Branched hybridization chain reaction-using highly dimensional DNA nanostructures for label-free, reagent-less, multiplexed molecular diagnostics.

The specific and multiplexed detection of DNA underpins many analytical methods, including the detection of microorganisms that are important in the medical, veterinary, and environmental sciences. To achieve such measurements generally requires enzyme-mediated amplification of the low concentrations of the target nucleic acid sequences present, together with the precise control of temperature, as well as the use of enzyme-compatible reagents. This inevitably leads to compromises between analytical performance and the complexity of the assay. The hybridization chain reaction (HCR) provides an attractive alternative, as a route to enzyme-free DNA amplification. To date, the linear nucleic acid products, produced during amplification, have not enabled the development of efficient multiplexing strategies, nor the use of label-free analysis. Here, we show that by designing new DNA nanoconstructs, we are able, for the first time, to increase the molecular dimensionality of HCR products, creating highly branched amplification products, which can be readily detected on label-free sensors. To show that this new, branching HCR system offers a route for enzyme-free, label-free DNA detection, we demonstrate the multiplexed detection of a target sequence (as the initiator) in whole blood. In the future, this technology will enable rapid point-of-care multiplexed clinical analysis or in-the-field environmental monitoring.

Ultrasonic waves in uniaxially stressed multilayered and one-dimensional phononic structures: Guided and Floquet wave analysis.

This paper shows that acoustoelasticity in one-dimensional (1D) multilayered isotropic hyperelastic materials can be understood through the analysis of elastic wave velocities as a function of applied stress. This theoretical framework is used for eigenvalue analyses in stressed elastic structures through a reformulation of the stiffness matrix method, obtaining modal solutions, as well as reflection and transmission coefficients for different multilayered configurations. Floquet wave analysis for the stressed 1D structures is supported using numerical results.

Hyperelastic Tuning of One-Dimensional Phononic Band Gaps Using Directional Stress.

In this paper, we show that acoustoelasticity in hyperelastic materials can be understood using the framework of nonlinear wave mixing, which, when coupled with an induced static stress, leads to a change in the phase velocity of the propagating wave with no change in frequency. By performing Floquet wave eigenvalue analysis, we also show that band gaps for periodic composites, acting as 1-D phononic crystals, can be tuned using this static stress. In the presence of second-order elastic nonlinearities, the phase velocity of propagating waves in the phononic structure changes, leading to observable shifts in the band gaps. Finally, we present numerical examples as evidence that the band gaps are tuned by both the direction of the stress and its magnitude.

Breaking the Symmetry of Momentum Conservation Using Evanescent Acoustic Fields.

Although the conservation of momentum is a fundamental law in physics, its constraints are not fulfilled for wave propagation at material boundaries, where incident waves give rise to evanescent field distributions. While nonlinear susceptibility tensor terms can provide solutions in the optical regime, this framework cannot be applied directly to acoustic waves. Now, by considering a complete representation of wave interactions and scattering at boundaries, we are able to show a generic formalism of sum-frequency mixing for the whole scattering field including all evanescent waves. This general case was studied analytically and verified both numerically and experimentally for ultrasonic waves, showing that considering evanescent waves leads to an anomalous nonlinear interaction which enhances sum-frequency generation. This new interpretation not only provides a deeper understanding of the momentum conservation laws in acoustics but also promises translation of this new understanding into optics and photonics, to enhance nonlinear interactions.

Lipid topology and electrostatic interactions underpin lytic activity of linear cationic antimicrobial peptides in membranes.

Linear cationic antimicrobial peptides are a diverse class of molecules that interact with a wide range of cell membranes. Many of these peptides disrupt cell integrity by forming membrane-spanning pores that ultimately lead to their death. Despite these peptides high potency and ability to evade acquired bacterial drug resistance, there is a lack of knowledge on their selectivity and activity mechanisms. Such an understanding would provide an informative framework for rational design and could lead to potential antimicrobial therapeutic targets. In this paper, we use a high-throughput microfluidic platform as a quantitative screen to assess peptide activity and selectivity by precisely controlling exposure to vesicles with lipid compositions that mimic both bacterial and mammalian cell membranes. We explore the complexity of the lipid-peptide interactions governing membrane-disruptive behaviors and establish a link between peptide pore formation and both lipid-peptide charge and topological interactions. We propose a topological model for linear antimicrobial peptide activity based on the increase in membrane strain caused by the continuous adsorption of peptides to the target vesicle coupled with the effects of both lipid-peptide charge and topographical interactions. We also show the validity of the proposed model by investigating the activity of two prototypical linear cationic peptides: magainin 2 amide (which is selective for bacterial cells) and melittin (which targets both mammalian and bacterial cells indiscriminately). Finally, we propose the existence of a negative feedback mechanism that governs the pore formation process and controls the membrane's apparent permeability.

Acoustic suppression of the coffee-ring effect.

We study the influence of acoustic fields on the evaporative self-assembly of solute particles suspended inside sessile droplets of complex fluids. The self-assembly process often results in an undesirable ring-like heterogeneous residue, a phenomenon known as the coffee-ring effect. Here we show that this ring-like self-assembly can be controlled acoustically to form homogeneous disc-like or concentrated spot-like residues. The principle of our method lies in the formation of dynamic patterns of particles in acoustically excited droplets, which inhibits the evaporation-driven convective transport of particles towards the contact line. We elucidate the mechanisms of this pattern formation and also obtain conditions for the suppression of the coffee-ring effect. Our results provide a more general solution to suppress the coffee-ring effect without any physiochemical modification of the fluids, the particles or the surface, thus potentially useful in a broad range of industrial and analytical applications that require homogenous solute depositions.

Interfacing low-energy SAW nebulization with Liquid Chromatography-Mass Spectrometry for the analysis of biological samples.

Soft ionization methods for the introduction of labile biomolecules into a mass spectrometer are of fundamental importance to biomolecular analysis. Previously, electrospray ionization (ESI) and matrix assisted laser desorption-ionization (MALDI) have been the main ionization methods used. Surface acoustic wave nebulization (SAWN) is a new technique that has been demonstrated to deposit less energy into ions upon ion formation and transfer for detection than other methods for sample introduction into a mass spectrometer (MS). Here we report the optimization and use of SAWN as a nebulization technique for the introduction of samples from a low flow of liquid, and the interfacing of SAWN with liquid chromatographic separation (LC) for the analysis of a protein digest. This demonstrates that SAWN can be a viable, low-energy alternative to ESI for the LC-MS analysis of proteomic samples.

Microrheology with optical tweezers: measuring the relative viscosity of solutions 'at a glance'.

We present a straightforward method for measuring the relative viscosity of fluids via a simple graphical analysis of the normalised position autocorrelation function of an optically trapped bead, without the need of embarking on laborious calculations. The advantages of the proposed microrheology method are evident when it is adopted for measurements of materials whose availability is limited, such as those involved in biological studies. The method has been validated by direct comparison with conventional bulk rheology methods, and has been applied both to characterise synthetic linear polyelectrolytes solutions and to study biomedical samples.

Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays.

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 µm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

Spatially selecting a single cell for lysis using light-induced electric fields.

An optoelectronic tweezing (OET) device, within an integrated microfluidic channel, is used to precisely select single cells for lysis among dense populations. Cells to be lysed are exposed to higher electrical fields than their neighbours by illuminating a photoconductive film underneath them. Using beam spot sizes as low as 2.5 µm, 100% lysis efficiency is reached in <1 min allowing the targeted lysis of cells.

Acoustically controlled enhancement of molecular sensing to assess oxidative stress in cells.

We demonstrate a microfluidic platform for the controlled aggregation of colloidal silver nanoparticles using surface acoustic waves (SAWs), enabling surface enhanced Raman scattering (SERS) analysis of oxidative damage in cells. We show that by varying the frequency and the power of the acoustic energy, it is possible to modulate the aggregation of the colloid within the sample and hence to optimise the SERS analysis.

Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies.

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-on-a-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system.

Nebulisation on a disposable array structured with phononic lattices.

We demonstrate the use of a phononic crystal to enable the nebulisation of liquid droplets from low-cost disposable arrays, using surface acoustic waves (SAW). The SAWs were generated using interdigitated transducers (IDT) on a piezoelectric surface (LiNbO(3)) and the acoustic waves were coupled into a disposable phononic crystal structure, referred to as a superstrate. Using its excellent reflecting properties, the phononic structures confined the acoustic field within the superstrate, resulting in the concentration of the acoustic energy, in a manner controllable by the excitation frequency. We show that this capability mitigates against coupling losses incurred by the use of a disposable superstrate, greatly reducing the time needed to nebulise a drop of water with respect to an unstructured superstrate for a given power. We also demonstrate that by changing the excitation frequency, it is possible to change the spatial position at which the acoustic energy is concentrated, providing a means to specifically nebulise drops across an array. These results open up a promising future for the use of phonofluidics in high-throughput sample handling applications, such as drug delivery or the "soft" transfer of samples to a mass spectrometer in the field of proteomics.

Integrated immunoassay using tuneable surface acoustic waves and lensfree detection.

The diagnosis of infectious diseases in the Developing World is technologically challenging requiring complex biological assays with a high analytical performance, at minimal cost. By using an opto-acoustic immunoassay technology, integrating components commonly used in mobile phone technologies, including surface acoustic wave (SAW) transducers to provide pressure driven flow and a CMOS camera to enable lensfree detection technique, we demonstrate the potential to produce such an assay. To achieve this, antibody functionalised microparticles were manipulated on a low-cost disposable cartridge using the surface acoustic waves and were then detected optically. Our results show that the biomarker, interferon-γ, used for the diagnosis of diseases such as latent tuberculosis, can be detected at pM concentrations, within a few minutes (giving high sensitivity at a minimal cost).

Phononic crystal structures for acoustically driven microfluidic manipulations.

The development of microfluidic systems is often constrained both by difficulties associated with the chip interconnection to other instruments and by limitations imposed by the mechanisms that can enable fluid movement and processing. Surface acoustic wave (SAW) devices have shown promise in allowing samples to be manipulated, although designing complex fluid operations involves using multiple electrode transducers. We now demonstrate a simple interface between a piezoelectric SAW device and a disposable microfluidic chip, patterned with phononic structures to control the acoustic wave propagation. The surface wave is coupled from the piezoelectric substrate into the disposable chip where it interacts with the phononic lattice. By implementing both a phononic filter and an acoustic waveguide, we illustrate the potential of the technique by demonstrating microcentrifugation for particle and cell concentration in microlitre droplets. We show for the first time that the interaction of the fluid within this metamaterial phononic lattice is dependent upon the frequency of the acoustic wave, providing a route to programme complex fluidic functions into a microchip (in much the same way, by analogy, that a holographic element would change the phase of a light wave in optical tweezers). A practical realisation of this involves the centrifugation of blood on the chip.

The use of surface-enhanced Raman scattering for detecting molecular evidence of life in rocks, sediments, and sedimentary deposits.

Raman spectroscopy is a versatile analytical technique capable of characterizing the composition of both inorganic and organic materials. Consequently, it is frequently suggested as a payload on many planetary landers. Only approximately 1 in every 10(6) photons are Raman scattered; therefore, the detection of trace quantities of an analyte dispersed in a sample matrix can be much harder to achieve. To overcome this, surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) both provide greatly enhanced signals (enhancements between 10(5) and 10(9)) through the analyte's interaction with the locally generated surface plasmons, which occur at a "roughened" or nanostructured metallic surface (e.g., Cu, Au, and Ag). Both SERS and SERRS may therefore provide a viable technique for trace analysis of samples. In this paper, we describe the development of SERS assays for analyzing trace amounts of compounds present in the solvent extracts of sedimentary deposits. These assays were used to detect biological pigments present in an Arctic microoasis (a small locale of elevated biological productivity) and its detrital regolith, characterize the pigmentation of microbial mats around hydrothermal springs, and detect fossil organic matter in hydrothermal deposits. These field study examples demonstrate that SERS technology is sufficiently mature to be applied to many astrobiological analog studies on Earth. Many current and proposed imaging systems intended for remote deployment already posses the instrumental components needed for SERS. The addition of wet chemistry sample processing facilities to these instruments could yield field-deployable analytical instruments with a broadened analytical window for detecting organic compounds with a biological or geological origin.

Tuneable surface acoustic waves for fluid and particle manipulations on disposable chips.

We establish a powerful new acoustic technique to programme complex fluidic functions such as droplet movement, merging, mixing and concentration, on a disposable superstrate.

Surface acoustic wave nebulization of peptides as a microfluidic interface for mass spectrometry.

We describe the fabrication of a surface acoustic wave (SAW) device on a LiNbO(3) piezoelectric transducer for the transfer of nonvolatile analytes to the gas phase at atmospheric pressure (a process referred to as nebulization or atomization). We subsequently show how such a device can be used in the field of mass spectrometry (MS) detection, demonstrating that SAW nebulization (SAWN) can be performed either in a discontinuous or pulsed mode, similar to that for matrix assisted laser desorption ionization (MALDI) or in a continuous mode like electrospray ionization (ESI). We present data showing the transfer of peptides to the gas phase, where ions are detected by MS. These peptide ions were subsequently fragmented by collision-induced dissociation, from which the sequence was assigned. Unlike MALDI mass spectra, which are typically contaminated with matrix ions at low m/z, the SAWN generated spectra had no such interference. In continuous mode, the SAWN plume was sampled on a microsecond time scale by a linear ion trap mass spectrometer and produced multiply charged peptide precursor ions with a charge state distribution shifted to higher m/z compared to an identical sample analyzed by ESI. The SAWN technology also provides the opportunity to re-examine a sample from a flat surface, repeatedly. The process can be performed without the need for capillaries, which can clog, reservoirs, which dilute the sample, and electrodes, which when in direct contact with sample, cause unwanted electrochemical oxidation. In both continuous and pulsed sampling modes, the quality of precursor ion scans and tandem mass spectra of peptides was consistent across the plume's lifetime.

Signal enhancement of surface enhanced Raman scattering and surface enhanced resonance Raman scattering using in situ colloidal synthesis in microfluidics.

We demonstrate the enhanced analytical sensitivity of both surface enhanced Raman scattering (SERS) and surface enhanced resonance Raman scattering (SERRS) responses, resulting from the in situ synthesis of silver colloid in a microfluidic flow structure, where both mixing and optical interrogation were integrated on-chip. The chip-based sensor was characterized with a model Raman active label, rhodamine-6G (R6G), and had a limit of detection (LOD) of ca. 50 fM (equivalent to single molecule detection). The device was also used for the determination of the natural pigment, scytonemin, from cyanobacteria (as an analogue for extraterrestrial life existing in extreme environments). The observed LOD of approximately 10 pM (ca. <400 molecules) demonstrated the analytical advantages of working with freshly synthesized colloid in such a flow system. In both cases, sensitivities were between 1 and 2 orders of magnitude greater in the microfluidic system than those measured using the same experimental parameters, with colloid synthesized off-chip, under quiescent conditions.