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Mesoporous SiO2 nanoparticles, MP-SiO2 NPs, are functionalized with the boronic acid ligand units. The pores of the MP-SiO2 NPs are loaded with the anticancer drug mitoxantrone, and the pores are capped with the anticancer drug gossypol. The resulting two-drug-functionalized MP-SiO2 NPs provide a potential stimuli-responsive anticancer drug carrier for cooperative chemotherapeutic treatment. In vitro experiments reveal that the MP-SiO2 NPs are unlocked under environmental conditions present in cancer cells, e.g., acidic pH and lactic acid overexpressed in cancer cells. The effective unlocking of the capping units under these conditions is attributed to the acidic hydrolysis of the boronate ester capping units and to the cooperative separation of the boronate ester bridges by the lactate ligand. The gossypol-capped mitoxantrone-loaded MP-SiO2 NPs reveals preferential cytotoxicity toward cancer cells and cooperative chemotherapeutic activities toward the cancer cells. The MCF-10A epithelial breast cells and the malignant MDA-MB-231 breast cancer cells treated with the gossypol-capped mitoxantrone-loaded MP-SiO2 NPs revealed after a time-interval of 5 days a cell death of ca. 8% and 60%, respectively. Also, the gossypol-capped mitoxantrone-loaded MP-SiO2 NPs revealed superior cancer-cell death (ca. 60%) as compared to control carriers consisting of β-cyclodextrin-capped mitoxantrone-loaded (ca. 40%) under similar loading of the mitoxantrone drug. The drugs-loaded MP-SiO2 NPs reveal impressive long-term stabilities.

A new sensitive surface-enhanced Raman scattering (SERS) assay for detection of cholinesterase inhibitors such as organophosphorous pesticides using silver colloidal nanoparticles was developed and optimized. Acetylcholinesterase (AChE) mediated the hydrolysis of acetylthiocholine to produce thiocholine, which interacted with the silver nanoparticles to give a specific SERS spectrum. Variation in enzyme activity due to inhibition was measured from changes in intensity of a characteristic peak (772 cm −1 ) of the SERS spectrum that was directly correlated with the concentration of produced thiocholine. The method was demonstrated for the detection of paraoxon as reference AChE inhibitor. Limit of detection of paraoxon for 5 min incubation at 25 °C was 1.8 × 10 −8 M. This assay can be utilized for the detection of trace amounts of any AChE inhibitor.

Fluorescent semiconductor nanocrystals (q-dots) benefit from practical features such as high fluorescence intensity, broad excitation band and emission diameter dependency. These unique spectroscopic characterizations make q-dots excellent candidates for new fluorescent labels in multi-chromatic analysis, such as Flow-Cytometry (FCM). In this work we shall present new possibilities of multi-labeling and multiplex analysis of pathogenic bacteria, by Flow-Cytometry (FCM) analysis and new specific IgG-q-dots conjugates. We have prepared specific conjugates against B. anthracis spores (q-dots585-IgGalphaB. anthracis and q-dots655-IgGalphaB.anthracis). These conjugates enabled us to achieve double staining of B. anthracis spores which improve the FCM analysis specificity versus control Bacillus spores. Moreover, multiplexed analysis of B. anthracis spores and Y. pestis bacteria was achieved by using specific antibodies labeled with different q-dots to obtain: q-dots585-IgGalphaB. anthracis and q-dots655-IgGalphaY.pestis, each characterized by its own emission peak as a marker. Specific and sensitive multiplex analysis for both pathogens has been achieved, down to 10(3) bacteria per ml in the sample.

Anticancer drug gossypol cross-links phenylboronic acid-modified acrylamide copolymer chains to form a hydrogel matrix. The hydrogel is dissociated in an acidic environment (pH 4.5), and its dissociation is enhanced in the presence of lactic acid (an α-hydroxy carboxylic acid) as compared to formic acid. The enhanced dissociation of the hydrogel by lactic acid is attributed to the effective separation of the boronate ester bridging groups through the formation of a stabilized complex between the boronic acid substituent and the lactic acid. Because lactic acid exists in cancer cells in elevated amounts and the cancer cells' environment is acidic, the cross-linked hydrogel represents a stimuli-responsive matrix for the controlled release of gossypol. The functionality is demonstrated and characterized by rheology and other spectroscopic means.

Photosensitizer/electron acceptor molecular cross-linked Au-nanoparticle arrays are assembled on indium-doped tin oxide (ITO) electrodes by a layer-by-layer deposition process. A Ru(II)−tris-(2,2‘-bipyridine)-cyclobis(paraquat-p-phenylene) catenane (1) or Zn(II)-protoporphyrin IX−bis(N-methyl-N‘-undecanoate-4,4‘-bipyridinium) (2) are used as molecular cross-linkers for the generation of Au-nanoparticle (13 ± 1 nm) arrays of a controlled number of layers. The Au-nanoparticle arrays are characterized by absorbance spectroscopy and by electrochemical means. The electrodes functionalized with 1- or 2-cross-linked Au-nanoparticle arrays are used in photoelectrochemical experiments. The resulting action spectra of the photocurrents follow the absorbance spectra of the respective chromophores. Mechanistic studies indicate that the photocurrents originate from intramolecular electron-transfer quenching of the photoexcited state of the photosensitizer by the electron acceptor units, leading to the formation of int...

Reconstitution of an apo-flavoenzyme with a relay-FAD-cofactor dyad yields an electrically contacted enzyme, “electroenzyme”. This is exemplified by the reconstitution of apo-glucose oxidase, apo-GOx, with a ferrocene–tethered FAD-cofactor. The resulting semisynthetic protein stimulates the bioelectrocatalyzed oxidation of glucose. Kinetic analysis of the ferrocene–FAD reconstituted enzyme reveals that its electrical communication with an electrode support is superior as compared to a protein randomly functionalized by ferrocene relay units. A pyrroloquinolinoquinone (PQQ)–FAD cofactor monolayer is assembled on a Au-electrode. Apo-GOx is reconstituted on the solid support to yield an aligned enzyme electrode of unprecendently efficient electrical contact. The electron-transfer turnover rate with the electrode is estimated to be 900±150 s−1, a value close to the electron transfer rate between native GOx and molecular oxygen. The effective electrical communication of the integrated enzyme electrode stimulates the efficient bioelectrocatalyzed oxidation of glucose, and results in high current densities and high sensitivity for glucose. The effective electrical contact of the enzyme-electrode yields also a specific glucose sensing electrode that is not perturbed by oxygen or interferrants. The possible application of the enzyme-electrode as an invasive biosensor is addressed.

A molecular rectifier and a bioelectrocatalytic assembly for the reduction of NO3- is provided by reconstitution of a de novo protein with two FeIII -protoporphyrin IX units. The function of the de novo protein can be tuned and tailored within the synthetic protocol.

Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers. A catalyst-NAD(+)-dyad for the binding of the NAD(+)-dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems. NAD(+)-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD+ monolayer. The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N6-(2-aminoethyl)-NAD+ to the monolayer. The interface modified with the PQQ-NAD(+)-dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer. The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH. Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C. 1.9.6.1 from E. coli) or CoII-protoporphyrin IX reconstituted myoglobin (CoII-Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode. The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules. The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and CoII-Mb show catalytic activities for NO3- reduction and acetylene-dicarboxylic acid hydrogenation, respectively. Two FeIII-protoporphyrin IX units are reconstituted into a four alpha-helix bundle de novo protein assembled as a monolayer on a Au-electrode. Vectorial electron transfer proceeds in the synthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the FeIII-protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate.

Electrical communication of the redox-active center of enzymes with an electrode surface is a fundamental element for the development of amperometric biosensor devices. Different methods to assemble enzyme-electrodes exhibiting electrical contact between the redox protein and electrode surface are discussed with specific examples for tailoring glucose sensing electrodes. By one approach, a multilayer array of glucose oxidase is assembled on a Au-electrode. The number of enzyme layers is controlled by the synthetic methodology to assemble the electrode. Electrical contact between the enzyme array and the electrode is established by chemical modification of the protein layer with N -(2-methyl-ferrocene)-caproic acid, acting as an electrode is established by chemical enzyme layers associated with the electrode allows one to control the sensitivity of the resulting enzyme electrode. A further means of enhancing the sensitivities of enzyme electrodes involves the application of rough Au-electrodes as base-support to assemble the enzyme network. The high surface area of these electrodes (roughness factor ≈ 20) allows the increase of the biocatalyst content in a single monolayer, and the resulting amperometric responses of the electrodes are ca. 8-fold enhanced compared to enzyme layers assembled on smooth electrodes of identical geometrical areas. A novel method to electrically wire flavoenzymes with electrode surfaces was developed by reconstitution of the apo-flavoenzyme with a ferrocene-tethered FAD diad. Reconstitution of apo-glucose oxidase with the ferrocene-FAD diad yields an active bioelectrocatalyst of direct electrical communication with the electrode, ‘electroenzyme’. The reconstitution methodology was further applied to tailor enzyme-electrodes of superior properties for electrical contact with the electrodes. A pyrroloquinoline quinone-FAD diad monolayer was assembled on a Au-electrode. Apo-glucose oxidase was reconstituted on the surface with the FAD-cofactor site to yield the aligned biocatalyst on the electrode. The pyrroloquinoline quinone. PQQ, redox unit acts as an electron relay that electrically contacts the FAD redox-site of the enzyme with the electrode. The surface reconstituted enzyme exhibits direct electrical communication with the electrode and acts as bioelectrocatalyst for the oxidation of glucose. The electrical communication of the reconstituted glucose oxidase on the PQQ-FAD monolayer is extremely efficient. The experimental current density at a glucose concentration of 80 mM is 300 ± 100 μ A · cm −2 . This value overlaps the theoretical current density of glucose oxidase electrode (290 ± 60 μ A · cm −2 ) taking into account the limiting turnover-rate of the enzyme, 900 ± 150 s −1 (at 35°C). The extremely efficient electrical contact of the reconstituted enzyme and the electrode yields an enzyme-electrode that is insensitive to oxygen and is not affected by glucose-sensing interferants such as ascorbic acid. The application of the different enzyme-electrode configurations as bioelectronic devices for the determination of glucose is addressed.

Microperoxidase-11, MP-11, is assembled as a monolayer on an Au electrode. The resulting MP-11 monolayer electrode mediates the electrocatalyzed reduction of CoII–protoporphyrin IX reconstituted myoglobin, CoII–Mb. The electrocatalyzed reduction of CoII–Mb proceeds via the formation of a complex between the MP-11 monolayer and CoII–Mb. The association constant of the complex formed between MP-11 and CoII–Mb is Ka = 1.6 × 105M–1. Cross-linking of the complex formed between CoII–Mb and the MP-11 monolayer with glutaric dialdehyde yields a stable integrated electrocatalytic electrode for the stereospecific hydrogenation of acetylenedicarboxylic acid to maleic acid (current yield is ca. 80%). The electrocatalyzed hydrogenation of acetylenedicarboxylic acid reveals an isotope effect, kH/kD ≈ 2.7, suggesting the insertion of the substrate to a CoIII–H species in the rate-limiting step of the hydrogenation process.

Functionalized monolayer electrodes provide the grounds for bioelectronic and optobioelectronic devices. Reconstitution of apo-glucose oxidase, apo-GOx, onto a pyrroloquinoline quinone-FAD diad, assembled as a monolayer on a Au-electrode, yields an aligned bioelectrocatalytically active enzyme on the electrode surface. The resulting reconstituted enzyme electrode exhibits superior electrical contact with the electrode surface and acts as an amperometric glucose sensing electrode. The enzyme electrode operates under oxygen and is unaffected by interfering substrates such as ascorbic acid. Photoswitchable redox proteins integrated with electrode surfaces act as active systems for the amplified amperometric transduction of recorded optical signals. Chemical modification of glucose oxidase by photoisomerizable nitrospiropyran units or reconstitution of apo-GOx with a photoisomerizable nitrospiropyran-FAD diad, yield photoisomerizable glucose oxidase with photoswitchable biocatalytic features. Assembly of the photoactive enzymes as monolayers on the Au-electrode results in functionalized surfaces for the cyclic ‘ON-OFF’ amplified amperometric transduction of recorded optical signals. A further method to photostimulate the electrical contact between a redox protein and an electrode involves the functionalization of the electrode with a photoisomerizable monolayer interface. A mixed monolayer consisting of pyridine and nitrospiropyran units was used to photoregulate the association and dissociation of cytochrome c to and from the monolayer assembly. The photostimulated electrical contact of cytochrome c with the monolayer electrode was employed to mediate the bioelectrocatalyzed reduction of oxygen in the presence of cytochrome oxidase, COx. The latter system provides an assembly for the cyclic amperometric transduction of recorded optical signals.

Eosin-modified reconstructed Co(II)-myoglobin, Eo 2− -Co(II)-Mb acts as photoenzyme for selective H 2 -evolution, hydrogenation and CO 2 -fixation.

The study of direct electrical communication between reaction centers of enzymes and electrodes bridges fundamental work on electron transfer through proteins and practical work on biosensors.1,2 Such electrical communication has been realized earlier through modification of enzymes with electron relays,3,4 through modification of their peripheral oligosaccharides with relays pendant on termini of flexible spacer chains,5 and through relays in electron-conducting hydrogels within which enzymes were covalently bound.6 Recently, electrical communication, through a defined molecular path, was achieved by reconstituting apo-glucose and apo-D-amino acid oxidases with a ferrocenemodified FAD cofactor.7 Here we report a novel method to assemble an enzyme electrode by reconstitution of apo-glucose oxidase on a pyrroloquinoline quinone/FAD (PQQ/FAD) monolayer associated with an Au-electrode. The resulting enzyme layer reveals efficient electrical contact with the electrode and stimulates effectively the electrobiocatalyzed oxidation of glucose. The monolayer of glucose oxidase (GOx) electrically connected to the Au-electrode, was formed as shown in Scheme 1. A base cystamine submonolayer was bound to a roughened gold foil having a solution-exposed surface area of 6 ( 2 cm2 (0.4 ( 0.04 cm2 geometric area, 15 ( 5 roughness coefficient).8 The base layer was first reacted with pyrroloquinoline quinone (PQQ),9 and the resulting PQQ submonolayer was then reacted with N6-(2-aminoethyl)-FAD to yield the PQQ/FAD diad layer. The diad exhibited at pH ) 7 the two expected redox waves, one characteristic of the PQQ/PQQH2 functions at -0.125 V (vs. SCE) and the second characteristic of FAD/FADH2 at -0.50 V (vs. SCE). Integration of the redox waves obtained by voltammetry showed the presence of 5.5 × 10-10 mol cm-2 of both PQQ/PQQH2 and FAD/FADH2, both in excess of the 1.7 × 10-12 mol cm-2 of GOx that can be maximally packed in a monolayer. Treatment of the submonolayer with apo-glucose oxidase10 produced the reconstituted electrically connected and densely packed GOx monolayer. Because part of the FAD/ FADH2 functions was now bound within the electrically insulating glycoprotein of the enzyme, the FAD/FADH2 redox waves were suppressed, while the PQQ/PQQH2 waves were not.11 The surface coverage of the Au-surface by the reconstituted GOx was determined by microgravimetric, quartz crystal microbalance (QCM) analysis. The PQQ/FAD diad was assembled onto Au-electrodes (0.2 cm2, roughness factor 3) associated with a quartz crystal (9 MHz). From the observed frequency change of the crystal upon reconstitution of apo-GOx on the surface (∆f ) -150 Hz), the surface coverage of the enzyme was calculated to be 1.5 × 10-12 mol cm-2, consistent * Author to whom correspondence should be addressed: fax 972-26527715 or 972-2-6585345. (1) (a) Heller, A. Acc. Chem. Res. 1990, 23, 128. (b) Heller, A. J. Phys. Chem. 1992, 96, 3579. (2) Willner, I.; Willner, B. React. Polym. 1994, 22, 267. (3) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615. (4) (a) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114, 10 965. (b) Willner, I.; Lapidot, N.; Riklin, A.; Kasher, R.; Zahavy, E.; Katz, E. J. Am. Chem. Soc. 1994, 116, 1428. (c) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. AdV. Mater. 1993, 5, 912. (5) Schuhmann, W.; Ohara, T.; Heller, A.; Schmidt, H.-L. J. Am. Chem. Soc. 1991, 113, 1394. (6) (a) De Lumley-Woodyear, T.; Rocca, P.; Lindsay, J.; Dror, Y.; Freeman, A. Anal. Chem. 1995, 67, 1332. (b) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66, 2451. (c) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1993, 65, 3512. (d) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5970. (e) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5976. (f) Gregg, B. A.; Heller, A. Anal. Chem. 1990, 62, 258. (7) Riklin, A.; Katz, E.; Willner, I.; Stocker A.; Buckmann, A. F. Nature 1995, 376, 672. (8) The Au-rough-electrodes were prepared according to the published procedure (cf., Riklin, A.; Willner, I. Anal. Chem. 1995, 67, 4118). (9) (a) Willner, I.; Riklin, A. Anal. Chem. 1994, 66, 1535. (b) Katz, E.; Schlereth, D. D.; Schmidt, H.-L. J. Electroanal. Chem. 1994, 367, 59. (10) Reconstitution of the enzyme on the electrode was accomplished by shaking the PQQ/FAD monolayer electrode with apo-GOx, 4 mg mL-1 in 0.1 M phosphate buffer, pH ) 7.0, for 4 h at 25 °C and 12 h at 4 °C. The electrode was rinsed by shaking the resulting electrode for 1 h in 0.1 M phosphate buffer, pH ) 7.0, at 0 °C. (11) Coulometric assay of the FAD/FADH2 and PQQ/PQQH2 waves after reconstitution with apo-GOx reveal that only ca. 10% of the FAD units are electrically accessible and the remainder are electrically insulated. We assume that not only FAD/FADH2 units bound within the protein but also external free FAD sites are electrically shielded by the reconstituted protein. Scheme 1. Reconstitution of Glucose Oxidase onto a PQQ/FAD Monolayer Au-Electrode and Direct Electrocatalyzed Oxidation of Glucose by the Modified Electrode 10321 J. Am. Chem. Soc. 1996, 118, 10321-10322