Perry G. Schiro

Bioconjugation of ultrabright semiconducting polymer dots for specific cellular targeting.

Semiconducting polymer dots (Pdots) represent a new class of ultrabright fluorescent probes for biological imaging. They exhibit several important characteristics for experimentally demanding in vitro and in vivo fluorescence studies, such as their high brightness, fast emission rate, excellent photostability, nonblinking, and nontoxic feature. However, controlling the surface chemistry and bioconjugation of Pdots has been a challenging problem that prevented their widespread applications in biological studies. Here, we report a facile yet powerful conjugation method that overcomes this challenge. Our strategy for Pdot functionalization is based on entrapping heterogeneous polymer chains into a single dot, driven by hydrophobic interactions during nanoparticle formation. A small amount of amphiphilic polymer bearing functional groups is co-condensed with the majority of semiconducting polymers to modify and functionalize the nanoparticle surface for subsequent covalent conjugation to biomolecules, such as streptavidin and immunoglobulin G (IgG). The Pdot bioconjugates can effectively and specifically label cellular targets, such as cell surface marker in human breast cancer cells, without any detectable nonspecific binding. Single-particle imaging, cellular imaging, and flow cytometry experiments indicate a much higher fluorescence brightness of Pdots compared to those of Alexa dye and quantum dot probes. The successful bioconjugation of these ultrabright nanoparticles presents a novel opportunity to apply versatile semiconducting polymers to various fluorescence measurements in modern biology and biomedicine.

Protein Quantification at the Single Vesicle Level Reveals That a Subset of Synaptic Vesicle Proteins Are Trafficked with High Precision

Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.

Sensitive and High-Throughput Isolation of Rare Cells from Peripheral Blood with Ensemble-Decision Aliquot Ranking†

This paper describes an approach called ensemble decision aliquot ranking (eDAR) for isolating rare cells from peripheral blood. eDAR has a recovery of over 93% (n=9) with a zero false positive rate (n=8), and provides direct easy access to individual isolated live cells for downstream single-cell manipulation and analysis. We anticipate eDAR will enable new studies of various types of rare cells that circulate in blood.

An Automated High-Throughput Counting Method for Screening Circulating Tumor Cells in Peripheral Blood

Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel, and interrogated by a line-confocal microscope. On the basis of the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 min. We applied this method in analyzing CTCs from 90 stage IV breast cancer patient samples and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by the U.S. Food and Drug Administration at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n = 9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast cancer patients ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast cancer patients that were positive for Her2 or CD44(+)/CD24(-), which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.

Optical gradient flow focusing

This paper describes a new method for carrying out flow cytometry, which employs optical gradient forces to guide and focus particles in the fluid flow. An elliptically shaped Gaussian beam was focused at the center of a microchannel to exert radiation pressure on suspended nanoparticles that are passing through the channel, such that these particles are guided to the center of the channel for efficient detection and sorting. To verify the efficiency of this optical-gradient-flow-focusing method, we present numerical simulations of the trajectories of the nanoparticles in both electroosmotic flow (EOF) and pressure-driven flow (PDF).

Fabrication improvements for thermoset polyester (TPE) microfluidic devices

Thermoset polyester (TPE) microfluidic devices were previously developed as an alternative to poly(dimethylsiloxane) (PDMS) devices, fabricated similarly by replica molding, yet offering stable surface properties and good chemical compatibility with some organics that are incompatible with PDMS. This paper describes a number of improvements in the fabrication of TPE chips. Specifically, we describe methods to form TPE devices with a thin bottom layer for use with high numerical aperture (NA) objectives for sensitive fluorescence detection and optical manipulation. We also describe plasma-bonding of TPE to glass to create hybrid TPE-glass devices. We further present a simple master-pretreatment method to replace our original technique that required the use of specialized equipment.

Determining the number of specific proteins in cellular compartments by quantitative microscopy

This protocol describes a method for determining both the average number and variance of proteins, in the few to tens of copies, in isolated cellular compartments such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number, but lack information on the variance, or they are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling of the cellular compartment with fluorescent primary-secondary antibody complexes, total internal reflection fluorescence microscopic imaging of the cellular compartment, digital image analysis and deconvolution of the fluorescence intensity data. A minimum of 2.5 d is required to complete the labeling, imaging and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.

New generation of ensemble-decision aliquot ranking based on simplified microfluidic components for large-capacity trapping of circulating tumor cells.

Ensemble-decision aliquot ranking (eDAR) is a sensitive and high-throughput method to analyze circulating tumor cells (CTCs) from peripheral blood. Here, we report the next generation of eDAR, where we designed and optimized a new hydrodynamic switching scheme for the active sorting step in eDAR, which provided fast cell sorting with an improved reproducibility and stability. The microfluidic chip was also simplified by incorporating a functional area for subsequent purification using microslits fabricated by standard lithography method. Using the reported second generation of eDAR, we were able to analyze 1 mL of whole-blood samples in 12.5 min, with a 95% recovery and a zero false positive rate (n = 15).

Method for the Accurate Preparation of Cell-Spiking Standards

Preparation of calibration standards for cell enumeration is critical in characterizing the performance of any method or apparatus intended for recovering rare cells. Diluting a cell suspension serially is prone to statistical sampling errors as the cell suspension becomes more dilute, whereas transferring and injecting cells individually into a diluent with a micromanipulator is time-consuming. We developed a simple and robust method using a surface-modified glass capillary to siphon and eject cells. One-dimensional confinement of cells offered by the capillary made cell enumeration by visual counting simple and rapid, and cell ejection from the capillary was near 100% when the appropriate surface coating and cell solution was used. The residence time of cells in the capillary, however, could affect the percentage of cells that was ejected from the capillary. To characterize the performance of this method, we enumerated the ejected cell using both visual counting under a microscope and automated detection using a chip-based flow cytometer.

High-throughput fluorescence-activated nanoscale subcellular sorter with single-molecule sensitivity.

Recent single-cell and single-molecule studies have shown that a variety of subpopulations exist within biological systems, such as synaptic vesicles, that have previously been overlooked in common bulk studies. By isolating and enriching these various subpopulations, detailed analysis with a variety of analytical techniques can be done to further understand the role that various subpopulations play in cellular dynamics and how alterations to these subpopulations affect the overall function of the biological system. Previous sorters lack the sensitivity, sorting speed, and efficiency to isolate synaptic vesicles and other nanoscale systems. This paper describes the development of a fluorescence-activated nanoscale subcellular sorter that can sort nearly 10 million objects per hour with single-molecule sensitivity. Utilizing a near-nanoscale channel system, we were able to achieve upward of 91% recovery of desired objects with a 99.7% purity.