Imaging chirality with surface second harmonic generation microscopy

Chirality is a fundamental construct in nature which arises from an antisymmetric arrangement of atoms, molecules, or larger structures, resulting in the formation of nonsuperimposable mirror images. Bulk chiral effects can easily be measured using circular dichroism (CD) or optical rotary dispersion (ORD). However, the imaging of chirality originating from molecular surface films cannot be obtained with these linear optical methods. By using chiral second harmonic generation (C-SHG), with its inherent surface sensitivity and ability to discriminate between the symmetry of surface adsorbed species in combination with a counter-propagating optical geometry, we have developed the first nonlinear chiral microscope. In the study presented here, the intrinsic chirality of R- and S-(+)-1,1'-bi-2-naphthol (RBN, SBN) has been used to image a patterned planar supported lipid bilayer (PSLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using C-SHG. Spatial resolution of the patterned PSLB is visible when either RBN or SBN is intercalated into the membrane. No image is observed when a racemic mixture of RBN and SBN is present. The C-SHG images are compared with those obtained from fluorescence microscopy to verify the C-SHG imaging technique. The results presented here demonstrate that C-SHG possesses the requisite surface selectivity and sensitivity to detect interfacial chirality and provides a direct route for the visualization of chirality originating from molecular surface films.     

In situ monitoring of protein adsorption on a nanoparticulated gold film by attenuated total reflection surface-enhanced infrared absorption spectroscopy

In situ surface enhanced infrared absorption spectroscopy (SEIRAS) with an attenuated total reflection (ATR) configuration has been used to monitor the adsorption kinetics of bovine hemoglobin (BHb) on a Au nanoparticle (NP) film. The IR absorbance for BHb molecules on a gold nanoparticle film deposited on a Si hemispherical optical window is about 58 times higher than that on a bare Si optical window and the detection sensitivity has been improved by 3 orders of magnitude. From the IR signal as a function of adsorption time, the adsorption kinetics and thermodynamics can be explored in situ. It is found that both the electrostatic interaction and the coordination bonds between BHb residues and Au NP film surface affect the adsorption kinetics. The maximum adsorption can be obtained in solution pH 7.0 (close to the isoelectric point of the protein) due to the electrostatic interaction among proteins. In addition, the isotherm of BHb adsorption follows well the Freundlich adsorption model.     

Sensitive Method for Enantioseparation of Rivastigmine with Highly Sulfated Cyclodextrin as Chiral Selector by Capillary Electrophoresis

A sensitive method for enantioseparation of a basic drug rivastigmine and determination of its optical impurityby capillary electrophoresis with highly sulfated β-cyclodextrin(HS-β-CD)as the chiral selector is described.Ingeneral,enantioseparation of basic chiral compounds is carried out in acidic condition(pH 2.5)to prevent analytesfrom adsorption on the capillary wall.However,in the case of rivastigmine,the detection sensitivity was too limitedto determine the optical impurity of S-rivastigmine lower than 1% when buffer pH was 2.5.It was found that thedetection sensitivity was improved 1.6 times just by raising the buffer pH value from 2.5 to 5.8.The poor columnefficiency due to the adsorption of the analytes on the capillary wall was resolved by using dynamical coating of thecapillary wall with the linear polyacrylamide solution.The experirnental parameters such as the concentration ofHS-β-CD,buffer pH and buffer ionic strength were optimized,respectively.The method was validated in terms ofrepeatability,linearity,limit of detection(LOD)and limit of quantitation(LOQ).Using the present method,the op-tical purity of nonracemic rivastigmine with the enantiomeric excess(ee)value of 99.14% was determined.     

Phenethylamine@Pillar[5]arene Biointerface for Highly Enantioselective Adsorption of Protein

In the life system, the biointerface plays an important role in cell adsorption, platelet adsorption and activation. Therefore, the study of protein adsorption on the biointerface is of great significance for understanding life phenomena and treatment in vitro. In this paper, a chiral biointerface was constructed by the virtue of host‐guest interaction between a water‐soluble pillar[5]arene (WP5) and phenethylamine (PEA) over a gold surface for adsorption of lysozyme proteins. From the experimental results it was identified that the host‐guest biointerface has a high adsorption capacity and strong chiral selectivity. Furthermotre, it was identified that the host‐guest interaction plays the decisive role in the enhancement of chirality of the interface, which was much beneficial for increasing protein adsorption and amplifying the capacity of chiral discrimination. Therefore, this work provides a new idea for the construction of biointerface materials with high protein adsorption capacity and high chiral selectivity through supramolecular interaction, which will have potential applications in the fields of biosensors, biocatalysts, biomaterials.     

Bioresponsive hydrogel microlenses

We report investigations of bioresponsive hydrogel microlenses as a new protein detection technology. Stimuli-responsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microgels have been synthesized via free-radical precipitation polymerization. These hydrogel microparticles were then functionalized with biotin via EDC coupling. Hydrogel microlenses were prepared from the particles via Coulombic assembly onto a silane-modified glass substrate. Arrays containing both pNIPAm-co-AAc microgels (as an internal control) and biotinylated pNIPAm-co-AAc microgels were then used to detect multivalent binding of both avidin and polyclonal anti-biotin. Protein binding was determined by monitoring the optical properties of the microlenses using a brightfield optical microscopy technique. The microlens method is shown to be very specific for the target protein, with no detectable interference from nonspecific protein binding. Finally, the reversibility of the hydrogel microlens assay has been studied in the case of anti-biotin to determine the potential application of the microlens assay technology in a displacement-type assay. These results suggest that the microlens method may be an appropriate one for label-free detection of proteins or small molecules via displacement of tethered protein--ligand pairs.     

Supramolecular assembly facilitating adsorbate-induced chiral electronic states in a metal surface

Through the application of optically active second-harmonic generation measurements (OA-SHG) we have demonstrated that the adsorption of amino acids cysteine (HSCH(2)CHNH(2)COOH) and penicillamine (HSC(CH3)(2)CHNH(2)COOH) from solution can induce chiral electronic states in an initially achiral polycrystalline Au film. The chiral induction is strongly dependent upon the pH of the deposition solution; adsorption of penicillamine and cysteine under acidic conditions (pH = 3) induces the same level of optical activity, whereas at pH = 11, the optical activity induced by cysteine is reduced by ca. 50% and penicillamine does not induce optical activity at all. The pH dependence indicates that the presence of interadsorbate hydrogen bonds, and consequently the supramolecular assembly of the adsorbates, facilitates the induction of chiral electronic states in the Au surface. This observation demonstrates that the symmetry properties of the extended structure of the self-assembled layer, and not the local adsorption geometry of the isolated adsorbed moiety, play the lead role in the induction of chiral metallic electronic states. The dependence of the chiral induction on COOH groups is identical to that observed in studies of optical activity in chiral thiol-protected nanoparticles, suggesting a common mechanism for the chiral perturbation in extended films and nanoparticles.     

Ellipsometric approach for the real-time detection of label-free protein adsorption by second harmonic generation

Second harmonic generation (SHG) was performed using a novel ellipsometric detection approach to selectively probe the real-time surface binding kinetics of an unlabeled protein. The coherence of nonlinear optical processes introduces new possibilities for exploiting polarization that are unavailable with incoherent methods, such as absorbance and fluorescence. Adsorption of bovine serum albumin (BSA) at silica/aqueous solution interfaces resulted in changes in the polarization state of the frequency-doubled light through weak, dynamic interactions with a coadsorbed nonlinear optical probe molecule (rhodamine 6G). Using a remarkably simple instrumental approach, signals arising exclusively from surface interactions with BSA were spatially isolated and selectively detected with high signal-to-noise. The relative intensities acquired during the kinetics experiments using both circularly and linearly polarized incident beams were in excellent agreement with the responses predicted from SHG ellipsometry polarization measurements. Analysis of the polarization-dependent SHG generated during BSA adsorption at glass/aqueous solution interfaces provided direct evidence for slow conformational changes within the protein layer after adsorption, consistent with protein denaturation. This polarization selection approach is sufficiently general to be easily extended to virtually all coherent nonlinear optical processes and a variety of different surface interactions and architectures.     

Monte Carlo modeling of chiral adsorption on nanostructured chiral surfaces and slit pores

Adsorptive separation of chiral molecules is a powerful technique that has long been used in the chemical and pharmaceutical industries. An important challenge in this field is to design and optimize new adsorbents to provide selective discrimination of enantiomers. In this article, we introduce an off-lattice model of chiral adsorption on nanostructured surfaces and slit pores with the aim of predicting their enantioslective properties. The concept presented here involves finding the optimal chiral pattern of active sites on the pore walls that maximizes the difference between the binding energies of the enantiomers. Our initial effort focuses on chiral molecules that do not have specific interactions with the pore surface. One candidate meeting this requirement is 1,2-dimethylcyclopropane (DMCP), a chiral hydrocarbon whose interaction with a model pore surface was described using the Lennard-Jones potential. To model the adsorption of DMCP, we used the Monte Carlo simulation method. It was demonstrated that the separation of the enantiomers of DMCP is hardly obtainable because of the smoothness of the potential energy surface for molecules physisorbed in the pore. However, the simulated results allowed the identification of key factors that influence the binding of the enantiomers of DMCP to the pore walls with a special distribution of active sites. This information will be useful in future considerations of the adsorption of more complex chiral molecules.     

Axially Chiral Facial Amphiphiles with a Dihydronaphthopentaphene Structure as Molecular Tweezers for SWNTs

Syntheses of chiral 6,15‐dihydronaphtho[2,3‐c]pentaphene derivatives of opposite configurations are reported. Starting from anthracene, the strategy involves two key steps: a Diels–Alder reaction on a prochiral dianthraquinone, and an enantiomeric resolution using (?)‐menthol. The final molecules exhibit very strong optical activity, as shown by their circular dichroism spectra, and are examples of chiral facial amphiphiles. Their adsorption at the surface of single‐walled carbon nanotubes (SWNTs) has also been studied, and has been found to occur preferentially on 0.8–1.0 nm diameter nanotubes among the population of a high‐pressure CO conversion (HiPco) SWNT sample (0.8–1.2 nm). The synthesised facial amphiphiles act as nano‐tweezers for the diameter‐selective solubilisation of SWNTs in water. The expected optical activities of the SWNT samples solubilised by each of the chiral amphiphiles have been studied by circular dichroism spectroscopy, but the results are not yet conclusive.     

Biocompatible polymers for antibody support on gold surfaces

The elimination or minimization of non-specific protein adsorption from serum is critical for the use of surface plasmon resonance (SPR) sensors for in vitro and in vivo analysis of complex biological solutions. The ultimate goals in this application are to minimize non-specific adsorption of protein and to maximize analyte signal. A reduction of the non-specific protein adsorption from serum of up to 73% compared to carboxymethylated-dextran 500 kDa (CM-dextran) was achieved following a survey of eight biocompatible polymers and 10 molecular weights of CM-dextran. These coatings minimize non-specific adsorption on the sensor while also serving as immobilization matrices for antibody fixation to the probes. Polymers including polysaccharides: CM-dextrans, CM-hyaluronic acid, hyaluronic acid, and alginic acid were investigated. Humic acid, polylactic acid, polyacrylic acid, orthopyridyldisuldfide–polyethyleneglycol–N-hydroxysuccinimide (OPSS–PEG–NHS), and a synthesized polymer; polymethacrylic-acid-co-vinyl-acetate (PMAVA) were also used. The non-specific protein adsorption reduction was measured over a 14 day period at 0 °C for each polymer. Calibration curves using some of these polymers were constructed to show the performance and low detection limit possibilities of these new antibody supports. For many of the polymers, this is the first demonstration of employment as an antibody support for an optical or surface active sensor. CM-dextran is the polymer offering the largest signal for the antigen detection. However, the biocompatible polymers demonstrate a greater stability to non-specific binding in serum. These biocompatible polymers offer different alternatives for CM-dextran.     

Density functional theory study of enantiospecific adsorption at chiral surfaces

Density functional theory calculations are carried out for the adsorption of a chiral molecule, (S)- and (R)-HSCH(2)CHNH(2)CH(2)P(CH(3))(2), on a chiral surface, Au(17 11 9)(S)(). The S-enantiomer is found to bind more strongly than the R-enantiomer by 8.8 kJ/mol, evidencing that the chiral nature of the kink sites at the Au(17 11 9) surface leads to enantiospecific binding. The adsorption of two related chiral molecules, HSCH(2)CHNH(2)COOH ("cysteine") and HSCH(2)CHNH(2)CH(2)NH(2), does not, however, lead to enantiospecific binding. The results of the density functional calculations are broken down into a local binding model in which each of the chiral molecule's three contact points with the surface provides a contribution to the overall adsorption bond strength. The enantiospecific binding is demonstrated to originate from the simultaneous optimization of these three local bonds. In the model, the deformation energy costs of both the molecule and the surface are further included. The model reveals that the molecule may undergo large deformations in the attempt to optimize the three bonds, while the surface deforms to a lesser extent. The most favorable binding configurations of each enantiomer are, however, characterized by small deformation energies only, justifying a local binding picture.     

Evaluation of quail egg white riboflavin binding protein as a chiral selector in capillary electrophoresis by applying a modified partial filling technique.

A preliminary evaluation of the enantioselective properties of quail egg yolk riboflavin binding protein (qRfBP) was carried out in capillary electrophoresis by using the complete filling technique. The most promising results obtained by this screening of nineteen chiral drugs were singled out with the aim of optimizing enantiomer separations by applying the partial filling technique, which allows operating at much higher protein concentrations without detection problems. The building of the separation zone in the partial filling technique has been modified in order to enable on-line monitoring, before each run, of the actual protein plug application velocity and, consequently, the building of a plug of the desired length. The electrophoretic conditions chosen gave opposite migration directions for the chiral selector and the analytes, with qRfBP migrating away from the detector. A polyvinyl alcohol-coated capillary was first totally filled with protein and the optimal plug length was obtained by further applying negative pressure together with positive voltage for the time needed. Separations of basic drugs were optimized by using protein concentrations ranging from 200 microM up to 900 microM and different plug lengths, while the running buffer pH (6.0), temperature (25 degrees C) and operating voltage (+20 kV) were kept constant. The enantioresolution of all solutes was affected by both the chiral selector concentration and protein plug length. Baseline separations were obtained for oxprenolol, prilocaine and bupivacaine.     

Application of liquid pre-column capillary electrophoresis technique to the study of interaction between drug enantiomers and human serum albumin

Based on the chiral separation of several basie drugs, dimetindene, tetryzoline, theodrenaline and verapamil, the liquid pre-colunm capillary electrophoresis (LPC-CE) technique was established. It was used to determine free concentrations of drug enantiomers in mixed solutions with human serum albumin (HSA). To prevent HSA entering the CE chiral separation zone, the mobility differences between HSA and drugs under a specific pH condition were employed in the LPC. Thus, the detection confusion caused by protein was totally avoided. Further study of binding constants determination and protein binding competitions was carried out. The study proves that the LPC technique could be used for complex media, particularly the matrix of protein coexisting with a variety of drugs.     

Reduction of nonspecific protein binding on surface plasmon resonance biosensors

Reduction of the nonspecific serum protein adsorption on a gold surface to levels low enough to allow the detection of biomarkers in complex media has been achieved using the N-hydroxysuccinimide (NHS) ester of 16-mercaptohexadecanoic acid. Carboxymethylated dextran (CM dextran), which is widely used, nonspecifically adsorbs enough proteins to mask the signal from target biomarkers in complex solutions such as serum or blood. The use of short-chain thiols greatly reduces the amount of nonspecific protein adsorption. Mixed layers of 11-mercaptoundecanoic acid or the NHS ester of 11-mercaptoundecanoic acid mixed layers with either 11-mercaptoundecanol or undecanethiol, and 16-mercaptohexadecanoic acid or the NHS ester of 16-mercaptohexadecanoic acid with hexadecanethiol, were also investigated for nonspecific protein binding properties as well as for biomarker signal response. The NHS ester of 16-mercaptohexadecanoic acid exhibits the largest signal for the biomarker myoglobin (including CM dextran) while offering a significantly diminished amount of nonspecific binding. The sensor has also been shown to detect interleukin-6 in cell culture media containing protein concentrations of at least 4 mg/mL.     

On the zopiclone enantioselective binding to human albumin and plasma proteins. An electrokinetic chromatography approach

In this work, a methodology for the chiral separation of zopiclone (ZPC) by electrokinetic chromatography (EKC) using carboxymethylated-β-cyclodextrin as chiral selector has been developed and applied to the evaluation of the enantioselective binding of ZPC enantiomers to HSA and total plasma proteins. Two mathematical approaches were used to estimate protein binding (PB), affinity constants (K(1)) and enantioselectivity (ES) for both enantiomers of ZPC. Contradictory results in the literature, mainly related to plasma protein binding reported data, suggest that this is an unresolved matter and that more information is needed. Discrepancies and coincidences with previous data are highlighted.     

Thermodynamic origin of the chiral recognition of tryptophan on teicoplanin and teicoplanin aglycone stationary phases

The D-, L-tryptophan binding and the chiral recognition properties of the teicoplanin and teicoplanin aglycone (TAG) chiral stationary phase (CSPs) were compared at various column temperatures. The solute adsorption isotherms (bi-Langmuir model) were determined for both the two CSPs using the perturbation method. It was demonstrated that the sugar units were involved in the reduction of the apparent enantioselectivity through two phenomena: (i) the inhibition of some enantioselective contacts with low-affinity binding regions of the aglycone and (ii) a decrease in the stereoselective properties of the aglycone high-affinity binding pocket. The phenomenon (ii) was governed by both a decrease in the ratio of the enantiomer adsorption constant and a strong reduction of the site accessibility for D- and L-tryptophan. In addition, a temperature effect study was performed to investigate the chiral recognition mechanism at the aglycone high-affinity pocket. An enthalpy-entropy compensation analysis derived from the Grunwald model as well as the comparison with the literature data demonstrated that the enantioselective binding mode was dependent on an interface dehydration process. The change in the enantioselective process observed between the TAG and teicoplanin CSP was characterized by a difference of ca. 2-3 ordered water molecules released from the species interface.     

Triplet excited States as chiral reporters for the binding of drugs to transport proteins

The triplet excited state of flurbiprofen methyl ester (FBPMe) has been used as a chiral reporter for the two binding sites of human serum albumin (HSA). The occupation level of the binding sites has been estimated from regression analysis of the triplet decays at several [FBPMe]/[HSA] ratios. The data agree with two high affinity binding sites (I and II) that are populated to a different extent. A remarkable stereodifferentiation has been found in the drug triplet lifetimes within the protein microenvironment.     

High salt stability and protein resistance of poly(L-lysine)-g-poly(ethylene glycol) copolymers covalently immobilized via aldehyde plasma polymer interlayers on inorganic and polymeric substrates

The electrostatic adsorption onto charged surfaces of comb copolymers comprising a polyelectrolyte backbone and pendent PEG side chains, such as poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG), has in previous studies provided protein-repellent thin coatings, particularly on metal oxide surfaces. A drawback of this approach is, however, the instability of such adsorbed layers under extreme pH values or high ionic strength. We have overcome this limitation in the present study by covalently immobilizing PLL-g-PEG copolymers onto aldehyde plasma-modified substrates. Silicon wafers, optical waveguide chips, and perfluorinated ethylene-co-propylene (FEP) polymer substrates were first coated with a thin plasma polymer layer using a propionaldehyde plasma, followed by covalent immobilization of PLL-g-PEG via reductive amination between amine groups of the PLL backbone with aldehyde groups on the plasma-deposited interlayer. The stability in high salt media and the protein resistance of different molecular architectures of immobilized PLL-g-PEG layers were quantitatively investigated by XPS, an optical waveguide technique (OWLS), and ToF-SIMS. The adsorption of bovine serum albumin was found to be below the detection limit (<2 ng/cm(2)), as for electrostatically adsorbed PLL-g-PEG layers. However, after 24 h of exposure of covalently immobilized layers of PLL-g-PEG to high ionic strength buffer (2400 mM NaCl), no significant change in the protein resistance was observed, whereas under the same conditions electrostatically adsorbed PLL-g-PEG coatings lost their protein resistance. Moreover, covalent immobilization via an aldehyde plasma interlayer enabled the application of PLL-g-PEG layers onto substrates such as FEP onto which electrostatic binding is not possible. These findings create a generic platform for the covalent immobilization of PLL-g-PEG onto a wide variety of substrates.     

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3-b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor

A stereoselective assay for the optical isomers [(S) and (R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4H-thieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25-250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single (S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5-100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.     

Catalytic hydrogenation of itaconic acid in a biotinylated Pyrphos–rhodium(I) system in a protein cavity

The construction of a chiral catalyst system embedded at a specific site in a protein has been studied. The preparation of the biotinylated Pyrphos–Rh(I) complex attached to the binding site in avidin and its application to the asymmetric hydrogenation of itaconic acid have been investigated. By introducing the chiral Pyrphos–Rh(I) moiety into the constrained environment of the protein cavity it was found that the enantioselectivity of the system was significantly influenced by the tertiary conformation within the avidin cavity. The effects of reaction conditions such as temperature, hydrogen pressure, and the pH value of the buffer on enantioselectivity are reported.