Immobilization of biotinylated bacteriophages on biosensor surfaces

Abstract

Bacteriophages are viruses that recognize specific receptors on the bacterium surface to which they bind and inject genetic material. The specificity of this recognition opens remarkable possibilities for biosensor development. The chemical attachment of T4 bacteriophages onto gold surfaces is being reported. This attachment leverages the genetic biotinylation of the capsid heads of bacteriophages, and the natural affinity of the biotin/streptavidin system. The development of a streptavidin-immobilization chemistry that minimizes non-specific binding of the target bacterium is first described. The attachment of genetically biotinylated T4 bacteriophages onto these streptavidin-coated surfaces is then reported. Such chemical immobilization results in a 15-fold improvement of attachment when compared to the simple physisorption of the wild-type phage onto bare gold. The attachment procedure was then used to investigate the effect of a biotinylated phage-terminated surface on the growth of the host bacteria. This assessment was conducted in an electric cell-substrate impedance sensing device. The streptavidin-mediated attachment of biotinylated phages significantly delays the growth of the host bacteria by up to 17.2 h. In comparison, non-specific binding of wild-type phages onto the streptavidin surface is found to cause a lesser growth delay of 13 h.

Introduction

Increasing public health concerns related to bacterial diseases, as well as the need to monitor food and water supplies have prompted interest in the development of low-cost and low-footprint pathogen detection systems. The detection and identification of pathogens in food products, drinking water supplies and hospitals continue to mostly rely on conventional microbiological culture techniques. These tests are based on assessing a bacterium's ability to grow in plates or tubes containing a variety of media (solid or liquid) under various conditions. While detection of a small number of bacteria is possible by incubation, growth of bacteria to numbers sufficient for identification can take several days. In addition, further biochemical and serological tests are required to confirm the identity of the agent.

Polymerase chain reaction (PCR) may also be used to amplify a small amount of genetic material from bacteria [1], [2]. Alternatively, bacterial identification using enzyme-linked immunosorbent assay (ELISA) is conducted by testing antibody–antigen interaction with the targeted bacterium and can be performed within a working day [3]. Combined PCR-ELISA increases sensitivity of the conventional PCR method [4], [5]. However, these techniques still require an enrichment step during which bacteria are grown to the levels required for detection. In addition, problems associated with enzyme inhibition and DNA extraction have made direct detection of low numbers of bacteria in foods by PCR difficult to achieve.

Therefore, there has been sustained interest towards the development of biosensing systems that would circumvent the limitations of conventional techniques. A typical biosensor platform couples a physical transducer (electrochemical, mechanical, thermal, or optical) with a specific recognition probe such as an enzyme, nucleic acid, cell, antibody or artificial receptor. The interaction between the probe and the target is converted to a quantifiable signal by the transducer. Micromechanical resonators [6], [7], quartz crystal microbalances (QCM) [8], [9], [10], surface plasmon resonance (SPR) [11], [12], [13], [14], [15], and amperometry [16] have been demonstrated as potent platforms for such applications. For example, Salmonella has been detected by immobilizing polyclonal antibodies on a quartz crystal acoustic wave device using Langmuir–Blodgett films [17]. Another example includes the use of SPR to effectively detect Escherichia coli bacteria [18]. The development of these technologies as viable biosensing systems requires the use of a recognition probe offering high levels of specificity, selectivity, and stability. Antibodies are frequently used as recognition receptor systems for the specific detection of antigens. While antibodies may offer some degree of selectivity and speficity (especially monoclonal antibodies), they suffer from environmental instabilities and require arduous and cost-intensive methods for their production, isolation and purification. In addition, polyclonal antobodies are limited by their heterogenicity towards other species, strains or molecules. Thus, there is a need for alternative probe selection [19], [20].

Bacteriophages (or phages) offer such potential as alternative probes for specific biosensing. They are viruses that recognize specific receptors on the bacterial surface to which they bind and inject their genetic material. Such injection allows replication of the phage and release of a new generation while killing the bacteria. These viruses recognize target bacteria through functional receptors located on their tail extremity (Fig. 1(a)) [21]. This recognition is routinely employed in phage typing where a group of phages are used to differentiate between different bacteria. This unique level of specificity also presents remarkable possibilities for biosensor development. For instance, the use of a lytic phage for the SPR detection of Staphylococcus aureus was recently reported [22]. However, the attachment of phages was accomplished by simple non-oriented physisorption of the viruses onto the sensor surface.

Alternatively, chemical attachment of phages onto sensor surfaces could significantly improve the stability and performance of the overall platform, and enable their employment in applications where patterning of the probing element is required. The chemical biotinylation of phages has already been shown to significantly increase the efficiency of phage-based biosorbents when compared to simple physical adsorption [23]. In the case of microsensors, bacteriophages bound from the head capsid protein onto the sensor surface would also allow the tail fibers to face the medium, enabling a more efficient capture of the bacteria.

The biotin–avidin complex has been used for binding purposes in previously reported bacterial sensing applications [24], [25]. Edgar et al. [26] has recently reported the genetic modification of the T7 bacteriophage to display a small peptide on the major capsid protein that is subsequently biotinylated by the biotin-ligase protein (BLP) present in the host bacterium. The biotin was attached postranslationally by the BLP to a specific lysine residue in the tagged peptide. Such genetic biotinylation opens the possibility of leveraging the affinity of the streptavidin/biotin system for the attachment of the phages onto particles and surfaces. In addition, as opposed to phages biotinylated by chemical procedures [23], the biotin is in these cases exclusively present on the phage capsid, and not on its tail, potentially allowing the oriented attachment of the phage onto surfaces.

We here describe such development of a surface attachment scheme that involves T4 bacteriophages that were similarly engineered to express a biotin binding domain on a capsid protein using a phage display technique. The constructed recombinant bacteriophage was reported to retain infectivity, burst size and latent period comparable to its wild-type counterpart. While the details of this biotinylation procedure is to be reported elsewhere [27], we here specifically report the streptavidin-mediated attachment of these recombinant biotinylated phages onto gold electrodes (Fig. 1(b)), as well as the use of this attachment in an impedance biosensing device (Fig. 1(c)). We observe that such streptavidin-mediated attachment of biotinylated phages improves by a factor of 15 the attachment of the viruses onto electrode surfaces, and significantly delays the growth of the host bacterium in comparison with electrodes functionalized using wild-type phages.

Such antibody-free chemical attachment of phages onto sensor surfaces could therefore be used in numerous other sensing transduction techniques, and enable the design of highly sensitive and highly specific platforms for the detection and identification of the host pathogenic organism.