Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips.

CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA. PAPERCLIP.

Characterization of autoimmune inflammation induced prostate stem cell expansion.

BACKGROUND: The presence of inflammation in prostate cancer (PCa) and benign prostate hyperplasia (BPH) has been well described but the cellular mechanisms by which inflammation modulates the prostate are currently unclear. Prostate stem cells (PSC) not only maintain prostate homeostasis but also are considered to be the cell of origin of PCa and an important contributor to BPH. However, the impact of inflammation on PSC is not well understood. Therefore, we initiated studies to evaluate the effect of inflammation on PSC. METHOD: Ovalbumin specific CD8(+) T cells were intravenously delivered to intact and castrated prostate ovalbumin expressing transgenic-3 (POET-3) mice to induce inflammation. Lin (CD45/CD31)(-) Sca1(+) CD49f(+) cells (LSC) and progenitor cells within LSC were determined by flow cytometry. Sorted LSC were subjected to a prostate sphere forming assay to evaluate PSC clonal propagation, proliferation, immediate differentiation, and self-renewal ability. Density of individual spheres was measured by a cantilever-based resonator weighing system. Morphology and characterization of prostate spheres was determined by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Finally, immediate PSC differentiation in sphere formation was determined by immunofluorescence for epithelial cytokeratin markers cytokeratin (CK) 5 and CK8. RESULT: Data presented here demonstrate a significant expansion of the proliferative (BrdU(+) ) LSC population, including CK5(+) , p63(+) , CK18(+) cells, as well as intermediate cells (CK5(+) /CK8(+) ) in inflamed prostates. Histological images reveal that PSC from inflamed prostates produce significantly larger spheres, indicating that the enhanced proliferation observed in LSC is sustained in vitro in the absence of inflammatory mediators. In addition, cultures from inflamed PSC yielded increased number of tubule-like spheres. These tube-like spheres grown from PSCs isolated from inflamed mice exhibited stratification of a CK8(+) luminal-like layer and a CK5(+) basal-like layer. Notably, the numbers of spheres formed by inflamed and non-inflamed PSC were equal, suggesting that even though proliferation is enhanced by inflammation, the homeostatic level of PSC is maintained. CONCLUSION: Induction of inflammation promotes PSC expansion and immediate differentiation through highly proliferative progenitor cells while the homeostasis of PSC is maintained.

Concurrent Detection of Cellular and Molecular Cancer Markers Using an Immunomagnetic Flow System.

We report a detection system for simultaneous measurement of cellular and molecular markers of cancer. Magnetic beads conjugated with antibodies against a specific antigen are used to capture both free molecules and whole cells overexpressing the antigen. The target-bound beads then flow through a microfluidic chamber where they are drawn to a glass surface by an external magnetic field. The cells and molecules captured on the surface are quantitatively analyzed using fluorescent microscopy. The system was characterized by detecting free folate receptor (FR) and an FR+ cancer cell line (KB) in culture media. The system detected as low as 10 pM of FR and captured 87% of the spiked KB cells at a volumetric throughput of 3 mL/min. We further demonstrated the detection of 100 KB cells and 200 pM FR spiked into healthy human blood to simulate detection of rare cells and protein biomarkers present in a cancer patient's blood sample. The FR concentration was measured to be 244 pM (including the intrinsic FR present in the blood), and the total number of KB cells in the sample was estimated to be 98. The potential of this approach in clinical diagnostics was also demonstrated by detecting both FR+ cells and free FR in an ascites sample obtained from an ovarian cancer patient. Because of the system's capability to detect multiple targets at the same time, its high throughput, and its overall simplicity, we expect it to be highly useful in a wide range of research settings.

Efficient capture of circulating tumor cells with low molecular weight folate receptor-specific ligands.

Retrieval of circulating tumor cells (CTC) has proven valuable for assessing a patient's cancer burden, evaluating response to therapy, and analyzing which drug might treat a cancer best. Although most isolation methods retrieve CTCs based on size, shape, or capture by tumor-specific antibodies, we explore here the use of small molecule tumor-specific ligands linked to magnetic beads for CTC capture. We have designed folic acid-biotin conjugates with different linkers for the capture of folate receptor (FR) + tumor cells spiked into whole blood, and application of the same technology to isolate FR + CTCs from the peripheral blood of both tumor-bearing mice and non-small cell lung patients. We demonstrate that folic acid linked via a rigid linker to a flexible PEG spacer that is in turn tethered to a magnetic bead enables optimal CTC retrieval, reaching nearly 100% capture when 100 cancer cells are spiked into 1 mL of aqueous buffer and ~ 90% capture when the same quantity of cells is diluted into whole blood. In a live animal model, the same methodology is shown to efficiently retrieve CTCs from tumor-bearing mice, yielding cancer cell counts that are proportional to total tumor burden. More importantly, the same method is shown to collect ~ 29 CTCs/8 mL peripheral blood from patients with non-small cell lung cancer. Since the ligand-presentation strategy optimized here should also prove useful in targeting other nanoparticles to other cells, the methods described below should have general applicability in the design of nanoparticles for cell-specific targeting.

Diffractometric detection of proteins using microbead-based rolling circle amplification.

We present a robust, sensitive, fluorescent- or radiolabel-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15 microm wide alternating lines and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe, and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor.

Deterministic culturing of single cells in 3D.

Models using 3D cell culture techniques are increasingly accepted as the most biofidelic in vitro representations of tissues for research. These models are generated using biomatrices and bulk populations of cells derived from tissues or cell lines. We present an alternate method to culture individually selected cells in relative isolation from the rest of the population under physiologically relevant matrix conditions. Matrix gel islands are spotted on a cell culture dish to act as support for receiving and culturing individual single cells; a glass capillary-based microfluidic setup is used to extract each desired single cell from a population and seed it on top of an island. Using examples of breast and colorectal cancers, we show that individual cells evolve into tumors or aspects of tumors displaying different characteristics of the initial cancer type and aggressiveness. By implementing a morphometry assay with luminal A breast cancer, we demonstrate the potential of the proposed approach to study phenotypic heterogeneity. Results reveal that intertumor heterogeneity increases with time in culture and that varying degrees of intratumor heterogeneity may originate from individually seeded cells. Moreover, we observe that a positive relationship exists between fast growing tumors and the size and heterogeneity of their nuclei.

High-purity isolation of rare single cells from blood using a tiered microchip system.

We present a two-tiered microchip system to capture and retrieve rare cells from blood samples with high purity. The first module of the system is a high throughput microfluidic interface that is used to immunomagnetically isolate targeted rare cells from whole blood, and discard > 99.999% of the unwanted leukocytes. The second module is a microwell array that furthers the purification by magnetically guiding each cell into a separate well concurrently, and allows individual retrieval of each cell. We demonstrate the design of the system as well as its characterization by experiments using model cell lines that represent circulating fetal trophoblasts. Our results show that single cells can be retrieved with efficiencies and purities as high as 100% within 145 mins.

Immunomagnetic diffractometry for detection of diagnostic serum markers.

We describe an integrated approach for detection of diagnostic markers using in situ assembled optical diffraction gratings in combination with immunomagnetic capture. Folate receptor (FR), a serum protein indicative of various cancers, was chosen as a model system to demonstrate the potential of the method. Magnetic beads coupled to FR antibody were used to capture FR from serum. The FR-bound magnetic beads self-assembled onto microcontact-printed folate-coupled BSA (F-BSA) patterns to form diffraction gratings which served to detect FR by measuring the diffraction intensities caused by laser illumination. The FR-containing beads, upon binding to the F-BSA surface, served as intrinsic signal enhancement agents, circumventing the need for additional enzymatic signal amplification or fluorescent labeling steps. With this approach, a detection sensitivity of 700 fM (20 pg/mL) was achieved. The potential use of this approach in clinical diagnostics was demonstrated by measuring FR concentration in blood samples obtained from cancer patients.

Separation and dual detection of prostate cancer cells and protein biomarkers using a microchip device.

Current efforts for the detection of prostate cancer using only prostate specific antigen are not ideal and indicate a need to develop new assays - using multiple targets - that can more accurately stratify disease states. We previously introduced a device capable of the concurrent detection of cellular and molecular markers from a single sample fluid. Here, an improved design, which achieves affinity as well as size-based separation of captured targets using antibody-conjugated magnetic beads and a silicon chip containing micro-apertures, is presented. Upon injection of the sample, the integration of magnetic attraction with the micro-aperture chip permits larger cell-bead complexes to be isolated in an upper chamber with the smaller protein-bead complexes and remaining beads passing through the micro-apertures into the lower chamber. This enhances captured cell purity for on chip quantification, allows the separate retrieval of captured cells and proteins for downstream analysis, and enables higher bead concentrations for improved multiplexed ligand targeting. Using LNCaP cells and prostate specific membrane antigen (PSMA) to model prostate cancer, the device was able to detect 34 pM of spiked PSMA and achieve a cell capture efficiency of 93% from culture media. LNCaP cells and PSMA were then spiked into diluted healthy human blood to mimic a cancer patient. The device enabled the detection of spiked PSMA (relative to endogenous PSMA) while recovering 85-90% of LNCaP cells which illustrated the potential of new assays for the diagnosis of prostate cancer.

Circulating tumor cell detection using a parallel flow micro-aperture chip system.

We report on-chip isolation and detection of circulating tumor cells (CTCs) from blood samples using a system that integrates a microchip with immunomagnetics, high-throughput fluidics and size-based filtration. CTCs in a sample are targeted via their surface antigens using magnetic beads functionalized with antibodies. The mixture is then run through a fluidic chamber that contains a micro-fabricated chip with arrays of 8 µm diameter apertures. The fluid runs parallel to the microchip while a magnetic field is generated underneath to draw the beads and cells bound to them toward the chip surface for detection of CTCs that are larger than the apertures and clear out free beads and other smaller particles bound to them. The parallel flow configuration allows high volumetric flow rates, which reduces nonspecific binding to the chip surface and enables multiple circulations of the sample fluid through the system in a short period of time. In this study we first present models of the magnetic and fluidic forces in the system using a finite element method. We then verify the simulation results experimentally to determine an optimal flow rate. Next, we characterize the system by detecting cancer cell lines spiked into healthy human blood and show that on average 89% of the spiked MCF-7 breast cancer cells were detected. We finally demonstrate detection of CTCs in 49 out of 50 blood samples obtained from non-small cell lung cancer (NSCLC) patients and pancreatic cancer (PANC) patients. The number of CTCs detected ranges from 2 to 122 per 8 mL s of blood. We also demonstrate a statistically significant difference between the CTC counts of NSCLC patients who have received therapy and those who have not.

On-demand weighing of single dry biological particles over a 5-order-of-magnitude dynamic range.

We report a simple and highly versatile system to select and weigh individual dry biological particles. The system is composed of a microtweezer to pick and place individual particles and a cantilever-based resonator to weigh them. The system can weigh entities that vary from a red blood cell (~10(-11) g) to the eye-brain complex of an insect (~10(-6) g), covering a 5-order-of-magnitude mass range. Due to its versatility and ease of use, this weighing method is highly compatible with established laboratory practices. The system can provide complementary mass information for a wide variety of individual particles imaged using scanning electron microscopy and determine comparative weights of individual biological entities that are attached to microparticles as well as weigh fractions of individual biological entities that have been subjected to focused ion beam milling.

A humidity-sensitive hydrogel-Bacillus spore composite for micropatterning of biomolecular gradients.

A composite material consisting of Bacillus subtilis spores suspended in a humidity sensitive hydrogel can be used to pattern biomolecules in different concentrations directly onto glass surfaces using a mechanical micromanipulator. By altering the relative humidity surrounding the composite gel during deposition, surface concentration of patterned biomolecules can be controlled and varied to create user-defined, biomolecular surface concentrations.

Laser-treated hydrophobic paper: an inexpensive microfluidic platform.

We report a method for fabricating inexpensive microfluidic platforms on paper using laser treatment. Any paper with a hydrophobic surface coating (e.g., parchment paper, wax paper, palette paper) can be used for this purpose. We were able to selectively modify the surface structure and property (hydrophobic to hydrophilic) of several such papers using a CO(2) laser. We created patterns down to a minimum feature size of 62±1 µm. The modified surface exhibited a highly porous structure which helped to trap/localize chemical and biological aqueous reagents for analysis. The treated surfaces were stable over time and were used to self-assemble arrays of aqueous droplets. Furthermore, we selectively deposited silica microparticles on patterned areas to allow lateral diffusion from one end of a channel to the other. Finally, we demonstrated the applicability of this platform to perform chemical reactions using luminol-based hemoglobin detection.

Real-time detection of airborne viruses on a mass-sensitive device.

We present real-time detection of airborne Vaccinia viruses using quartz crystal microbalance (QCM) in an integrated manner. Vaccinia viruses were aerosolized and neutralized using an electrospray aerosol generator, transported into the QCM chamber, and captured by a QCM crystal. The capture of the viruses on the QCM crystal resulted in frequency shifts proportional to the number of viruses. The capture rate varied linearly with the concentration of initial virus suspensions (8.5x10(8)-8.5x10(10) particlesml) at flow rates of 2.0 and 1.1 lmin. This work demonstrates the general potential of mass sensitive detection of nanoscale biological entities in air.

Bright-field analysis of phi29 DNA packaging motor using a magnetomechanical system.

We report a simple and robust magnetomechanical system for direct visual observation of the DNA packaging behavior of the bacteriophage phi29 in real time. The system comprises a micron-sized magnetic bead attached to the free end of the viral DNA, a magnet and a bright-field microscope. We show that the phi29 DNA packaging activity can be observed and dynamically analyzed at the single molecular level in bright field with a relatively simple system. With this system we also visually demonstrate the phi29 motor transporting a cargo 10 000 times the viral size.

Ribozyme-mediated signal augmentation on a mass-sensitive biosensor.

Mass-based detection methods such as the quartz crystal microbalance (QCM) offer an attractive option to label-based methods; however the sensitivity is generally lower by comparison. In particular, low-molecular-weight analytes can be difficult to detect based on mass addition alone. In this communication, we present the use of effector-dependent ribozymes (aptazymes) as reagents for augmenting small ligand detection on a mass-sensitive device. Two distinct aptazymes were chosen: an L1-ligase-based aptazyme (L1-Rev), which is activated by a small peptide (MW approximately 2.4 kDa) from the HIV-1 Rev protein, and a hammerhead cleavase-based aptazyme (HH-theo3) activated by theophylline (MW = 180 Da). Aptazyme activity was observed in real time, and low-molecular-weight analyte detection has been successfully demonstrated with both aptazymes.

Micromechanical detection of proteins using aptamer-based receptor molecules.

We report label-free protein detection using a microfabricated cantilever-based sensor that is functionalized with DNA aptamers to act as receptor molecules. The sensor utilizes two adjacent cantilevers that constitute a sensor/reference pair and allows direct detection of the differential bending between the two cantilevers. One cantilever is functionalized with aptamers selected for Taq DNA polymerase while the other is blocked with single-stranded DNA. We have found that the polymerase-aptamer binding induces a change in surface stress, which causes a differential cantilever bending that ranges from 3 to 32 nm depending on the ligand concentration. Protein recognition on the sensor surface is specific and has a concentration dependence that is similar to that in solution.