Here we describe a robust, microfluidic technique to generate and analyze 3D tumor spheroids, which resembles tumor microenvironment and can be used as a more effective preclinical drug testing and screening model. Monodisperse cell-laden alginate droplets were generated in Polydimethylsiloxane (PDMS) microfluidic devices that combine T-junction droplet generation and external gelation for spheroid formation. The proposed approach has the capability to incorporate multiple cell types. For the purposes of our study, we generated spheroids with breast cancer cell lines (MCF-7 drug sensitive and resistant) and co-culture spheroids of MCF-7 together with a fibroblast cell line (HS-5). The device has the capability to house 1000 spheroids on chip for drug screening and other functional analysis. Cellular viability of spheroids in the array part of the device was maintained for two weeks by continuous perfusion of complete media into the device. The functional performance of our 3D tumor models and a dose dependent response of standard chemotherapeutic drug, Doxorubicin (Dox) and standard drug combination Dox and Paclitaxel (PCT) was analyzed on our chip-based platform. Altogether, our work provides a simple and novel, in vitro platform to generate, image and analyze uniform, 3D monodisperse Alginate hydrogel tumors for various Omic studies and therapeutic efficiency screening, an important translational step before in vivo studies.

In this communication, we describe the integration of microarray sensor technology with logic capability for screening combinations of proteins and DNA in a biological sample. In this system, we have demonstrated the use of a single platform amenable to both protein detection and protein-DNA detection using molecular logic gates. The pattern of protein and DNA inputs results in fluorescence outputs according to a truth table for AND and INHIBIT gates, thereby demonstrating the feasibility of performing medical diagnostics using a logic gate design. One possible application of this technique would be for the direct screening of various medical conditions that are dependent on combinations of diagnostic markers.Molecular logic gates and molecular computational systems have used a variety of recognition mechanisms including proteins, 1-5 biochemical pathways in living cells, 6-15 and DNA [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] . Molecular logic and computation may also be applied to medical diagnostics. For example, if abnormal results were detected during medical examination, they could be interpreted using Boolean logic resulting in intelligent diagnostics. In this communication, we report the integration of microarray sensor technology with logic capability for screening combinations of proteins and DNA in a biological sample. In this system, the reporter and receptor molecules perform simple logic operations by coupling multiple molecular recognition inputs to a fluorescence signal output.Previously developed molecular AND gates by the de Silva group allowed the creation of a "lab on a molecule" prototype, which responded to inputs of the electrolytes Na + , H + , and Zn 2+ in water leading to an enhanced fluorescence signal as output. 28 In addition, the NOTIF logic function was previously demonstrated in designed synthetic peptide networks that mimic some basic logic functions of more complex biological networks. 32 In this communication, we demonstrate both AND and INHIBIT (NOTIF) logic gates that respond to the presence of both protein and DNA in a sample. Such a system potentially could be used for performing smart diagnostics. For instance, increased airway obstruction in patients with chronic obstructive pulmonary disease (COPD) or bronchial asthma is frequently associated with bacterial respiratory infections, especially Haemophilus influenzae (H. influenzae), and is accompanied by the secretion of proinflammatory cytokines, such as IL-8 protein. [32][33][34][35][36][37] It is possible to describe the resulting logic network of bacterial DNA and IL-8 protein using a Boolean operator truth table ( Figure 1A).Our system is designed to determine if both a protein and a nucleic acid sequence are present in a sample, or if only protein is present. A schematic illustration of the protein and protein-DNA sensors is shown in Figures 2A and B, respectively. The fiberoptic microarray was prepared by loading monoclonal antibody (MAb)-functionalized microspheres into microwells crea...

With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, we have applied a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection event that can be greatly amplified via isothermal Rolling Circle Amplification (RCA). By merging the single-molecule detection power of RCA reaction with microfluidic technology we were able to demonstrate that identification of specific protein markers can be achieved on tumor cell surface in miniaturized nano-liter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer.

While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

Here we present a microfluidic method for the analysis of single cell secretions. The method co-encapsulates cells with microspheres conjugated with capture antibodies and detection fluorescence-labeled antibodies. The secreted substance captured on the microsphere surface and detected via detection antibodies generating a localized fluorescent signal on a microsphere surface. Using this method, CD4+CD25+ regulatory T cells were encapsulated and assayed to detect IL-10 secreting cell in population.

We describe herein a newly developed optical microbiosensor for the diagnosis of hepatitis C virus (HCV) by using a novel photoimmobilization methodology based on a photoactivable electrogenerated polymer film deposited upon surface-conductive fiber optics, which are then used to link a biological receptor to the fiber tip through light mediation. This fiber-optic electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide on the silica surface of the fiber optics. Monomers are then electropolymerized onto the conductive metal oxide surface; thereafter, the fibers are immersed in a solution containing HCV-E2 envelope protein antigen and illuminated with UV light (wavelength approximately 345 nm). As a result of the photochemical reaction, a thin layer of the antigen becomes covalently bound to the benzophenone-modified surface. The photochemically modified fiber optics were tested as immunosensors for the detection of anti-E2 protein antibody analyte that was measured through chemiluminescence reaction. The biosensor was tested for sensitivity, specificity, and overall practicality. Our results suggest that the detection of anti-E2 antibodies with this microbiosensor may enhance significantly HCV serological standard testing especially among patients during dialysis, which were diagnosed as HCV negative, by standard immunological tests, but were known to carry the virus. If transformed into an easy to use procedure, this assay might be used in the future as an important clinical tool for HCV screening in blood banks.

We describe a high-density microarray for simultaneous detection of proteins and DNA in a single test. In this system, Rolling Circle Amplification (RCA) was used as a signal amplification method for both protein and nucleic acid detection. The microsphere sensors were tested with synthetic DNA and purified recombinant protein analytes. The target DNA sequence was designed from a highly conserved gene that encodes the outer membrane protein P6 (OMP-P6) of both typeable and nontypeable strains of Haemophilus influenzae. The proinflammatory mediators IL-6 and IL-8 were selected as target proteins. Capture antibodies were first immobilized on fluorescently-encoded microspheres. The microspheres were then loaded into the etched microwells of an imaging optical fiber bundle. A sandwich assay was performed for target proteins IL-6 and IL-8 using biotin-labeled secondary antibodies. Biotinylated capture DNA probes were then attached to the detection antibodies via an avidin bridge. A padlock probe, complementary to the target sequence, was subsequently hybridized to the capture probe. In the presence of the target sequence, the padlock probe was ligated and this circular sequence was used for RCA. Following RCA, multiple fluorescently-labeled signal probes were hybridized to each amplified sequence and the microarray was imaged using an epi-fluorescence microscope. With this assay, detection limits down to 10 fM and 1 pM were achieved for proteins and target DNA, respectively. In addition to this new approach for detecting both protein and DNA in a single test using RCA, the limit of detection for IL-8 and IL-6 was improved by three orders of magnitude compared to similar microsphere-based assays.

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.