Biomolecule-embedded metal-organic frameworks as an innovative sensing platform

Technological advancements combined with materials research have led to the generation of enormous types of novel substrates and materials for use in various biological/medical, energy, and environmental applications. Lately, the embedding of biomolecules in novel and/or advanced materials (e.g., metal-organic frameworks (MOFs), nanoparticles, hydrogels, graphene, and their hybrid composites) has become a vital research area in the construction of an innovative platform for various applications including sensors (or biosensors), biofuel cells, and bioelectronic devices. Due to the intriguing properties of MOFs (e.g., framework architecture, topology, and optical properties), they have contributed considerably to recent progresses in enzymatic catalysis, antibody-antigen interactions, or many other related approaches. Here, we aim to describe the different strategies for the design and synthesis of diverse biomolecule-embedded MOFs for various sensing (e.g., optical, electrochemical, biological, and miscellaneous) techniques. Additionally, the benefits and future prospective of MOFs-based biomolecular immobilization as an innovative sensing platform are discussed along with the evaluation on their performance to seek for further development in this emerging research area.     

Glyptal as a support for enzyme immobilisation

Glyptal, a polyester obtained from phthalic anhydride and glycerol, was used as a support for protein immobilisation. Hydrazide groups were introduced in the polymer and then converted to azide groups, through which protein was covalently immobilised. Amyloglucosidase was used as a model and an insoluble water derivative was synthesised retaining 24 % of the specific activity of the native enzyme. Some properties of this immobilised enzyme were studied: Km (4.54 g.l^–1 using starch as substrate), optimal temperature (55°C) and half life (8 days). Furthermore, ferromagnetic-azide-glyptal derivative showed to be useful for the amyloglucosidase immobilisation.     

Amplified electrochemical aptasensor taking AuNPs based sandwich sensing platform as a model

Here, we report a sensitive amplified electrochemical impedimetric aptasensor for thrombin, a kind of serine protease that plays important role in thrombosis and haemostasis. For improving detection sensitivity, a sandwich sensing platform is fabricated, in which the thiolated aptamers are firstly immobilized on a gold substrate to capture the thrombin molecules, and then the aptamer functionalized Au nanoparticles (AuNPs) are used to amplify the impedimetric signals. Such designed aptamer/thrombin/AuNPs sensing system could not only improve the detection sensitivity compared to the reported impedimetric aptasensors but also provide a promising signal amplified model for aptamer-based protein detection. In this paper, we realize a sensitive detection limit of 0.02 nM, with a linear range of 0.05-18 nM. Meanwhile, the effect of 6-mercaptohexanol (MCH) and 2-mercaptoethanol (MCE) on the modification of the electrode is investigated.     

Au-nanoparticles as an electrochemical sensing platform for aptamer-thrombin interaction

A novel electrochemical method for the detection of bioaffinity interactions based on a gold-nanoparticles sensing platform and on the usage of stripping voltammetry technique was developed. The oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. As a model, thrombin-thrombin binding aptamer couple was chosen. The aptamer was immobilized on a screen-printed electrode modified with gold-nanoparticles by avidin-biotin technology. Cathodic peak area was found proportional to thrombin quantity specifically adsorbed onto electrode surface. Sigmoid calibration curve as is typical for immunoassay was obtained, with thrombin detection limit of 10(-9)M. Linear range corresponds from 10(-8) to 10(-5)M thrombin concentration or 2 x 10(-14) to 2 x 10(-11)mol/electrode (R=0.996). Binding of thrombin to an aptamer has also been detected using the ferricyanide/ferrocyanide redox couple as electrochemical indicator.     

Polyvinyl alcohol-glutaraldehyde network as a support for protein immobilisation

Polyvinyl alcohol-glutaraldehyde (PVA-glut) network was synthesised in bead and disc forms and used for protein immobilisation. Xanthine oxidase, a-amylase and amyloglucosidase were covalently fixed on the beads yielding preparations with specific activities retention of 72.3%, 1.6% and 1.4%, respectively. Km of xanthine oxidase PVA-glut beads (24 ± 4 µM) was slightly higher than that estimated for the soluble enzyme (16 ± 2 mM). Antigens from Schistosoma mansoni were covalently fixed onto PVA-glut discs and ELISA was carried out. The relationship between the amount of fixed antigen and the results obtained by ELISA showed a hyperbolic curve and the saturation of antigen-antibody complex was achieved at the plateau.This revised version was published online in October 2005 with corrections to the Cover Date.     

Enzyme integrated silicate-Pt nanoparticle architecture: a versatile biosensing platform

A novel 3-D nanoarchitectured platform based on Pt nanoparticles (nPts) is developed for the sensing of sub-nanomolar levels of hydrogen peroxide and for the fabrication of amperometric biosensor for uric acid, cholesterol and glucose. The nPts have been immobilized on the thiol functional group containing sol-gel silicate 3-D network derived from 3-mercaptopropyltrimethoxysilane (MPTS). The nanoparticles on the 3-D architecture have size distribution between 7 and 10nm. The nPts on the platform efficiently catalyze the oxidation of H(2)O(2) at the potential of +0.45 V in the absence of enzymes and redox mediators. This nanoarchitectured platform is highly sensitive and can detect H(2)O(2) at sub-nanomolar levels (0.1 nM) in neutral solution. The nanoarchitectured platform does not suffer from interference due to other common easily oxidizable interfering agents. Excellent reproducibility, long-term storage and operational stability are observed. This platform is used to determine H(2)O(2) concentration in rainwater and for the fabrication of biosensors. Amperometric oxidase-based biosensing platforms are developed by integrating the enzymes and nPts with the silicate network for the sensing of uric acid cholesterol and glucose. The enzyme encapsulated 3-D architecture retains the enzymatic activity and efficiently detects enzymatically generated H(2)O(2) without any interference. These biosensors are stable and show excellent sensitivity and fast response time. A linear response was obtained for a wide concentration range of all analytes. The practical utilization of the biosensor for the measurement of uric acid, cholesterol and glucose in serum sample is demonstrated. The biological sample analysis was validated with clinical laboratory measurements.     

A bioinspired autonomous swimming robot as a tool for studying goal-directed locomotion

The bioinspired approach has been key in combining the disciplines of robotics with neuroscience in an effective and promising fashion. Indeed, certain aspects in the field of neuroscience, such as goal-directed locomotion and behaviour selection, can be validated through robotic artefacts. In particular, swimming is a functionally important behaviour where neuromuscular structures, neural control architecture and operation can be replicated artificially following models from biology and neuroscience. In this article, we present a biomimetic system inspired by the lamprey, an early vertebrate that locomotes using anguilliform swimming. The artefact possesses extra- and proprioceptive sensory receptors, muscle-like actuation, distributed embedded control and a vision system. Experiments on optimised swimming and on goal-directed locomotion are reported, as well as the assessment of the performance of the system, which shows high energy efficiency and adaptive behaviour. While the focus is on providing a robotic platform for testing biological models, the reported system can also be of major relevance for the development of engineering system applications.     

Microfluidic chip-based nanoelectrode array as miniaturized biochemical sensing platform for prostate-specific antigen detection

A microfluidic biosensor chip with an embedded three-electrode configuration is developed for the study of the voltammetric response of a nanoelectrode array with controlled inter-electrode distance in a nanoliter-scale sample volume. The on-chip three-electrode cell consists of a 5 × 5 array of Au working nanoelectrodes with radii between 60 and 120 nm, a Cl(2)-plasma-treated Ag/AgCl reference electrode, and a Au counter electrode. The nanoelectrode array is fabricated by creating high-aspect-ratio pores through an alumina insulating layer using an I(2) gas-assisted focused-ion-beam (FIB) milling, ion beam sculpting, and electrodeposition of Au. The glass substrate with the electrode pattern is assembled with a polydimethylsiloxane (PDMS) microchannel slab giving a volume of 180 nL for each channel. Cyclic voltammetry calibration with a standard redox species exhibits a significant increase of current density by two orders of magnitude compared to that obtained from a microelectrode. On-chip functionalization of the nanoelectrodes with a prostate-specific antigen (PSA) biosensor complex and detection of PSA based on a competitive immunoassay method are performed. The detection limit is approximately 10 pg/mL (～270 fM), which corresponds to roughly 30,000 copies of PSA in the microchannel test volume.     

Hydrophobised sawdust as a carrier for immobilisation of the hydrocarbon-oxidizing bacterium Rhodococcus ruber

Pine sawdust treated by a series of hydrophobising agents (drying oil, organosilicon emulsion, n-hexadecane and paraffin) was examined as carrier for adsorption immobilisation of hydrocarbon-oxidizing bacterial cells Rhodococcus ruber. It was shown that hydrophobising agents based on drying oil turned out to be optimal (among the other modifiers examined) for the preparation of sawdust carriers suitable for the efficient immobilisation. The results obtained demonstrate promising possibilities in developing a wide range of available and cheap, biodegradable cellulose-containing carriers that possess varying surface hydrophobicity.     

Signal-enhancer molecules encapsulated liposome as a valuable sensing and amplification platform combining the aptasensor for ultrasensitive ECL immunoassay

An innovatory ECL immunoassay strategy was proposed to detect the newly developing heart failure biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP). Firstly, this strategy used small molecules encapsulated liposome as immune label to construct a sandwich immune sensing platform for NT-proBNP. Then the ECL aptasensor was prepared to collect and detect the small molecules released from the liposome. Finally, based on the ECL signal changes caused by the small molecules, the ECL signal indirectly reflected the level of NT-proBNP antigen. In this experiment, the cocaine was chosen as the proper small molecule that can act as signal-enhancer to enhance the ECL of Ru(bpy)(3)(2+). The cocaine-encapsulated liposomes were successfully characterized by TEM. The quantificational calculation proved the ～5.3×10(3) cocaine molecules per liposome enough to perform the assignment of signal amplification. The cocaine-binding ECL aptasensor further promoted the work aimed at amplifying signal. The performance of NT-proBNP assay by the proposed strategy exhibited high sensitivity and high specificities with a linear relationship over 0.01-500 ng mL(-1) range, and a detection limit down to 0.77 pg mL(-1).     

Ricebran based activated carbon as a carrier material for immobilisation of P. pictorum

In wastewater treatment microbial cultures immobilised on various matrices are used to protect the microbes from confronting shock loads of organic pollutants. Because of the beneficial effect of activated carbon, it is generally used as a carrier material in comparison to other matrices. In this study mutant strain of P. pictorum (MU 174) was immobilised on ricebran based activated carbon. The effect of contact time, pH, particle size, mass of activated carbon, temperature and ionic strength on adsorption of MU 174 on activated carbon were investigated. The adsorption kinetic parameters like K _p , K _ad and H were also determined.Authors are grateful to Dr. K.V. Raghavan, Director, CLRI for his keen interest in publishing this work. Financial assistance by CSIR/UGC is gratefully acknowledged by Miss S. Chitra.     

Construction of simultaneous SPR and QCM sensing platform

To construct a novel simultaneous SPR and QCM sensing system, an AT-cut quartz crystal is fabricated by sputtering 250 nm of ITO on one side of the quartz plate over a 5-nm thick underlay of titanium, while a 50-nm thick layer of gold is sputter-deposited on the other side to induce a total light reflection of an incident laser beam on the thin gold layer. The signals of the sensing system are detected using a Handy-SPR and QCA922 when dropping 200 μL of Milli-Q water into the sensing cell. A decrease in the SPR reflected light intensity is clearly identified. In the same experiment, the changes in the resonant frequency and resistance are about 2 kHz and 200 Ω, respectively. Furthermore, the oscillation stabilities of the resonant frequency and resistance are about 50 Hz and 2 Ω, respectively, which are sufficient to detect a large mass change on the QCM/SPR chip.     

Effects of sustained immobilisation on aphids

Immobilisation of aphids of three species, Schizaphis graminum, Myzus persicae and Dactynotus ambrosiae, was accomplished using low temperature (6 ± 1°C; or nitrogen (N₂), argon (Ar), or carbon dioxide (CO₂) gases at 25°C for 1,3,6, or 24 h. Mortality was recorded and also times until treated aphids first walked or probed. Cold immobilisation caused least mortality and allowed rapid recovery of walking and probing abilities. CO₂ treatment, even when accompanied by cyclic administration of bottled air, caused excessive mortality when used beyond 6 h, and long recovery times. After 24 h immobilisation with CO₂ aphids seldom walked or probed. N₂ or Ar gases, when administered with alternating cycles of bottled air, produced a nontoxic, hypoxic immobilisation from which aphids recovered fairly rapidly to walk and probe, apparently normally.     

Cellulosic exopolysaccharide produced by Zoogloea sp. as a film support for trypsin immobilisation

Trypsin (E.C. 3.4.21.4) was covalently immobilised onto a membrane of a cellulosic exopolysaccharide produced by Zoogloea sp. in sugarcane molasses. Carbonyl groups were introduced into the matrix by sodium metaperiodate oxidation and the enzyme was immobilised either directly or through bovine serum albumin (BSA) as a spacer. The trypsin-membrane and trypsin–BSA-membrane retained, respectively, 37.2% and 9.16% of the specific activity of the native enzyme acting on N-benzoil-dl-arginine-p-nitroanilide (BAPNA). No activity decrease was observed in both preparations after seven reutilisations as well as they showed to be more thermal stable than the native enzyme. The trypsin–BSA-membrane presented the same initial activity (99%) after 54 days stored in 0.1 M Tris–HCl buffer, pH 8.0, at 4 °C but the trypsin-membrane lost 15% of activity. Furthermore, the trypsin–BSA-membrane lost 31% of activity after reuse at 9 days interval during 54 days of storage at 4 °C whereas the trypsin-membrane lost 69% of activity under the same conditions. These results showed an additional application for this biofilm, namely, to act as a reusable matrix for trypsin immobilisation and the presence of BSA improved the derivative performance.     

Direct visualization of IgM antibodies bound to tissue antigens using a monoclonal anti-type III collagen IgM as a model system

A mouse monoclonal IgM antibody directed against human Type III collagen was utilized to immunolocalize Type III collagen by transmission and scanning electron microscopy without the use of an electron-dense conjugate. Because bound IgM can be directly visualized, primary or secondary antibody conjugates, such as ferritin, HRP, colloidal gold, etc., are unnecessary in this method. Immunolocalization to Type III collagen in the matrix of human skin and to fibrils formed in vitro using only IgM antibody reveals uninterrupted IgM binding which exactly matches the banding period of the collagen fibrils. In contrast, colloidal gold-conjugated secondary antibody complexes directed against primary IgM binding sites reveal less precise labeling. The data suggest that direct visualization of primary monoclonal IgM antibodies may be useful in a wide variety of highly specific ultrastructural immunolocalization studies without requiring the use of electron-dense conjugates.     

A low-volume platform for cell-respirometric screening based on quenched-luminescence oxygen sensing

Cell viability assays represent an important technology in modern cell biology, drug discovery and biotechnology, where currently there is a high demand for simple, sensitive and cost-effective screening methods. We have developed a new methodology and associated tools for cell-based screening assays, which are based on the measurement of the rates of oxygen uptake in cells by luminescence quenching. Sealable microchamber devices matching the footprint of a standard 96-well plate were developed and used in conjunction with long-decay phosphorescent oxygen probes. These devices permit cell non-invasive, real-time monitoring of cellular respiration and a rapid, one-step, kinetic assessment of multiple samples for cell viability, drug/effector action. These assays can be carried out on conventional fluorescence plate readers, they are suitable for different types of cells, including adherent and slow-respiring cells, require small sample volumes and cell numbers, and are amenable for high throughput screening. Monitoring of as little as 300 mammalian cells in 3 microl volume has been demonstrated.