedical fields such as cell culture and biomolecule synthesis as well as those cases that require constant temperature conditions. Here we report on a simple and versatile approach for in situ fabrication of a smart hydrogel microvalve within a microchip for thermostatic control. The thermo-responsive hydrogel microvalve enables the "on-off" switch by sensing temperature fluctuations to control the fluid flux as well as the fluid heat exchange for self-regulation of the temperature at a constant range. Such temperature self-regulation is demonstrated by integrating the microvalve-incorporated microchip into the flow circulation loop of a micro-heat-exchanging system for thermostatic control. Moreover, the microvalve-incorporated microchip is employed for culturing cells under temperature self-regulation. The smart microvalve shows great potential as a temperature controller for applications that require thermostatic conditions. This approach offers a facile and flexible strategy for in situ fabricating hydrogel microvalves within microchips as chemostats and microreactors for biomedical applications.

Monitoring conformational changes in peroxisome proliferator-activated receptor α by a genetically encoded photoamino acid, cross-linking, and mass spectrometry.

Chemical cross-linking combined with an enzymatic digestion and mass spectrometric analysis of the reaction products has evolved into an alternative strategy to structurally resolve protein complexes. We investigated conformational changes in peroxisome proliferator-activated receptor α (PPARα) upon ligand binding. Using E. coli cells with a special tRNA/aminoacyl-tRNA synthetase pair, two PPARα variants were prepared in which Leu-258 or Phe-273 were site-specifically replaced by the genetically encoded photoreactive amino acid p-benzoylphenylalanine (Bpa). PPARα variants were subjected to UV-induced cross-linking, both in the absence and in the presence of ligands. After the photo-cross-linking reaction, reaction mixtures were enzymatically digested and peptides were analyzed by mass spectrometry. The inter-residue distances disclosed by the photochemical cross-links served to monitor conformational changes in PPARα upon agonist and antagonist binding. The data obtained with our strategy emphasize the potential of genetically encoded internal photo-cross-linkers in combination with mass spectrometry as an alternative method to monitor in-solution 3D-protein structures.