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.
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. Sizedependent 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.
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.
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 (K m ) from the acetylcholine–acetylcholinesterase reaction is also demonstrated.
Silicon nanowire field-effect transistors (Si-NW FETs) have been demonstrated as a versatile class of potentiometric nanobiosensors for real time, label-free, and highly sensitive detection of a wide ...range of biomolecules. In this review, we summarize the principles of such devices and recent developments in device fabrication, fluid integration, surface functionalization, and biosensing applications. The main focus of this review is on CMOS compatible Si-NW FET nanobiosensors.
A new surface functionalization scheme for nano‐Bio field effect transistors (FETs) using biocompatible polyelectrolyte thin films (PET) is developed. PET assemblies on Si nanowires (Si‐NWs) are ...driven by electrostatic interactions between the positively charged polymer backbone and negatively charged Si/SiO2 surface. Such assemblies can be directly coated from PET aqueous solutions and result in a uniform nanoscale thin film, which is more stable compared to the conventional amine silanization. Short oligo‐ethylene glycol chains are grafted on the PETs to prevent nonspecific protein binding. Moreover, the reactive groups of the polymer chains can be further functionalized to other chemical groups in specific stoichiometry for biomolecules detection. Therefore, it opens a new strategy to precisely control the functional group densities on various biosensor surfaces at the molecular level. In addition, such assemblies of the polymers together with the bound analytes can be removed with the pH stimulation resulting in regeneration of a bare sensor surface without compromising the integrity and performance of the Si‐NWs. Thus, it is believed that the developed PET coating and sensing systems on Si‐NW FETs represent a versatile, promising approach for regenerative biosensors which can be applied to other biosensors and will benefit real device applications, enhancing sensor lifetime, reliability, and repeatability.
Functionalized polyelectrolytes are successfully synthesized and assembled on silicon nanowire field effect transistors with the aim of creating regenerative nano‐electronic biosensors. Such coating has the advantage of direct solution coating and can be applied on a wafer scale. It minimizes protein nonspecific adsorption and precisely controls the surface functional group densities at molecular level.
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.
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.
Electronic nose (e-nose) is a useful tool for gas detection in environment monitoring, food industry and medical diagnosing. It utilizes sensor arrays to generate recognition pattern in identifying ...odor or specific analyte. Here a novel multimode e-nose is demonstrated for the detection of volatile organic compounds (VOCs) by integrating both gravimetric-sensitive sensor and electric-sensitive sensor. In this system, film bulk acoustic resonator (FBAR) is used to gain mass related information and silicon nanowire field effect transistor (Si-NW FET) to collect electrical related information for the analytes. The electronic nose is applied to discriminate the ethanol-hexane mixture and the concentration of each gas can be successfully calculated by simple math without using complex mathematic model. The hybrid e-nose system shows potential for the analysis of VOC mixture in the future.
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