Rheology at the micro-scale: new tools for bio-analysis

We present a simple and non-invasive experimental procedure to measure the linear viscoelastic properties of cells by passive particle tracking microrheology. In order to do this, a generalised Langevin equation is adopted to relate the timedependent thermal fluctuations of a probe sensor, immobilised to the cell’s membrane, to the frequency-dependent viscoelastic moduli of the cell. The method has been validated by measuring the linear viscoelastic response of a soft solid and then applied to cell physiology studies. It is shown that the viscoelastic moduli are related to the cell’s cytoskeletal structure, which in this work is modulated either by inhibiting the actin/myosin-II interactions by means of blebbistatin or by varying the solution osmolarity from iso- to hypo-osmotic conditions. The insights gained from this form of rheological analysis promises to be a valuable addition to physiological studies; e.g. cell physiology during pathology and pharmacological response

Highly Efficient Spatially Offset Raman Spectroscopy to Profile Molecular Composition in Bone

Spatially offset Raman spectroscopy (SORS) offers the prospect of collecting spectral information detailing the molecular composition of biomaterials at greater depths below the surface layers than are normally probed by conventional Raman spectroscopy. By collecting off-axial scattered light, the technique overcomes the large background from in-line light within scattering media. In this paper we present a configuration which enables the highly efficient collection of spectral markers, indicative of bone health, including Raman signatures to assess phosphate, collagen and carbonate content, at millimeter depths. We demonstrate the effectiveness of the technique by performing spectral decompositions to analyze the molecular distribution of these markers non-invasively, using in vitro model systems, comprising bone and tissue, in situ

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

Multiple plasmon resonances from gold nanostructures

Understanding and controlling plasmon resonances from metallic nanoscale structures have been the focus of much attention recently, with applications including local surface plasmon resonance sensing, surface enhanced Raman spectroscopy, and negative refractive index materials. In this letter the authors demonstrate the fabrication of uniform arrays of split rings from gold and show that such structures are capable of supporting multiple plasmon resonances. The authors show that up to five plasmon resonances can be identified and use finite difference time domain modeling and absorption spectroscopy to fully characterize and identify each resonance. The implications of higher order surface plasmon resonances for sensing are discussed

Micromirror Angle Dependence with Etchant Choice on <100> Silicon Via Wet Etching

In creating mirrored silicon structures for micro-optics, the smoothness of the surface and etch rate are crucial parameters. We demonstrate a method of creating both 45° and 90° etch-planes from monocrystalline silicon for use as retro-reflective sidewalls in a microfluidic device. The technique uses the same photolithographic pattern orientation, but with two different etchants. Etching on direction in Si(100) with potassium hydroxide (KOH) gives vertical surfaces (where e.g. the high surface tension influences etching of crystallographic silicon planes), whilst tetramethylammonium hydroxide (TMAH) gives 45° sidewalls. We illustrate the use of these fabricated structures by creating arrays of micromirrors that enable an optical beam to be reflected parallel back and forth from 45° and -45° tilted vertical structures. This device has potential uses in optofluidic spectroscopic applications, where there is a need to increase the effective pathlength of a beam through a sample whilst keeping the device as small as possible

Micromirror Angle Dependence with Etchant Choice on <100> Silicon Via Wet Etching

In creating mirrored silicon structures for micro-optics, the smoothness of the surface and etch rate are crucial parameters. We demonstrate a method of creating both 45° and 90° etch-planes from monocrystalline silicon for use as retro-reflective sidewalls in a microfluidic device. The technique uses the same photolithographic pattern orientation, but with two different etchants. Etching on &lt;;100&gt; direction in Si(100) with potassium hydroxide (KOH) gives vertical surfaces (where e.g. the high surface tension influences etching of crystallographic silicon planes), whilst tetramethylammonium hydroxide (TMAH) gives 45° sidewalls. We illustrate the use of these fabricated structures by creating arrays of micromirrors that enable an optical beam to be reflected parallel back and forth from 45° and -45° tilted vertical structures. This device has potential uses in optofluidic spectroscopic applications, where there is a need to increase the effective pathlength of a beam through a sample whilst keeping the device as small as possible

Gradient microfluidics enables rapid bacterial growth inhibition testing

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (&lt;4 days, compared to weeks in a culture flask)

Effect of Laser Irradiation on Cell Function and Its Implications in Raman Spectroscopy

Lasers are instrumental in advanced bioimaging and Raman spectroscopy. However, they are also well known for their destructive effects on living organisms, leading to concerns about the adverse effects of laser technologies. To implement Raman spectroscopy for cell analysis and manipulation, such as Raman-activated cell sorting, it is crucial to identify nondestructive conditions for living cells. Here, we evaluated quantitatively the effect of 532-nm laser irradiation on bacterial cell fate and growth at the single-cell level. Using a purpose-built microfluidic platform, we were able to quantify the growth characteristics, i.e., specific growth rates and lag times of individual cells, as well as the survival rate of a population in conjunction with Raman spectroscopy. Representative Gram-negative and Gram-positive species show similar trends in response to a laser irradiation dose. Laser irradiation could compromise the physiological function of cells, and the degree of destruction is both dose and strain dependent, ranging from reduced cell growth to a complete loss of cell metabolic activity and finally to physical disintegration. Gram-positive bacterial cells are more susceptible than Gram-negative bacterial strains to irradiation-induced damage. By directly correlating Raman acquisition with single-cell growth characteristics, we provide evidence of nondestructive characteristics of Raman spectroscopy on individual bacterial cells. However, while strong Raman signals can be obtained without causing cell death, the variety of responses from different strains and from individual cells justifies careful evaluation of Raman acquisition conditions if cell viability is critical. / IMPORTANCE: In Raman spectroscopy, the use of powerful monochromatic light in laser-based systems facilitates the detection of inherently weak signals. This allows environmentally and clinically relevant microorganisms to be measured at the single-cell level. The significance of being able to perform Raman measurement is that, unlike label-based fluorescence techniques, it provides a “fingerprint” that is specific to the identity and state of any (unlabeled) sample. Thus, it has emerged as a powerful method for studying living cells under physiological and environmental conditions. However, the laser's high power also has the potential to kill bacteria, which leads to concerns. The research presented here is a quantitative evaluation that provides a generic platform and methodology to evaluate the effects of laser irradiation on individual bacterial cells. Furthermore, it illustrates this by determining the conditions required to nondestructively measure the spectra of representative bacteria from several different groups