Miniaturizing High Throughput Droplet Assays For Ultrasensitive Molecular Detection On A Portable Platform

Digital droplet assays – in which biological samples are compartmentalized into millions of femtoliter-volume droplets and interrogated individually – have generated enormous enthusiasm for their ability to detect biomarkers with single-molecule sensitivity. These assays have untapped potential for point-of-care diagnostics but are mainly confined to laboratory settings due to the instrumentation necessary to serially generate, control, and measure millions of compartments. To address this challenge, we developed an optofluidic platform that miniaturizes digital assays into a mobile format by parallelizing their operation. This technology has three key innovations: 1. the integration and parallel operation of hundred droplet generators onto a single chip that operates \u3e100x faster than a single droplet generator. 2. the fluorescence detection of droplets at \u3e100x faster than conventional in-flow detection using time-domain encoded mobile-phone imaging, and 3. the integration of on-chip delay lines and sample processing to allow serum-to-answer device operation. By using this time-domain modulation with cloud computing, we overcome the low framerate of digital imaging, and achieve throughputs of one million droplets per second. To demonstrate the power of this approach, we performed a duplex digital enzyme-linked immunosorbent assay (ELISA) in serum to show a 1000x improvement over standard ELISA and matching that of the existing laboratory-based gold standard digital ELISA system. This work has broad potential for ultrasensitive, highly multiplexed detection, in a mobile format. Building on our initial demonstration, we explored the following: (i) we demonstrated that the platform can be extended to \u3e100x multiplexing by using time-domain encoded light sources to detect color-coded beads that each correspond to a unique assay, (ii) we demonstrated that the platform can be extended to the detection of nucleic acid by implementing polymerase chain reaction, and (iii) we demonstrated that sensitivity can be improved with a nanoparticle-enhanced ELISA. Clinical applications can be expanded to measure numerous biomarkers simultaneously such as surface markers, proteins, and nucleic acids. Ultimately, by building a robust device, suitable for low-cost implementation with ultrasensitive capabilities, this platform can be used as a tool to quantify numerous medical conditions and help physicians choose optimal treatment strategies to enable personalized medicine in a cost-effective manner

Common-path multimodal optical microscopy

We have developed a common-path multimodal optical microscopy system that is capable of using a single optical source and a single camera to image amplitude, phase, and fluorescence features of a biological specimen. This is achieved by varying either contrast enhancement filters at the Fourier plane and/or neutral density/fluorescence filters in front of the CCD camera. The feasibility of the technique is demonstrated by obtaining brightfield, fluorescence, phase-contrast, spatially filtered, brightfield + fluorescence, phase +fluorescence, and edge-enhanced+fluorescence images of the same Drosophila embryo without the need for image registration and fusion. This comprehensive microscope has the capability of providing both structural and functional information and may be used for applications such as studying live-cell dynamics and in high throughput microscopy and automated microscopy

Nonlinear photoacoustics for measuring the nonlinear optical absorption coefficient

We report a novel photoacoustic Z-scan (PAZ-scan) technique that combines the advantages offered by the conventional Z-scan method and the sensitivity of the photoacoustic detection. The sample is scanned through the focused laser beam and the generated photoacoustic signal is recorded using a 10 MHz focused ultrasound transducer. Since the signal strength is directly proportional to the optical absorption, PAZ-scan displays nonlinear behavior depicting the nonlinear optical absorption of the material. Among many advantages, our experiments on mouse blood show that PAZ-scan can potentially be used as a standard technique to calibrate contrast agents used in theranostics in general and photoacoustics in particular

Passive all-optical diode using asymmetric nonlinear absorption

Saturable and reverse saturable absorptions are well-known phenomena, originating from the imaginary component of the third order nonlinear optical susceptibility. We note that structures with an axially asymmetric nonlinear absorption can be easily realized from saturable and reverse saturable absorption materials arranged in tandem. In this paper, the basic transmission behavior of such a structure is worked out. Detailed numerical simulations demonstrate passive all-optical diode behavior, and the results are verified experimentally. The principle will work for all light polarizations, has no phase-matching restrictions, and can be extended to a large number of available nonlinear media for possible applications

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies

Medical image processing using transient Fourier holography in bacteriorhodopsin films

Real time image processing is demonstrated by recording and reconstructing the transient photoisomerizative grating formed in the bR film using Fourier holography. Desired spatial frequencies including both high and low band in the object beam are reconstructed by controlling the reference beam intensity. The results are in agreement with a theoretical model based on photoisomerization grating. We exploit this technique to process mammograms in real-time for identification of microcalcifications buried in the soft tissue for early detection of breast cancer. A feature of the technique is the ability to transient display of selected spatial frequencies in the reconstructing process which enables the radiologists to study the features of interest

Phase contrast imaging using photothermally induced phase transitions in liquid crystals

Phase contrast imaging is performed for live biological species using photothermal induced birefringence in dye doped liquid crystals. Using typical 4-f configuration, when liquid crystal cell is at back focal plane of Fourier lens, low spatial frequencies at center of Fourier spectrum are intense enough to induce local liquid crystal molecules into isotropic phase, whereas high spatial frequencies on the edges are not intense enough and remain in anisotropic phase. This results in π/2 phase difference between high and low spatial frequencies. This simple, inexpensive, all-optical, user-friendly, self-adaptive phase contrast imaging technique using low-power laser offers several distinct advantages

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB

Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis. The 15 nm cis-opening and a 3.5 nm trans-constriction describe a conical shape that allows the characterization of a wide range of proteins. Molecular dynamics showed proteins are captured by the electroosmotic flow, and the overall resistance is largely dominated by the narrow trans constriction region of the nanopore. Conveniently, proteins in the 35-125 kDa range remain trapped within the conical lumen of the nanopore for a time that can be tuned by the external bias. Contrary to cylindrical nanopores, in YaxAB, the current blockade decreases with the size of the trapped protein, as smaller proteins penetrate deeper into the constriction region than larger proteins do. These characteristics are especially useful for characterizing large proteins, as shown for pentameric C-reactive protein (125 kDa), a widely used health indicator, which showed a signal that could be identified in the background of other serum proteins. </p

Toward fast malaria detection by secondary speckle sensing microscopy

Diagnosis of malaria must be rapid, accurate, simple to use, portable and low cost, as suggested by the World Health Organization (WHO). Despite recent efforts, the gold standard remains the light microscopy of a stained blood film. This method can detect low parasitemia and identify different species of Plasmodium. However, it is time consuming, it requires well trained microscopist and good instrumentation to minimize misinterpretation, thus the costs are considerable. Moreover, the equipment cannot be easily transported and installed. In this paper we propose a new technique named “secondary speckle sensing microscopy” (S3M) based upon extraction of correlation based statistics of speckle patterns generated while illuminating red blood cells with a laser and inspecting them under a microscope. Then, using fuzzy logic ruling and principle component analysis, good quality of separation between healthy and infected red blood cells was demonstrated in preliminary experiments. The proposed technique can be used for automated high rate detection of malaria infected red blood cells