An observation about circular shortest paths: Dealing with additional constraints using branch and bound

Circular shortest paths represent a powerful methodology for image segmentation. The circularity condition ensures that the contour found by the algorithm is closed, a natural requirement for regular objects. Several implementations have been proposed in the past that either promise closure with high probability or ensure closure strictly, but with a mild computational efficiency handicap. Circularity can be viewed as a priori information that helps recover the correct object contour. Our "observation" is that circularity is only one among many possible constraints that can be imposed on shortest paths to guide them to a desirable solution. In this contribution, we illustrate this opportunity under a volume constraint but the concept is generally applicable. We also describe several adornments to the circular shortest path algorithm that proved useful in applications. © 2011 IEEE.

Droplet digital PCR as a useful tool for the quantitative detection of Enterovirus 71

Hand, foot and mouth disease (HFMD) is a contagious viral disease that frequently affects infants and children and present with blisters and flu-like symptoms. This disease is caused by a group of enteroviruses such as enterovirus 71 (EV71) and coxsackievirus A16 (CA16). However, unlike other HFMD causing enteroviruses, EV71 have also been shown to be associated with more severe clinical manifestation such as aseptic meningitis, brainstem and cerebellar encephalitis which may lead to cardiopulmonary failure and death. Clinically, HFMD caused by EV71 is indistinguishable from other HFMD causing enteroviruses such as CA16. Molecular diagnosis methods such as the use of real-time PCR has been used commonly for the identification of EV71. In this study, two platforms namely the real-time PCR and the droplet digital PCR were compared for the detection quantitation of known EV71 viral copy number. The results reveal accurate and consistent results between the two platforms. In summary, the droplet digital PCR was demonstrated to be a promising technology for the identification and quantitation of EV71 viral copy number.

Enhanced Nanoparticle Detection with Liquid Droplet Resonators

Whispering gallery mode particle sensing experiments are commonly performed with solid resonators, whereby the sensing volume is limited to the weak evanescent tail of the mode near the resonator surface. In this work we discuss in detail the sensitivity enhancements achievable in liquid droplet resonators wherein the stronger internal fields and convenient means of particle delivery can be exploited. Asymptotic formulae are derived for the relative resonance shift, line broadening and mode splitting of TE and TM modes in liquid droplet resonators. As a corollary the relative fraction of internal and external mode energy follows, which is shown to govern achievable sensitivity enhancements of solute concentration measurements in droplet sensors. Experimental measurements of nanoparticle concentration based on whispering gallery mode resonance broadening are also presented.

Detection of MegaTAL-induced HIV pol Mutation By Droplet Digital PCR

Thesis (Master's)--University of Washington, 2015

Detection of bacteriophage and respiratory viruses in droplets

Influenza is a widespread disease occurring in seasonal epidemics, and each year is responsible for up to 500,000 deaths worldwide. Influenza can develop into strains which cause severe symptoms and high mortality rates, and could potentially reach pandemic status if the virus’ properties allow easy transmission. Influenza is transmissible via contact with the virus, either directly (infected people) or indirectly (contaminated objects); via reception of large droplets over short distances (one metre or less); or through inhalation of aerosols containing the virus expelled by infected individuals during respiratory activities, that can remain suspended in the air and travel distances of more than one metre (the aerosol route). Aerosol transmission of viruses involves three stages: production of the droplets containing viruses; transport of the droplets and ability of a virus to remain intact and infectious; and reception of the droplets (via inhalation). Our understanding of the transmission of influenza viruses via the aerosol route is poor, and thus our ability to prevent a widespread outbreak is limited. This study explored the fate of viruses in droplets by investigating the effects of some physical factors on the recovery of both a bacteriophage model and influenza virus. Experiments simulating respiratory droplets were carried out using different types of droplets, generated from a commonly used water-like matrix, and also from an ‘artificial mucous’ matrix which was used to more closely resemble respiratory fluids. To detect viruses in droplets, we used the traditional plaque assay techniques, and also a sensitive, quantitative PCR assay specifically developed for this study. Our results showed that the artificial mucous suspension enhanced the recovery of infectious bacteriophage. We were able to report detection limits of infectious bacteriophage (no bacteriophage was detected by the plaque assay when aerosolised from a suspension of 103 PFU/mL, for three of the four droplet types tested), and that bacteriophage could remain infectious in suspended droplets for up to 20 minutes. We also showed that the nested real-time PCR assay was able to detect the presence of bacteriophage RNA where the plaque assay could not detect any intact particles. Finally, when applying knowledge from the bacteriophage experiments, we reported the quantitative recoveries of influenza viruses in droplets, which were more consistent and stable than we had anticipated. Influenza viruses can be detected up to 20 minutes (after aerosolisation) in suspended aerosols and possibly beyond. It also was detectable from nebulising suspensions with relatively low concentrations of viruses.

Highly sensitive surface enhanced Raman scattering (SERS) detection platforms formed by large area self-assembled Au nanorod arrays

This thesis explores a new method to fabricate SERS detection platforms formed by large area self-assembled Au nanorod arrays. For the fabrication of these new SERS platforms a new droplet deposition method for the self-assembly of Au nanorods was developed. The method, based in the controlled evaporation of organic suspensions of Au nanorods, was used for the fabrication of horizontal and vertical arrays of Au nanorods over large areas (100μm2). The fabricated nanorods arrays showed a high degree of order measured by SEM and optical microscopy over mm2 areas, but unfortunately they detached from the support when immersed in any analyte solutions. In order to improve adhesion of arrays to the support and clean off residual organic matter, we introduced an additional stamping process. The stamping process allows the immobilization of the arrays on different flexible and rigid substrates, whose feasibility as SERS platforms were tested satisfactory with the model molecule 4ABT. Following the feasibility study, the substrates were used for the detection of the food contaminant Crystal Violet and the drug analogue Benzocaine as examples of recognition of health menaces in real field applications.

Direct Sensing in Liquids Using Whispering-Gallery-Mode Droplet Resonators

Liquid droplets suspended by the tip of a thin wire, a glass capillary, or a needle form high-Q optical resonators, thanks to surface tension. Under gravity equilibrium conditions, the maximum drop diameter is approximately 1.5 mm for paraffin oil (volume ∼ 0.5 μL) using, for instance, a silica fiber with 250 μm thickness. Whispering gallery modes are excited by a free-space near-infrared laser that is frequency locked to the cavity resonance. The droplet cavity serves as a miniature laboratory for sensing of chemical species and particles.

Magnetic liquid marbles : Toward “Lab in a Droplet“

Liquid marbles exhibit great potential for use as miniature labs for small-scale laboratory operations, such as experiment and measurement. While important progress has been made recently in exploring their applications as microreactions, “on-line“ measurement of the components inside the liquid still remains a challenge. Herein, it is demonstrated that “on-line“ detection can be realized on magnetic liquid marbles by taking advantage of their unique magnetic opening feature. By partially opening the particle shell, electrochemical measurement is carried out with a miniaturized three-electrode probe and the application of this technique for quantitative measurement of dopamine is demonstrated. Fully opened magnetic liquid marble makes it feasible to detect the optical absorbance of the liquid in a transmission mode. With this optical method, a glucose assay is demonstrated. Moreover, when magnetic particle shell contains low melting point material, e.g., wax, the liquid marble shows a unique encapsulation ability to form a rigid shell after heating, which facilitates the storage of the non-volatile ingredients. These unique features, together with the versatile use as microreactors, enable magnetic liquid marbles to function as a miniature lab (or called “lab in a droplet“), which may find applications in clinical diagnostics, biotechnology, chemical synthesis, and analytical chemistry.

Analytical chemistry in a liquid film/droplet

The measurement of nitrogen dioxide at the parts-perbillion level is described. The experimental arrangement consists of two optical fibers placed on opposite sides of and in contact with a liquid film (14-57 μL in volume) supported on a U-shaped wire guide and two tubular conduits (one of which constitutes the means for the delivery of the liquid), light from a green (555 nm) light-emitting diode enters the liquid film, composed of Griess-Saltzman reagent. The transmitted light is measured by a referenced photodetection arrangement. Sample gas flows past the droplet at a low flow rate (typically 0.10-0.25 L/min). The response is proportional to the sampling period and the analyte concentration. The limit of detection for this nonoptimized arrangement is estimated to be <10 ppb by volume for a 5 min sample. Some unusual characteristics are observed. The initial absorbance, when most of the analyte/reaction product is still near the surface, is higher than that when the content of the droplet is fully mixed. The signal depends on the sample flow rate in a nonmonotonic fashion, first increasing and then decreasing with increasing sampling rate; the specific chemistry involved in the collection and determination of NO2 may be responsible.

Colorimetric determination of ambient ozone using indigo blue droplet

A simple and sensitive method based on a liquid droplet is described for the measurement of atmospheric ozone. A 30 μL drop of indigo blue solution is suspended in a flowing-air sampling stream. The ozone collected reacts with the indigo solution resulting in its decolorization. The colorimetric sensor is composed of two optical fibers and the source of monochromatic light was a red LED (625 nm). The calibration curve was constructed with ozone standard concentrations ranging from 37 - 123 ppbv. The detection limit achieved was 7.3 ppbv. The method considered here showed itself to be easy to apply with a fast response and a total analysis time of only 5 minutes.

Intracellular protein determination using droplet-based immunoassays

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 µM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells. © 2011 American Chemical Society.

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Schistosomiasis is a chronically debilitating helminth infection with a significant socio-economic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostic procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum infection which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas.

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.