Proteomics of protein trafficking by in vivo tissue-specific labeling.

Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.

Mass measurements during lymphocytic leukemia cell polyploidization decouple cell cycle- and cell size-dependent growth.

Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from ∼1% to ∼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2 Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.

Trapping of sub-100 nm nanoparticles using gigahertz acoustofluidic tweezers for biosensing applications.

In this study, we present a nanoscale acoustofluidic trap (AFT) that manipulates nanoparticles in a microfluidic system actuated by a gigahertz acoustic resonator. The AFT generates independent standing closed vortices with high-speed rotation. Via careful design and optimization of geometric confinements, the AFT was able to effectively capture and enrich sub-100 nm nanoparticles with a low power consumption (0.25-5 µW µm-2) and rapid trapping (within 30 s), showing significantly enhanced particle-operating ability as compared to its acoustic and optical counterparts; using specifically functionalized nanoparticles (SFNPs) to selectively capture target molecules from the sample, the AFT led to the molecular concentration enhancement of ∼200 times. We investigated the feasibility of the SFNP-assisted AFT preconcentration method for biosensing applications and successfully demonstrated the capability of this method for the detection of serum prostate-specific antigen (PSA). The AFT was prepared via a fully CMOS-compatible process and thus could be conveniently integrated on a single chip, with potential for "lab-on-a-chip" or point-of-care (POC) nanoparticle-based biosensing applications.

Cellphone-Enabled Microwell-Based Microbead Aggregation Assay for Portable Biomarker Detection.

Quantitative biomarker detection methods featured with rapidity, high accuracy, and label-free are demonstrated for the development of point-of-care (POC) technologies or "beside" diagnostics. Microbead aggregation via protein-specific linkage provides an effective approach for selective capture of biomarkers from the samples, and can directly readout the presence and amount of the targets. However, sensors or microfluidic analyzers that can accurately quantify the microbead aggregation are scared. In this work, we demonstrate a microwell-based microbeads analyzing system, by which online manipulations of microbeads including trapping, arraying, and rotations can be realized, providing a series of microfluidic approaches to layout the aggregated microbeads for further convenient characterizations. Prostate specific antigen is detected using the proposed system, demonstrating the limit of detection as low as 0.125 ng/mL (3.67 pM). A two-step reaction kinetics model is proposed for the first time to explain the dynamic process of microbeads aggregation. The developed microbeads aggregation analysis system has the advantages of label-free detection, high throughput, and low cost, showing great potential for portable biomarker detection.

Sensing the electrical activity of single ion channels with top-down silicon nanoribbons.

Using top-down fabricated silicon nanoribbons, we measure the opening and closing of ion channels alamethicin and gramicidin A. A capacitive model of the system is proposed to demonstrate that the geometric capacitance of the nanoribbon is charged by ion channel currents. The integration of top-down nanoribbons with electrophysiology holds promise for integration of electrically active living systems with artificial electronics.

Nanoelectronic Platform for Ultrasensitive Detection of Protein Biomarkers in Serum using DNA Amplification.

Silicon nanowire field effect transistors (NWFETs) are low noise, low power, ultrasensitive biosensors that are highly amenable to integration. However, using NWFETs to achieve direct protein detection in physiological buffers such as blood serum remains difficult due to Debye screening, nonspecific binding, and stringent functionalization requirements. In this work, we performed an indirect sandwich immunoassay in serum combined with exponential DNA amplification and pH measurement by ultrasensitive NWFET sensors. Measurements of model cytokine interleukin-2 concentrations from <20 fM to >200 pM were demonstrated, surpassing the conventional NWFET urease-based readout. Our approach paves way for future development of universal, highly sensitive, miniaturized, and integrated nanoelectronic devices that can be applied to a wide variety of analytes.

Smartphone-Enabled Colorimetric Trinitrotoluene Detection Using Amine-Trapped Polydimethylsiloxane Membranes.

A smartphone-enabled platform for easy and portably colorimetric analysis of 2,4,6-trinitrotoluene (TNT) using amine-trapped PDMS is designed and implemented. The amine-trapped polydimethylsiloxane (PDMS) is simply prepared by immersing the cured PDMS in aminosilane solutions forming an amine-containing polymer. After contacting with TNT-containing solutions, the colorless PDMS showed a rapid colorimetric change which can be easily identified by the naked eye. The amine-trapped PDMS was carefully optimized to achieve visible detection of TNT at concentrations as low as 1 µM. Using an integrated camera in the smartphone, pictures of colored PDMS membranes can be analyzed by a home-developed mobile application. Thus, the TNT amount can be precisely quantified. Direct TNT detection in real samples (e.g., drinking, tap, and lake waters) is demonstrated as well. The smartphone-enabled colorimetric method using amine-trapped PDMS membranes realizes a convenient and efficient approach toward a portable system for field TNT detections.

Metal-coated microfluidic channels: An approach to eliminate streaming potential effects in nano biosensors.

We report a method to suppress streaming potential using an Ag-coated microfluidic channel on a p-type silicon nanowire (SiNW) array measured by a multiplexed electrical readout. The metal layer sets a constant electrical potential along the microfluidic channel for a given reference electrode voltage regardless of the flow velocity. Without the Ag layer, the magnitude and sign of the surface potential change on the SiNW depends on the flow velocity, width of the microfluidic channel and the device's location inside the microfluidic channel with respect to the reference electrode. Noise analysis of the SiNW array with and without the Ag coating in the fluidic channel shows that noise frequency peaks, resulting from the operation of a piezoelectric micropump, are eliminated using the Ag layer with two reference electrodes located at inlet and outlet. This strategy presents a simple platform to eliminate the streaming potential and can become a powerful tool for nanoscale potentiometric biosensors.

Bioinspired Nanoparticulate Medical Glues for Minimally Invasive Tissue Repair.

Delivery of tissue glues through small-bore needles or trocars is critical for sealing holes, affixing medical devices, or attaching tissues together during minimally invasive surgeries. Inspired by the granule-packaged glue delivery system of sandcastle worms, a nanoparticulate formulation of a viscous hydrophobic light-activated adhesive based on poly(glycerol sebacate)-acrylate is developed. Negatively charged alginate is used to stabilize the nanoparticulate surface to significantly reduce its viscosity and to maximize injectability through small-bore needles. The nanoparticulate glues can be concentrated to ≈30 w/v% dispersions in water that remain localized following injection. With the trigger of a positively charged polymer (e.g., protamine), the nanoparticulate glues can quickly assemble into a viscous glue that exhibits rheological, mechanical, and adhesive properties resembling the native poly(glycerol sebacate)-acrylate based glues. This platform should be useful to enable the delivery of viscous glues to augment or replace sutures and staples during minimally invasive procedures.

Direct, rapid, and label-free detection of enzyme-substrate interactions in physiological buffers using CMOS-compatible nanoribbon sensors.

We demonstrate the versatility of Al2O3-passivated Si nanowire devices ("nanoribbons") in the analysis of enzyme-substrate interactions via the monitoring of pH change. Our approach is shown to be effective through the detection of urea in phosphate buffered saline (PBS), and penicillinase in PBS and urine, at limits of detection of <200 µM and 0.02 units/mL, respectively. The ability to extract accurate enzyme kinetics and the Michaelis-Menten constant (Km) from the acetylcholine-acetylcholinesterase reaction is also demonstrated.

Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction.

Secreted proteins including cytokines, chemokines, and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g., in the immunological system (Rothenberg, 2007), tumor microenvironment (Hanahan and Weinberg, 2011), or stem cell niche (Gnecchi etal., 2008). Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically identical cell population can give rise to diverse phenotypic differences (Niepel etal., 2009). Non-genetic heterogeneity is also emerging as a potential barrier to accurate monitoring of cellular immunity and effective pharmacological therapies (Cohen etal., 2008; Gascoigne and Taylor, 2008), but can hardly assessed using conventional approaches that do not examine cellular phenotype at the functional level. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

High-throughput secretomic analysis of single cells to assess functional cellular heterogeneity.

Secreted proteins dictate a range of cellular functions in human health and disease. Because of the high degree of cellular heterogeneity and, more importantly, polyfunctionality of individual cells, there is an unmet need to simultaneously measure an array of proteins from single cells and to rapidly assay a large number of single cells (more than 1000) in parallel. We describe a simple bioanalytical assay platform consisting of a large array of subnanoliter microchambers integrated with high-density antibody barcode microarrays for highly multiplexed protein detection from over a thousand single cells in parallel. This platform has been tested for both cell lines and complex biological samples such as primary cells from patients. We observed distinct heterogeneity among the single cell secretomic signatures that, for the first time, can be directly correlated to the cells' physical behavior such as migration. Compared to the state-of-the-art protein secretion assay such as ELISpot and emerging microtechnology-enabled assays, our approach offers both high throughput and high multiplicity. It also has a number of clinician-friendly features such as ease of operation, low sample consumption, and standardized data analysis, representing a potentially transformative tool for informative monitoring of cellular function and immunity in patients.

A portable chemotaxis platform for short and long term analysis.

Flow-based microfluidic systems have been widely utilized for cell migration studies given their ability to generate versatile and precisely defined chemical gradients and to permit direct visualization of migrating cells. Nonetheless, the general need for bulky peripherals such as mechanical pumps and tubing and the complicated setup procedures significantly limit the widespread use of these microfluidic systems for cell migration studies. Here we present a simple method to power microfluidic devices for chemotaxis assays using the commercially available ALZET® osmotic pumps. Specifically, we developed a standalone chemotaxis platform that has the same footprint as a multiwell plate and can generate well-defined, stable chemical gradients continuously for up to 7 days. Using this platform, we validated the short-term (24 hours) and long-term (72 hours) concentration dependent PDGF-BB chemotaxis response of human bone marrow derived mesenchymal stem cells.

Nanoparticle-based monitoring of cell therapy.

Exogenous cell therapy aims to replace/repair diseased or dysfunctional cells and promises to revolutionize medicine by restoring tissue and organ function. To develop effective cell therapy, the location, distribution and long-term persistence of transplanted cells must be evaluated. Nanoparticle (NP) based imaging technologies have the potential to track transplanted cells non-invasively. Here we summarize the most recent advances in NP-based cell tracking with emphasis on (1) the design criteria for cell tracking NPs, (2) protocols for cell labeling, (3) a comparison of available imaging modalities and their corresponding contrast agents, (4) a summary of preclinical studies on NP-based cell tracking and finally (5) perspectives and future directions.