Bioimprinting strategies: From soft lithography to biomimetic sensors and beyond

Imprinting is a straightforward, yet a reliable technique to develop dynamic artificial recognition materials—so called as synthetic antibodies. Surface imprinting strategies such as soft lithography allow biological stereotyping of polymers and sol–gel phases to prepare extremely selective receptor layers, which can be combined with suitable transducer systems to develop high performance biomimetic sensors. This article presents an overview of the remarkable technical advancements in the field of surface bioimprinting with particular emphasis on surface imprinted bioanalyte detection systems and their applications in rapid bioanalysis and biotechnology. Herein, we discuss a variety of surface imprinting strategies including soft lithography, template immobilization, grafting, emulsion polymerization, and others along with their biomimetic sensor applications, merits and demerits. The pioneering research works on surface patterned biosensors are described with selected examples of detecting biological agents ranging from small biomolecules and proteins to living cells and microorganisms.     

Manganese substituted Compound I of cytochrome P450 biomimetics: A comparative reactivity study of Mn-oxo versus Mn-oxo species

Manganese-oxo porphyrins have been well studied as biomimetic models of cytochromes P450 and are known to be able to catalyze substrate hydroxylation reactions. Recent experimental studies [J.Y. Lee, Y.-M. Lee, H. Kotani, W. Nam, S. Fukuzumi, Chem. Commun. (2009) 704] showed that Mn(V)-oxo porphyrins react rapidly with 10-methyl-9,10-dihydroacridine (AcrH₂) via a proton-coupled-electron-transfer followed by an electron transfer. In this work, we present a computational study on the reactivity patterns of Mn(V)-oxo and Mn(IV)-oxo with respect to AcrH₂. This study shows that although both oxidants are capable of hydroxylating AcrH₂, the Mn^V-oxo species is the more active oxidant. We have generalized these observations with thermodynamic cycles that explain the reaction mechanisms and electron transfer processes. For the Mn^V-oxo mechanism the reactions proceed with a fast spin state crossing from the ground state singlet to the triplet spin state prior to a hydrogen atom transfer followed by another electron transfer. The present results are fully consistent with previous studies on iron-oxo porphyrins and manganese-oxo porphyrins and shows that the interplay of low lying singlet and triplet spin state surfaces influences the reaction mechanisms and kinetics.     

Improved stability of the carbon nanotubes-enzyme bioconjugates by biomimetic silicification

Nano-materials have been applied in many fields due to their excellent characteristics, such as the high surface area-to-volume ratio, excellent physicochemical properties and biological compatibility. In this study, multi-walled carbon nanotubes (MWCNTs) were utilized to prepare MWCNTs-papain bioconjugates and then realized the immobilization of papain. MWCNTs functionalized with carboxyl- and amine- groups on their surface were used as immobilization carriers. The immobilization of papain on the functionalized MWCNTs through physical absorption was examined. The conjugates were denoted as MWCNTs-papain bioconjugates. To improve the stability, the bioconjugates were further coated by silica through the biomimetic silicification process that induced by papain (denoted as silica-coated bioconjugates). The as-prepared MWCNTs-papain bioconjugates and the silica-coated bioconjugates were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The preliminary results showed that the bioconjugates could retain most of the initial activity of papain. Compared to free papain and MWCNTs-papain bioconjugates, the silica-coated bioconjugates exhibited significantly improved thermal, pH and recycling stability. Comparisons of the kinetic parameters between MWCNTs-papain bioconjugates and the silica-coated bioconjugates revealed that the K_m value of the immobilized papain experienced a slight increase after silica coating, which suggested that the silica coating did not significantly hinder papain's access to substrate or release of product.     

Efficient Preparation of Giant Vesicles as Biomimetic Compartment Systems with High Entrapment Yields for Biomacromolecules

The ‘lipid‐coated ice‐droplet hydration method’ was applied for the preparation of milliliter volumes of a suspension of giant phospholipid vesicles containing in the inner aqueous vesicle pool in high yield either calcein, α‐chymotrypsin, fluorescently labeled bovine serum albumin or dextran (FITC‐BSA and FITC‐dextran; FITC=fluorescein isothiocyanate). The vesicles had an average diameter of ca. 7–11 μm and contained 20–50% of the desired molecules to be entrapped, the entrapment yield being dependent on the chemical structure of the entrapped molecules and on the details of the vesicle‐formation procedure. The ‘lipid‐coated ice droplet hydration method’ is a multistep process, based on i) the initial formation of a monodisperse water‐in‐oil emulsion by microchannel emulsification, followed by ii) emulsion droplet freezing, and iii) surfactant and oil removal, and replacement with bilayer‐forming lipids and an aqueous solution. If one aims at applying the method for the entrapment of enzymes, retention of catalytic activity is important to consider. With α‐chymotrypsin as first model enzyme to be used with the method, it was shown that high retention of enzymatic activity is possible, and that the entrapped enzyme molecules were able to catalyze the hydrolysis of a membrane‐permeable substrate which was added to the vesicles after their formation. Furthermore, one of the critical steps of the method that leads to significant release of the molecules from the water droplets was investigated and optimized by using calcein as fluorescent probe.     

Inorganic-binding peptides as tools for surface quality control

This paper highlights an innovative application of inorganic-binding peptides as quality control tools for detecting defects on inorganic surfaces of any shape. The approach involves attaching a fluorescent label to an inorganic-binding peptide and exploiting the peptide's high binding specificity to detect, by simple fluorescence microscopy, chemical composition defects of µm size and crystallographic state defects. Proof of concept was demonstrated by monitoring binding of a previously isolated ZnO-binding peptide to galvanized steel substrates. The approach was further validated for TiO₂ coatings and stainless steel, with two new, specific inorganic-binding peptides isolated by phage display.     

Catecholase activity of a μ-hydroxodicopper(II) macrocyclic complex: structures, intermediates and reaction mechanism

The monohydroxo-bridged dicopper(II) complex (1), its reduced dicopper(I) analogue (2) and the trans-μ-1,2-peroxo-dicopper(II) adduct (3) with the macrocyclic N-donor ligand [22]py4pz (9,22-bis(pyridin-2′-ylmethyl)-1,4,9,14,17,22,27,28,29,30- decaazapentacyclo -[22.2.1^14,7.1^11,14.1^17,20]triacontane-5,7(28),11(29),12,18,20(30), 24(27),25-octaene), have been prepared and characterized, including a 3D structure of 1 and 2. These compounds represent models of the three states of the catechol oxidase active site: met, deoxy (reduced) and oxy. The dicopper(II) complex 1 catalyzes the oxidation of catechol model substrates in aerobic conditions, while in the absence of dioxygen a stoichiometric oxidation takes place, leading to the formation of quinone and the respective dicopper(I) complex. The catalytic reaction follows a Michaelis–Menten behavior. The dicopper(I) complex binds molecular dioxygen at low temperature, forming a trans-μ-1,2-peroxo-dicopper adduct, which was characterized by UV–Vis and resonance Raman spectroscopy and electrochemically. This peroxo complex stoichiometrically oxidizes a second molecule of catechol in the absence of dioxygen. A catalytic mechanism of catechol oxidation by 1 has been proposed, and its relevance to the mechanisms earlier proposed for the natural enzyme and other copper complexes is discussed. Electronic Supplementary Material Supplementary material is available for this article at     

Supercontraction forces in spider dragline silk depend on hydration rate

Spider dragline silk is a model biological polymer for biomimetic research due to its many desirable and unusual properties. ‘Supercontraction’ describes the dramatic shrinking of dragline silk fibers when wetted. In restrained silk fibers, supercontraction generates substantial stresses of 40–50 MPa above a critical humidity of 70% relative humidity (RH). This stress may maintain tension in webs under the weight of rain or dew and could be used in industry for robotics, sensor technology, and other applications. Our own findings indicate that supercontraction can generate stress over a much broader range than previously reported, from 10 to 140 MPa. Here we show that this variation in supercontraction stress depends upon the rate at which the environment reaches the critical level of humidity causing supercontraction. Slow humidity increase, over several minutes, leads to relatively low supercontraction stress, while fast humidity increase, over a few seconds, typically results in higher supercontraction stress. Slowly supercontracted fibers take up less water and differ in thermostability from rapidly supercontracted fibers, as shown by thermogravimetric analysis. This suggests that spider silk achieves different molecular configurations depending upon the speed at which supercontraction occurs. Ultimately, rate-dependent supercontraction may provide a mechanism to tailor the properties of silk or biomimetic fibers for various applications.     

High-level cell-free expression and functional characterization of a novel aquaporin from Photobactetrium profundum SS9

AqpSS9 is a novel aquaporin derived from the deep sea bacterium Photobactetrium profundum SS9 and has attracted many attentions in developing water filtration biomimetic membranes. Functional characterization of AqpSS9 was carried out first by its expression in E. coli MM1211 (aqpZ⁻). Results showed that it was similar to bacterial aquaporin Z (AqpZ) and functioned as a real aquaporin. In-vitro expression of AqpSS9 were systematically investigated using three different modes: precipitate-based cell-free (P-CF) mode, the detergent-based cell-free (D-CF) mode and lipid-based cell-free expression mode (L-CF). D-CF mode showed more superiority than P-CF and L-CF mode, and the highest expression level of 571 mg/l was achieved by adding 0.7% Brij-78. Then AqpSS9 was purified by affinity chromatograph and incorporated into DOPC liposomes. Osmotic water permeability values (P_f) of reconstituted AqpSS9 proteoliposomes was measured as 310.7 ± 3.2 μm/s, which was about 3.5 times of empty control liposomes and comparable to reported Aqps. The AqpSS9 embedded layer-by-layer (LbL) membrane was fabricated and tested, which showed enhanced water permeability and salt rejection in comparison with the control membrane. This work demonstrated the good performance of AqpSS9 as a water channel protein, which may become an alternative candidate for biomimetic membrane construction for water filtration.     

Oxidation of methionines by oxochromium(V) cations: a kinetic and spectral study

The oxidation of methionine (Met) plays an important role during biological conditions of oxidative stress as well as for protein stability. By choosing [oxo(salen)chromium(V)] ions, [(salen)Cr(V)=O](+) (where salen = bis(salicylidene)ethylenediamine) as suitable biomimics for the peptide complexes that are formed during the reduction of Cr(VI) with biological reductants, the oxidation of methionine and substituted methionines with five [oxo(salen)chromium(V)] complexes in aqueous acetonitrile has been investigated by spectrophotometric, electron paramagnetic resonance (EPR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) methods. In aqueous solution [(salen)Cr(V)=O](+) ion is short lived, ligation of H(2)O to the Cr center takes place and [O=Cr(V)(salen)-H(2)O](+) adduct is the active oxidant. The reaction is found to be first order each in the oxidant and the substrate. The presence of water in the reaction system accelerates the reaction rate and an inactive, stable mu-oxo dimer is also formed during the course of the reaction. On the basis of spectral, kinetic and product analysis study a mechanism involving direct oxygen transfer from [O=Cr(V)(salen)-H(2)O](+) to methionine has been proposed as a suitable mechanism for the reaction.     

Self-organized Polymer Aggregates with a Biomimetic Hierarchical Structure and its Superhydrophobic Effect

Nature always gives us inspirations to fabricate functional materials by mimicking the structure design of biomaterials. In this article, we report that polymeric aggregates with morphology similar to the papilla on lotus leaf can be self-organized in the polymer solution by adding 16 wt% water into 5 mg/ml polycarbonate solution in N, N′-dimethylformamide. The hierarchically structured aggregates at micro- and nano-scale alone show superhydrophobic effect without the need of modification with low surface energy compound. Small amount of liquid can be wrapped by the aggregates to form the so-called liquid marble. Influence of the amount of water added into the solution on the morphology of resultant polymer aggregates was investigated. By using the hierarchical aggregates as the surface building blocks, superhydrophobic coating with a static water contact angle larger than 160° and sliding angle less than 5° (for a water drop of 5 μl) was formed. Other solutions, like acid, basic and blood plasma are also repelled on the coating.