Biosensors printed on bioactive paper

Biosensors printed on bioactive paper

The immobilization of biomacromolecules such as proteins, enzymes and DNA in various inert matrices is a research field that has been attracting considerable attention for many years and is motivated by fundamental, biomedical and industrial interests. For instance, permanent immobilization of biorecognition molecules on surfaces is a crucial step in developing biosensor materials and devices.

Developing bioassays that are simple, portable, disposable and inexpensive will provide important tools to rapidly detect toxic substances. This technology could also be extremely useful in monitoring environmental and food-based toxins in remote settings such as less industrialized countries where these tools are essential for the first stages of detecting disease settings and where the time and expense of using sophisticated instrumentation would be prohibitive.

To that end, researchers have developed simple, portable, disposable, and inexpensive paper-based solid-phase sensors to run multiple bioassays and controls simultaneously. Bioactive paper is any low-cost and easy-to-use paper product laced with biologically active chemicals that provides a rapid way to detect toxins like E. coli bacteria and salmonella, or pathogens such as SARS or influenza. Applications for bioactive paper range from food packaging and hospital masks to paper strips for detecting and purifying unsafe drinking water or checking for banned pesticides in crop produce.

Since the biorecognition elements used in these sensors are physically adsorbed onto the paper surfaces no permanent immobilization method such as covalent or affinity attachment or entrapment techniques have been employed.

"Due to the adsorption of the biomolecules on paper-based sensors they could be used only as lateral flow sensors and have not been amenable to dipstick sensing formats," John Brennan tells Nanowerk. "Developing a technique that permanently immobilizes biomolecules on solid surfaces, and is compatible with an automated coating or printing process, is a crucial step in the development of bioactive paper-based sensors. To achieve this goal it is necessary to develop protein immobilization methods that are compatible with automated coating and/or printing methods and which retain the biomolecule at the surface of the paper substrate."

In his latest work, Brennan, a professor in the Department of Chemistry and Canada Research Chair Bioanalytical Chemistry at McMaster University, together with his team explores the use of biocompatible sol-gel-derived materials for this purpose. Working with Canada's SENTINEL Bioactive Paper Network they developed a new inkjet method for printing bioactive inks on paper strips used to detect harmful substances.

The key advancement is the ability to use ink-jet printing of sol-gel derived materials as a method to deposit an enzyme and colorimetric reagents onto paper. This allows for automation of the printing process and clearly shows that biomolecules can withstand the ink-jet deposition process.

The team has reported their findings in a recent paper in Analytical Chemistry ("Development of a Bioactive Paper Sensor for Detection of Neurotoxins Using Piezoelectric Inkjet Printing of Sol-Gel-Derived Bioinks").

Long-term stability of AChE and DNTB within the layered coating (e.g., PVAm, silica, AChE+DTNB, silica) of the paper-based sensor. The sensor was stored at 4°C for 68 days and overspotted with 10 µL ATCh (300 µM). (a) When AChE was absent (control), and (b) when AChE was present. (c) Color formation when all bioinks were present and overspotted with 10 µL ATCh (300 µM) at day 1

The SENTINEL Bioactive Paper Network is a consortium formed in 2005 of 11 Canadian universities plus industry and government partners working toward development of bioactive paper that will detect, capture and deactivate water and airborne toxins. The Faculty of Engineering at McMaster University hosts SENTINEL's administrative center. The team chose the specific target of neurotoxins owing to the need for detection of such species in the developing world (organophosphate pesticides are still widely used there) and as bioterror agents. Dimatix Fujifilm is a partner in the Sentinel network and the printing was done using a Dimatix Fujifilm Materials Printer.

"The basic methodology builds on some 20 years of work on the entrapment of biomolecules into silica materials using sol-gel processing, which has been a major activity within my research group," explains Brennan. "The new feature of our work is the ability to print the components needed for entrapment of the protein as thin layers on paper. Previously it has been shown that the entrapment of biomolecules within sol-gel-derived materials allows proteins to retain their bioactivity for prolonged periods of time and that sol-gel-based materials are amenable to inkjet deposition – although not with proteins. However, until now, biocompatible sol-gel materials with entrapped proteins have not yet been deposited via inkjet printing and have not been incorporated within bioactive paper sensors."

Brennan and his colleagues introduced a novel sol-gel-based method for coating enzymes onto paper substrates using inkjet printing of various 'ink' layers to produce a bioactive paper sensor for the detection of acetylcholinesterase (AChE) substrates and inhibitors. As a signal generation method, they have utilized the well-known Ellman colorimetric assay. The use of poly(vinylamine) as a cationic capture agent on the paper significantly enhances the signal intensity and it also retains the signal over several months.

"Our data show that AChE can be printed between two biocompatible silica layers on paper and that the enzyme retains full activity for at least two months when stored at 4°C" Brennan points out.

Brennan notes that, in the longer term, the team will be working to develop other sensor strips that can be used to detect markers of food spoilage, and ultimately moving toward the detection of pathogens in food and beverages. In the short term, they are currently developing a second generation paper strip with all reagents present on the paper. The first stage bioactive paper strips still require the addition of some reagents prior to running the assay.

Some of the future directions will include development of multi-analyte sensor strips, integration of different types of (bio)chemical reagents onto paper to allow detection of different analytes, and working to develop a commercial technology around the bioactive paper platform. Challenges will include the development of rapid assay methods, good signal generation methods, and methods to keep biological reagents stable on the paper.

Bio-interface nanosensor visually reports damage to a material

Bio-interface nanosensor visually reports damage to a material

The term bio-interface describes the boundary between synthetic materials such as plastics, and biological systems. This rapidly growing research area, where biology and material sciences overlap, is creating new opportunities for the design, synthesis, and optimization of biologically-enabled and biologically-inspired materials. It involves manufacturing and characterization of functional surfaces for specific interactions with bio-systems and studies of the molecular and kinetic processes occurring at such interfaces, ranging from small molecule and biomolecular interactions, to cell adhesion, differentiation and tissue formation at the interface.

For example, the incorporation of proteins into polymers can result in hybrid materials that combine the properties of the polymer as a cost-effective and easy to process material with the highly evolved biological functionality of the protein, enabling new concepts for construction of sensors and biomedical materials.

While researchers so far have been focusing on altering the properties of a polymer by adding the functionality of a biomolecule, a group in California has now demonstrated the reverse situation, where changes in the polymer can alter the properties of the protein. They report a polymer–protein hybrid material in which changes in stress of the polymer matrix result in changes to the fluorescence properties of the protein complex, thus creating a material that visually reports damage.

Douglas S. Clark, a professor in the Department of Chemical Engineering at the University of California Berkeley and his lab have recently proven the concept of a protein-based nanosensor that is able to report deformation of the embedding polymer matrix. The team combined the structural properties of a chaperonin from a thermophilic organism with the spectral properties of fluorescent proteins in order to generate a protein complex that exhibits fluorescent energy resonance transfer (FRET) and is sensitive to structural deformation. Such self-reporting materials could be used in myriad applications where easy and early detection of damage is essential to avoid catastrophic failure of the material.

Clark and his team have reported their findings in a recent paper in Angewandte Chemie International Edition ("'Mechanical Nanosensor Based on FRET within a Thermosome: Damage-Reporting Polymeric Materials").

In their work, the scientists combined the structural properties of the thermosome (THS), a chaperonin (protein complex) from the thermophilic organism Thermoplasma acidophilum, with the spectral properties of fluorescent proteins to generate a protein complex that exhibits fluorescence resonance energy transfer (FRET) and is sensitive to structural deformation

Mechanical nanosensor based on FRET within a thermosome for damage-reporting polymeric materials

"Our concept for a THS-based stress sensor is based on the idea that guest proteins can be permanently entrapped into the cavities by covalent linker chemistry," writes Clark. "Entrapping a donor–acceptor pair of fluorescent proteins into the cavities gives the possibility for FRET to occur. The centers of the two cavities are approximately 7 nm apart, which is close to the Förster radius (4.9 nm) of enhanced cyan fluorescent protein (eCFP) and enhanced yellow fluorescent protein (eYFP), which are the most commonly used donor–acceptor FRET pair in molecular biology. We thus hypothesized that, by covalently crosslinking chains of a polymer matrix with the THS–guest complex, a sufficient force applied to the matrix will separate the two halves of the THS, which results in reduced FRET."

The coupling between the modified THS and the modified fluorescent proteins was carried out by simple incubation of a mixture of the proteins for several hours at room temperature in a coupling buffer. Subsequent fluorescence spectroscopy measurements proved that FRET occurs between the fluorescent proteins.

The researchers then went on to investigate the effect of mechanical deformation of the polymer on the biomechanical sensor. To this end, they strained samples of the polymer until they fractured and then imaged the area surrounding the fracture by multichannel confocal microscopy.

Clark's team found that the FRET efficiency in the non deformed material surrounding the cracks is surprisingly low. "However, the FRET signal increases 1.5-fold in regions corresponding to the locations of the cracks. The FLIM images show a shorter mean lifetime of the donor within the cracks compared to the surrounding matrix , which corresponds to a higher FRET efficiency. The lifetime difference suggests that changes in the microenvironment of the fluorophores within the cracks occurs, which most likely arises from a shortening of the donor–acceptor distance."

Further work is now underway in Clark's lab to examine the sensitivity of the sensor. Ideally, it will be able to report damage on a submicrometer scale before visible cracks become detectable by standard microscopic methods. These self-reporting materials could be used in myriad applications where easy and early detection of damage is essential to avoid catastrophic failure of the material.