Probing biomolecular interaction forces using an anharmonic acoustic technique for selective detection of bacterial spores
Introduction
Established biochemical methods for pathogen detection are primarily based on the detection of specific nucleotide sequences within the pathogen genome or on the detection of pathogen-specific surface epitopes using specific receptors (antibodies, peptides or aptamers). The most widely used methods in the two categories are the nucleic acid-based assays, such as polymerase chain reaction (PCR), and the antibody-based assays, such as enzyme-linked immunosorbent assays (ELISA). Although offering high sensitivity, and in some cases high selectivity, these methods can require time-consuming and skill-demanding sample preparation. Moreover, being label-dependent, they are confined to specialised laboratories with appropriate interrogation equipment. However, in clinical diagnosis, food and environmental monitoring, and detection of biowarfare pathogens, there is an increasing demand for rapid and easy-to-use detection platforms that can be implemented at the point-of-care (POC).
Labelled antibody-based tests, such as dip-stick method (Dewey et al., 1989) and lateral flow devices (LFD) (Lane et al., 2007), are relatively fast and easy-to-use but provide only qualitative or semi-quantitative data at limited sensitivity (Skottrup et al., 2008). Immunosensors based on platforms such as surface plasmon resonance (SPR) (Hoa et al., 2007), quartz crystal microbalance (QCM) (Cooper and Singleton, 2007) and cantilever-based sensors (Waggoner and Craighead, 2007), provide rapid and easy detection with high sensitivity and quantification wherein the binding of receptors (immobilized on the immunosensor surface) with pathogens via pathogen-specific surface epitopes results in a direct measurable signal without the requirement for labels. Yet, an inherent shortcoming in most sensing techniques used here is the inability to differentiate between specific (target) and non-specific (non-target) interactions, the latter resulting from the formation of non-specific bonds that, though weaker, cannot be easily dissociated. The resulting false positive responses can lead to misdiagnosis, consequent mistreatment (clinical) and false alarms (biosecurity).
Here we report a technique based on nonlinear acoustics that can be used to sense biomolecular interaction forces and hence distinguish the target from non-targets in an analyte. Acoustic nonlinearity has been used in non-destructive testing for measurement of strength of adhesive bonds (Fassbender and Arnold, 1996). The underlying concept here is that binding forces in the soft interface layer are nonlinear and result in a nonlinear modulation of transmitted or reflected ultrasonic waves that can be analysed to measure the binding forces (Hirsekorn, 2001). Similarly, surface-coupled streptavidin-coated polystyrene microbeads (SCPM) on thickness-shear mode (TSM) quartz crystal in air result in a significant enhancement to its nonlinear response (Ghosh et al., 2010). We employ this principle for the first time to investigate the interaction forces at the biological interface in an immunosensor. By studying the nonlinear acoustic response from a TSM quartz crystal with functionalized specific antibodies, we can sensitively detect Bacillus subtilis spores and distinguish them from physisorbed SCPM.
Section snippets
Theoretical basis
Supplementary Fig. 1 illustrates a spore attached to the sensor via a biomolecular tether, modeled as a spring. The high relative motion between the spore and the sensor surface at resonance causes significant strain in the tether, which causes forces to act on it. These forces are transmitted onto the sensor. The horizontal component of the transmitted force (Ftx) is only significant here as only the horizontal shear forces are transduced into charge due to piezoelectric effect of TSM quartz.
Experimental approach
We chose TSM AT-cut quartz as the sensing platform since its use in QCM sensors is well established and the TSM has good quality factor in liquid (approximately 2000 in our case). Following immobilization of B. subtilis spores by anti-spore IgG (as detailed in Section 7), the sensor was driven with a pure sinusoidal voltage of frequency close to its fundamental resonant frequency (f = 14.3 MHz) with linearly increasing amplitude for 2 min.
Results and discussion
Measurements were taken before and after the incubation of spores as described in Section 7. The increase in the amplitude of 3f response (current) was found to be significant after incubation of spores (Fig. 1a) given the number of spores captured on the sensor (Sensor 1) was approximately 6400, as observed using an optical microscope. This is equivalent in weight to 9.6 ng, using the experimentally determined value of 1.5 pg as the weight of a spore (McCormick and Halvorson, 1964). For similar
Comparison with other POC immunosensing methods
A nonlinear acoustic biosensor based on the described anharmonic detection technique (ADT) would have distinct advantages compared to existing POC immunosensors for a number of reasons.
First, since the signal is based on the force of interaction between the sensor and the coupled particles, specific and non-specific interactions can be distinguished using the shape of the signal profile for varying drive amplitude. The fabrication of a reliable reference sensor, as utilised in some resonant
Conclusion
The anharmonic detection technique (ADT) described here opens a new paradigm of biological detection. This technique discriminates between specific and non-specific interactions and has an additional level of selectivity over the efficacy of the receptor. Moreover, this enables quantitative measurement with high sensitivity with the potential for the method to be extended to the detection of a single bacterial spore. This also allows for a rapid, easy-to-use and cost-effective detection
Spore preparation and storage
Vegetative cultures of B. subtilis 168 (A. Moir) were maintained on nutrient agar plates (oxoid) incubated at 37 °C. Sporulation was induced by inoculating 400 mL supplemented nutrient broth (SNB) with 1 mL of a mid-log-phase culture. SNB medium consists of the following (per litre): Difco nutrient broth, 8.0 g; glucose, 1.0 g; KCl, 1.0 g; MgSO4·7H2O, 246 mg; CaCl2·2H2O, 147 mg; MnCl2·4H2O, 4 mg; and FeSO4·7H2O, 0.3 mg. The pH was adjusted to 7.2 prior to autoclaving. Cultures were incubated (37 °C, 225
Acknowledgement
This project was funded in part by the UK Engineering and Physical Sciences Research Council and the Cambridge Trusts.
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