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Diagnostic assays are commonly performed in multiple steps, where reagents are added at specific times and concentrations into a reaction chamber. The reagents require storage, preparation, and addition in the correct sequence and amount. These steps rely on trained technicians and instrumentation to perform each task. The reliance on such resources hinders the use of these diagnostic assays by lay users. We developed a tablet that can sequentially introduce prequantified lyophilized diagnostic reagents at specific time points for a multistep assay. We designed the tablet to have multiple layers using cellulose-grade polymers, such as microcrystalline cellulose and hydroxypropyl cellulose. Our formulation allows each layer to dissolve at a controlled rate to introduce reagents into the solution sequentially. The release rate is controlled by modulating the compression force or chemical formulation of the layer. Controlling the reagent release time is important because different assays have specific times when reagents need to be added. As proof of concept, we demonstrated two different assays with our tablet system. Our tablet detected nucleic acid target (

There is an increasing need for highly sensitive and quantitative diagnostics at the point-of-care. The lateral flow immunoassay (LFA) is one of the most widely used point-of-care diagnostic tests; however, LFAs generally suffer from low sensitivity and lack of quantification. To overcome these limitations, thermal contrast amplification (TCA) is a new method that is based on the laser excitation of gold nanoparticles (GNPs), the most commonly used visual signature, to evoke a thermal signature. To facilitate the clinical translation of the TCA technology, we present the development of a TCA reader, a platform technology that significantly improves the limit of detection and provides quantification of disease antigens in LFAs. This TCA reader provides enhanced sensitivity over visual detection by the human eye or by a colorimetric reader (e.g., BD Veritor System Reader). More specifically, the TCA reader demonstrated up to an 8-fold enhanced analytical sensitivity and quantification among LFAs for influenza, malaria, and Clostridium difficile. Systematic characterization of the laser, infrared camera, and other components of the reader and their integration into a working reader instrument are described. The development of the TCA reader enables simple, highly sensitive quantification of LFAs at the point-of-care.

Polymer microbeads are witnessing renewed interest for performing biomolecule recognition assays with distinct advantages over planar microarray technology. In this study, DNA hybridization assays are performed on the surfaces of 1-microm-diameter, synthetically modified polystyrene microbeads. The microbead surfaces contain varying amounts of poly(acrylic acid) as a source of carboxylate groups to which a DNA capture strand may bind. Through a series of controlled experiments in which the microbead carboxylate density and DNA:surface area ratios are systematically altered, we find that the density of carboxylate groups on the microbead surface may be the most important parameter affecting not only the total number of DNA strands that may bind to the microbead surface but, surprisingly, also the efficiency of DNA hybridization with complementary strands. These studies are aimed directly at understanding the physical interactions between DNA strands and an anionic microbead surface.

Further insight toward the complex association and dissociation events of macromolecules requires the development of a spectroscopic technique that can track individual components, or building blocks of these macromolecules, and the complexes which they form, in real time. Three-color fluorescence cross-correlation spectroscopy (3C-FCCS) has been shown to track assemblies of three spectrally labeled species in solution. Here, we clearly show that 3C-FCCS is capable of distinguishing beads barcoded with quantum dots from free quantum dots in the background despite the 800-to-1 difference in concentration of these two components. The validation of this spectroscopic technique in combination with the development of barcode labels would enable one to start to investigate complex association and dissociation kinetics of macromolecules and nanomaterials during the assembly process.

Quantum dot (QD) based contrast agents are currently being developed as probes for bioimaging and as vehicles for drug delivery. The ability to detect QDs, regardless of fluorescence brightness, in cells, tissues, and organs is imperative to their development. Traditional methods used to visualize the distribution of QDs in biological samples mainly rely on fluorescence imaging, which does not account for optically degenerate QDs as a result of oxidative quenching within the biological environment. Here, we demonstrate the use of silver staining for directly visualizing the distribution of QDs within biological samples under bright field microscopy. This strategy involves silver deposition onto the surface of QDs upon reduction by hydroquinone, effectively amplifying the size of QDs until visible for detection. The method can be used to detect non-fluorescent QDs and is fast, simple, and inexpensive.

Direct observation of single molecules and single molecular events inside living cells could dramatically improve our understanding of basic cellular processes (e.g., signal transduction and gene transcription) as well as improving our knowledge on the intracellular transport and fate of therapeutic agents (e.g., antisense RNA and gene therapy vectors). However, a key remaining question is whether single-molecule methodologies could be developed to study complex molecular processes in living cells. in contrast to clean and well-controlled conditions in-vitro, the intracellular environment contains a broad collection of biological macromolecules and fluorescent materials such as porphyrins and flavins. This complex environment is known to produce intense background fluorescence, commonly known as autofluorescence. Thus, a major concern is that this intracellular background could overwhelm the relatively weak signals arising from single molecules.We demonstrate that fluorescence detection of single molecules can be achieved by tightly focusing a laser beam into a living cell (see Figure 1). The observed background fluorescence is indeed higher than that in-vitro (e.g., pure biological buffer), but this background is continuous and stable, and does not significantly interfere with the measurement of single-molecule photon bursts. Specifically, we report single-molecule results on three types of extrinsic fluorescent molecules in cultured human HeLa cells (a cervical cancer cell line).