Electropolymerized films formed from the amphiphilic decyl esters of D- and L-tyrosine compared to L-tyrosine using the electrochemical quartz crystal microbalance

Using the electrochemical quartz crystal microbalance (EQCM), we compared thin films formed on Pt by electropolymerization of l-tyrosine to that of the amphiphilic monomers, decyl esters of d- and l-tyrosine (DEDT and DELT). Mass build-up and film properties were determined as a function of monomer concentration via frequency, f, motional resistance, R, and charge passage, Q, measurements. Films were found to occur by a combination of monomer electropolymerization and adsorption for DEDT and DELT, but only by electropolymerization for l-tyrosine. This difference in film formation process for the monomers is reflected in the net mass build-up for each film, as represented by calculated df/dQ values. For the adsorbing monomers DEDT and DELT, films possessed concentration dependent df/dQ values, more than 100-fold greater than that for l-tyrosine film formation under equivalent electropolymerization conditions. During the entire film growth process, all three films exhibited no significant energy dissipation properties (DeltaR invariant). Concentration dependent adsorption of significant levels of unpolymerized but self-assembled DEDT and DELT monomers account for the subsequent time dependent mass loss observed from the films maintained in buffer in the absence of monomer. Contact angle measurements demonstrated a pH dependent increase in the surface hydrophilicity of films electropolymerized from the DEDT, DELT, and l-tyrosine monomers but not films formed from phenol and 3-nitrophenol monomers. This behavior is consistent with the monomers' known changes in titration/charge state properties with increasing pH. This study provided insight into the film formation, stability, and surface hydrophilicity resulting from electropolymerization of these related tyrosine based monomers. This information is critical to assessing the utility these films may have in the development of new biomaterials and as biological macromolecule or cell immobilization strategies in biosensors.     

Quartz crystal microbalance bioaffinity sensor for biotin based on mixed self-assembled monolayers and metastable molecular complex receptor

A quartz crystal microbalance (QCM) sensor was proposed for the detection of small molecule biotin based on the mixed self-assembled monolayer (SAM) of thiols on gold substrate and the bioaffinity difference between an analyte (biotin) and an analogue compound (HABA) in binding avidin. Avidin formed a metastable complex with 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) immobilized on the crystal surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The frequency change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the frequency change and the biotin concentration. The use of mixed SAMs allows the stable attachment of bioreceptor molecules on the QCM, and enhances the amount of the immobilized molecules on the QCM, as a longer "space arm" in the mixed SAMs makes this monolayer membrane more accessible to capture the immobilized molecules. The proposed bioaffinity sensor has nice response to biotin in the range of 0.017-1.67 microg/mL. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.     

Selective enzymatic degradation of self-assembled particles from amphiphilic block copolymers obtained by the combination of N-carboxyanhydride and nitroxide-mediated polymerization

Combining controlled radical polymerizations and a controlled polypeptide synthetic technique, such as N-carboxyanhydride (NCA) ring-opening polymerization, enables the generation of well-defined block copolymers to be easily accessible. Here we combine NCA polymerization with the nitroxide-mediated radical polymerization of poly(n-butyl acrylate) (PBA) and polystyrene (PS), using a TIPNO and SG1-based bifunctional initiator to create a hybrid block copolymer. The polypeptide block consists of (block) copolymers of poly(L-glutamic acid) embedded with various quantities of L-alanine. The formed superstructures (vesicles and micelles) of the block copolymers possessed varying degrees of enzyme responsiveness when exposed to elastase and thermolysin, resulting in controlled enzymatic degradation dictated by the polypeptide composition. The PBA containing block copolymers possessing 50% L-alanine in the polypeptide block showed a high degradation response compared to polymers containing lower L-alanine quantities. The particles stabilized by copolypeptides with L-alanine near the hydrophobic block showed full degradation within 4 days. Particles containing polystyrene blocks revealed no appreciable degradation under the same conditions, highlighting the specificity of the system and the importance of synthetic polymer selection. However, when the degradation temperature was increased to 70 °C, degradation could be achieved due to the higher block copolymer exchange between the particle and the solution. A number of novel biohybrid structures are disclosed that show promise as enzyme-responsive materials with potential use as payload release vehicles, following their controlled degradation by specific, target, enzymes.     

Quartz crystal microbalance studies of multilayer glucagon fibrillation at the solid-liquid interface

We have used a quartz crystal microbalance with dissipation (QCM-D) to monitor the changes in layer thickness and viscoelastic properties accompanying multilayer amyloid deposition in situ for the first time. By means of atomic force microscope imaging, an unequivocal correlation is established between the interfacial nucleation and growth of glucagon fibrils and the QCM-D response. The combination of the two techniques allows us to study the temporal evolution of the interfacial fibrillation process. We have modeled the QCM-D data using an extension to the Kelvin-Voigt viscoelastic model. Three phases were observed in the fibrillation process: 1), a rigid multilayer of glucagon monomers forms and slowly rearranges; 2), this multilayer subsequently evolves into a dramatically more viscoelastic layer, containing a polymorphic network of micrometer-long fibrils growing from multiple nucleation sites; and 3), the fibrillar formation effectively stops as a result of the depletion of bulk-phase monomers, although the process can be continued without a lag phase by subsequent addition of fresh monomers. The robustness of the QCM-D technique, consolidated by complementary atomic force microscope studies, should make it possible to combine different components thought to be involved in the plaque formation process and thus build up realistic models of amyloid plaque formation in vitro.     

Quartz crystal microbalance for the determination of daminozide using molecularly imprinted polymers as recognition element

As the daminozide (DM) and its metabolite have been identified to be potentially carcinogenic, rapid detection method for them is necessary for food safety. A type of piezoelectric crystal sensor has been prepared by using a molecularly imprinted polymer (MIP) as recognition element. The molecularly imprinted polymer was prepared by hot-induced precipitation polymerization, and then the polymer particles were fixed on the surface of the electrode. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were employed to evaluate the obtained imprinted polymer particles and the MIP sensitive film coated on the electrode. The results showed that a typical time-response curve of the MIP-coated crystal to the DM solution had been given, frequency shifts versus logarithm changes of DM showed good linear correlation within the concentration range of 1.0x10(-9) to 10(-6) mg/mL (y=11.38 lg x+115.45, r=0.9872) and 1.0x10(-6) to 10(-1) mg/mL (y=25.22lgx+209.44, r=0.9938), respectively. The detection limit was 5.0x10(-8) mg/mL (S/N=3), which is lower than that of conventional methods. Further, computer simulation technology was employed to investigate the interaction between methacrylic acid and DM for elucidating the recognition mechanism. The influencing factor pH has also been investigated. The injection experiments of DM structurally related compounds indicated that the obtained sensor has high sensitivity, excellent selectivity, low cost, good reproducibility, and reusable property by combining with piezoelectric crystal and molecularly imprinted polymer.     

Quartz crystal microbalance (QCM) as a device for the screening of phage libraries

An immunosensing system based on a quartz crystal microbalance (QCM) is presented for the selection of both antigen specific recombinant antibodies and antigen specific human pancreatic secretory trypsin inhibitor (hPSTI) mutants isolated from large phage libraries. The QCM was integrated into a flow injection analysis system for the straightforward analysis of large sample numbers. Measurements were performed using a biotinylated antigen immobilized by streptavidin onto the gold surface of the quartz crystal and phages displaying recombinant antibodies or hPSTI mutants. The results obtained by the QCM were in accordance to those of a well established enzyme linked immunosorbent assay (ELISA). Therefore, the QCM is well suited for the detection of single high affinity clones isolated from large phage display libraries.     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Quartz crystal microbalance: a useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface

The quartz crystal microbalance (QCM) is a simple, cost effective, high-resolution mass sensing technique, based upon the piezoelectric effect. As a methodology, the QCM evolved a solution measurement capability in largely analytical chemistry and electrochemistry applications due to its sensitive solution-surface interface measurement capability. The technique possesses a wide detection range. At the low mass end, it can detect monolayer surface coverage by small molecules or polymer films. At the upper end, it is capable of detecting much larger masses bound to the surface. These can be complex arrays of biopolymers and biomacromolecules, even whole cells. In addition, the QCM can provide information about the energy dissipating properties of the bound surface mass. Another important and unique feature of the technique is the ability to measure mass and energy dissipation properties of films while simultaneously carrying out electrochemistry on solution species or upon film systems bound to the upper electrode on the oscillating quartz crystal surface. These measurements can describe the course of electropolymerization of a film or can reveal ion or solute transport within a film during changes in the film environment or state, including the oxidation state for an electroactive film driven by the underlying surface potential. The past decade has witnessed an explosive growth in the application of the QCM technique to the study of a wide range of molecular systems at the solution-surface interface, in particular, biopolymer and biochemical systems. In this report, we start with a brief historical and technical overview. Then we discuss the application of the QCM technique to measurements involving micellar systems, self-assembling monolayers and their phase transition behavior, molecularly imprinted polymers, chemical sensors, films formed using the layer-by-layer assembly technique, and biopolymer films and point out the utility of the electrochemical capabilities of the technique to characterizing film properties, especially electroactive polymer films. We also describe the wide range of surface chemistries and attachment strategies used by investigators to bring about surface attachment and multi-layer interactions of these thin film systems. Next we review the wide range of recent applications of the technique to: studies of complex biochemical and biomimetic systems, the creation of protein and nucleic acid biosensors, studies of attached living cells and whole cell biosensor applications. Finally, we discuss future technical directions and applications of the QCM technique to areas such as drug discovery.     

Enzymatic copolymerization alters the structure of unpolymerized mixtures of the biomimetic monomers: the amphiphilic decyl ester of L-tyrosine and L-tyrosineamide--an AFM investigation of nano- to micrometer-scale structure differences

Previously, we have shown that the amphiphilic decyl esters of both D- and L-tyrosine (DELT) self-assemble in aqueous solution above their critical micelle concentration values to form long rodlike structures that can be enzymatically polymerized. In the current study, we have examined the self-assembled structures of unpolymerized and enzymatically (horseradish peroxidase) copolymerized 1:1 molar mixtures of DELT with the nonamphiphilic comonomer L-tyrosineamide. The structures were examined following adsorption to gold-coated mica surfaces using optical microscopy and scanning electron microscopy, but primarily noncontact atomic force microscopy. Both unpolymerized and copolymerized 1:1 comonomer mixture aggregates produced amorphous to spherical shaped structures, exhibiting increased flexibility that contrasted with our previous observations of the more highly ordered long rodlike structures seen with the pure DELT. The unpolymerized comonomer aggregates were amorphous and of varying size. Interestingly, they contained occasional novel structures-smooth, sharp, nipplelike features that rose hundreds of nanometers above the smooth aggregate surface. However, upon enzymatic copolymerization, the structures are altered, forming nearly hemispherical aggregates in contact with each other on the surface. These structures possessed diameters of 1.51 +/- 0.24 microm. The copolymerized structures lacked any evidence of the sharp nipplelike features observed in the unpolymerized sample, but they did exhibit nanometer-scale detailed surface features, indicative of a higher degree of internal organization. The measured surface roughness of the copolymerized comonomer mixture was more than 10 times greater than the surface roughness of the unpolymerized comonomer mixture.     

Dissolution mechanism of supported phospholipid bilayer in the presence of amphiphilic drug investigated by neutron reflectometry and quartz crystal microbalance with dissipation monitoring

The influence and interaction of the ionizable amphiphilic drug amitriptyline hydrochloride (AMT) on a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) phospholipid bilayer supported on a silica surface have been investigated using a combination of neutron reflectometry and quartz crystal microbalance with dissipation monitoring. Adding AMT solutions with concentrations 3, 12, and 50 mM leaves the lipid bilayer mainly intact and we observe most of the AMT molecules attached to the head-group region of the outer bilayer leaflet. Virtually no AMT penetrates into the hydrophilic head-group region of the inner leaflet close to the silica surface. By adding 200 mM AMT solution, the lipid bilayer dissolved entirely, indicating a threshold concentration for the solubilization of the bilayer by AMT. The observed threshold concentration is consistent with the observation that various bilayer structures abruptly transform into mixed AMT-DOPC micelles beyond a certain AMT-DOPC composition. Based on our experimental observations, we suggest that the penetration of drug into the phospholipid bilayer, and subsequent solubilization of the membrane, follows a two-step mechanism with the outer leaflet being removed prior to the inner leaflet.     

Quartz crystal microbalance biosensor study of endothelial cells and their extracellular matrix following cell removal: Evidence for transient cellular stress and viscoelastic changes during detachment and the elastic behavior of the pure matrix

A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.     

Synthesis of tethered-polymer brush by atom transfer radical polymerization from a plasma-polymerized-film-coated quartz crystal microbalance and its application for immunosensors

This study synthesizes a tethered surface-grafted poly(acrylic acid) with quartz crystal microbalance (QCM) surfaces and provides detailed analysis of their properties and application. A tethered polyelectrolyte brush of poly(acrylic acid) is generated by first covering the substrate with a plasma-polymerized allyl alcohol (pp-AA) film, changing the polymerization initiators (bromination), and then grafting through atom transfer radical polymerization (ATRP) of tert-butyl acrylate (t-BA); these initiators are immobilized on a surface and exposed to a monomer. Finally, we convert the poly(t-BA) brush into poly(acrylic acid) through hydrolysis. We use the QCM technique to measure configuration change of the tethered poly(acrylic acid) grafted chains with two different degrees of polymerization (DP=50,200) in aqueous solutions at three different pH values (4.0, 4.8, and 5.4). The tethered poly(acrylic acid) grafted QCM shows that repeatable frequency responses are induced by pH change of solution. These frequency responses of large DP for pH are 20 times larger than responses of lower DP for pH. The frequency response of antibody immobilization on tethered poly(acrylic acid) grafted QCM (DP=200) and its frequency response of immunoreaction are 10 times larger than conventional immobilization methods by cysteamine with glutalaldehyde coupling of the antibody. The tethered poly(acrylic acid) grafted QCM can increase the frequency response for pH, the immobilization amount of antibody, and immunosensor response.     

Dynamic measurement of the surface stress induced by the attachment and growth of cells on Au electrode with a quartz crystal microbalance

The processes of adhesion, spreading and proliferation of human mammary cancer cells MCF-7 on two Au electrodes with different surface roughness (R(f) and R(f)=3.2 or 1.1) were monitored and clearly identified with the quartz crystal microbalance (QCM) technique. Analyses of the QCM responses on the resonant frequency shifts (Deltaf(0)) vs. the motional resistance changes (DeltaR(1)) revealed a significant surface-stress effect in the involved courses, in addition to a viscodensity effect and a relatively small mass effect (especially at the smooth electrode). Experiments of fluorescence microscopy, cyclic voltammetry and electrochemical impedance spectroscopy were conducted to investigate the cell population on the electrode vs. the electrode-surface roughness. Simplified equations are deduced to quantitatively evaluate the surface stress, and a novel QCM method for dynamically measuring the surface stress on an electrode in cell-culture course is thus described. It was found that the smoother surface (R(f)=1.1) gave a higher surface stress during cell attachment and less cell population on it than the rougher surface (R(f)=3.2). In addition, real-time QCM monitoring showed on the same electrode the surface stress induced by hepatic normal cells being notably higher than that caused by hepatic cancer cells at cell-attachment stage, suggesting that the surface-stress measurement can exhibit the difference of adhesion-performance between the healthy and ill-behaved cells.     

Modified polynucleotides. I. Investigation of the enzymatic polymerization of 5-alkyl-dUTP-s.

The chemical synthesis of 5-alkyl-dUTP-s and their participation as substrates in poly[d(A-6)] primed polymerization reactions with dATP by E. coli DNA polymerase I enzyme has been described. In comparison with dTTP, at saturating substrate concentrations, the rate of hypochromic effect was found to be 17.3% higher for dUTP and was lower by 27.4% for 5-ethyl-dUTP, 29.5% for 5-n-propyl-dUTP, 31.4% for 5-n-butyl-dUTP and by 85.0% for 5-n-pentyl-dUTP. No hypochromic effect could be observed, however, with 5-iso-propyl-, 5-tert.butyl- and 5-n-hexyl-dUTP-s. Polydeoxynucleotides have also been isolated from the reaction mixture and some of their structural properties determined.     

Histological and enzymatic studies on the renal tubules of some marine elasmobranchs

Renal tubules in the dog shark, leopard shark, and red skate were examined histologically and analyzed histochemically for enzymes. Cells of the distal and collecting tubules exhibit extensive interdigitations and large intercellular spaces, suggesting that these tubules are sites of sodium reabsorption. Although Na-K-ATPase is very scarce to nonexistent in the distal and collecting tubules, very intense carbonic anhydrase activity in these segments indicates that they secrete large amounts of hyrogen ion and reabsorb sodium by H⁺/Na⁺ exchange process. Epithelial cells of the necks are not interdigitated, tightly join adjacent cells, and have low enzyme activities. They seem to be passively permeable to the water. Necks are attached to the distal tubules with scant intervening stroma. It seems likely that the stroma has a high osmotic pressure resulting from absorption of solutes in the distal tubules. Water may be reabsorbed from necks to stroma because of a concentration gradient of the solutes distributed between these sites.     

Quartz crystal microbalance study of endothelial cell number dependent differences in initial adhesion and steady-state behavior: evidence for cell-cell cooperativity in initial adhesion and spreading

The quartz crystal microbalance (QCM) technique has been applied to the real time monitoring of endothelial cell (EC) adhesion and spreading on the QCM gold surface. We previously showed that the measured QCM Deltaf and DeltaR shifts were due to cells adhering to the gold crystal surface, requiring proteolytic enzyme treatment to be removed from the surface, in order for the Deltaf and DeltaR shifts to return to zero. In the present report, we demonstrate the quantitative dependence and saturation of the measured Deltaf and DeltaR shifts on the number of firmly attached ECs as measured by electronic counting of the cells. We demonstrate through a light microscope simulation experiment that the different Deltaf and DeltaR regions of the QCM temporal response curve correspond to the incident ECs contacting the surface, followed by their adhesion and spreading, which reflect cellular mass distribution and cytoskeletal viscoelasticity changes. Also, we demonstrate that the dose response curve of Deltaf and DeltaR values versus attached EC number is more sensitive and possesses less scatter for the hydrophilically treated surface compared to the native gold surface of the QCM. For both surfaces, a Deltaf and DeltaR versus trypsinized, attached EC number plot 1 h post-seeding exhibits a sigmoid curve shape whereas a similar plot 24 h post-seeding exhibits a hyperbolic curve shape. This number dependence suggests cell-cell cooperativity in the initial cell adhesion and spreading processes. These QCM data and our interpretation are corroborated by differences in cell appearance and spreading behavior we observed for ECs in a light microscope fluorescence simulation experiment of the cell density effect. For a stably attached EC monolayer at 24 h post-addition, steady-state Deltaf and DeltaR values are higher and exhibit saturation behavior for both the hydrophilically treated gold surface as compared to the untreated surface. The steady-state 24 h Deltaf and DeltaR values of stably attached ECs are shifted from the 1 h attached ECs. The 24 h values are characteristic of a more energy-dissipative structure. This is consistent with the time-dependent elaboration of surface contacts in anchorage-dependent ECs via the attachment of intregrins to underlying extracellular matrix. It is also in agreement with the known energy dissipation function of the ECs that cover the interior of blood vessels and are exposed to continuous pulsatile blood flow.     

Preparation of the methyl ester of hyaluronan and its enzymatic degradation

A methyl ester of hyaluronan in which the carboxyl groups were fully esterified was prepared using trimethylsilyl diazomethane. This derivative, while not depolymerized by hyaluronan lyases or hyaluronan hydrolases, was a substrate for both chondroitin ACI lyase (EC 4.2.2.5) from Flavobacterium heparinum and chondroitin ACII lyase (EC 4.2.2.5) from Arthrobacter aurescens. The major product isolated in these depolymerization reactions was methyl alpha-L-threo-hex-4-enepyranosyluronate-(1-->3)-2-acetamido-2-deoxy-alpha,beta-D-glucopyranoside as determined by 1H NMR spectroscopy and MALDITOF mass spectrometry.     

Ketoprofen resolution by enzymatic estirification and hydrolysis of the ester product

ImmobilizedCandida antarctica lipase was used to catalyze the separation of ketoprofen into its components by means of esterification followed by the enzymatic hydrolysis of the ester product. In this study, ketoprofen underwent esterification to ethanol in the presence of isooctane. When the reaction was complete, 58.3% of the ketoprofen had been transformed into an ester. The ketoprofen remaining in solution after the reation was complete consisted primarily of itsS-enantiomer (83.0%), while the 59.4% of the ketoprofen component of the ester consisted of itsR-enantiomer. We then subjected the ester product to enzymatic hydrolysis in the presence of the same enzyme and produced a ketoprofen product rich in theR-enantiomer; 77% of this product consisted of theR-enantiomer when 50% of the ester had been hydrolyzed, and 90% of it consisted of theR-enantiomer when 30% of the ester had been hydrolyzed. By contrast, theR-enantiomer levels only reached approximately 42 and 65%, respectively, when 50 and 30% of the racemic ester was hydrolyzed under the same conditions.     

Electrochemical quartz crystal microbalance studies on enzymatic specific activity and direct electrochemistry of immobilized glucose oxidase in the presence of sodium dodecyl benzene sulfonate and multiwalled carbon nanotubes

The electrochemical quartz crystal microbalance (EQCM) technique was utilized to monitor in situ the adsorption of glucose oxidase (GOD) and the mixture of GOD and sodium dodecyl benzene sulfonate (SDBS) onto Au electrodes with and without modification of multiwalled carbon nanotubes (MWCNTs) or SDBS/MWCNTs composite, and the relationship between enzymatic specific activity (ESA) and direct electrochemistry of the immobilized GOD was quantitatively evaluated for the first time. Compared with the bare gold electrode at which a little GOD was adsorbed and the direct electrochemistry of the adsorbed GOD was negligible, the amount and electroactivity of adsorbed GOD were greatly enhanced when the GOD was mixed with SDBS and then adsorbed onto the SDBS/MWCNTs modified Au electrode. However, the ESA of the adsorbed GOD was fiercely decreased to only 16.1% of the value obtained on the bare gold electrode, and the portion of adsorbed GOD showing electrochemical activity exhibited very low enzymatic activity, demonstrating that the electroactivity and ESA of immobilized GOD responded oppositely to the presence of MWCNTs and SDBS. The ESA results obtained from the EQCM method were well supported by conventional UV-vis spectrophotometry. The direct electrochemistry of redox proteins including enzymes as a function of their biological activities is an important concern in biotechnology, and this work may have presented a new and useful protocol to quantitatively evaluate both the electroactivity and ESA of trace immobilized enzymes, which is expected to find wider applications in biocatalysis and biosensing fields.