Session Q10: Focus Session: Physics of Biochips I

Unexpected phenomena in multiplex hybridization

We discuss the characteristics of multiplex hybridization using experimental (using a custom multi-color real-time evanescent wave platform) and computational methods. The hybridization of multiple nucleic acid sequences to the same probe challenges quantitative microarray interpretation. Multiplex hybridization follows the rules of competitive displacement, in which lower affinity species are displaced by higher affinity species. This phenomenon increases the time to equilibrium and is the underlying mechanism of specificity in molecular capture. A simple three-component kinetic model of hybridization quantitatively describes multiplex hybridization, where cross-hybrids are explicitly accounted for without \textit{a priori} information. Melting analysis is a recognized methodology to study the thermodynamics of DNA interactions and positively identify sequence-specific DNA targets in a multi-component environment. Melting analysis typically assumes that multi-component melts are superpositions of individual melts. We show that under probe-limiting conditions this assumption is not valid. Multi-target interactions with a single probe sequence result in three counter-intuitive phenomena: 1) suppression of all but one apparent melt transition 2) the bound concentration of a lower-affinity species increasing with temperature and 3) the ratio between bound concentrations of high and low affinity species decreasing with temperature.   

On-Chip Nanoplasmonic Biosensors with Actively Controlled Nanofluidic Surface Delivery

Performances of biosensors are often limited by the depletion zones created around the sensing area which impede the effective analyte transport. To overcome this limitation, we propose and demonstrate a novel nanoplasmonic-nanofluidic sensor with dramatic improvements in mass transport efficiency. Unlike previous approaches where the analytes simply stream pass over the surface, our platform enables targetted delivery of the analytes to the biosensor surface. Using our platform, we show 14-fold improvement in the mass transport rate constants. Considering that this rate constant appears in the exponential term, such an improvement means much superior analyte delivery to the sensing surface with respect to conventional fluidic schemes. Our detection platform is based on extraordinary light transmission effect (EOT) in suspended plasmonic nanohole arrays. The nanoholes here act as nanofluidic channels connecting the fluidic chambers on both sides of the sensors. To fabricate these nanostructures, we introduce a lift-off free plasmonic device fabrication technique based on positive resist electron beam lithography (EBL). The simplicity of this fabrication technique allows us to fabricate nanostructures with extremely high yield/reproducibility and minimal surface roughness.   

Vapor Sensors Using Olfactory Proteins Coupled to Carbon Nanotubes

We have constructed bio-nano devices which combine mammalian olfactory proteins with carbon nanotubes to create a new class of vapor sensors. Olfactory proteins are a specific class of G-protein coupled receptors, and require a cell membrane or similar environment for proper function. Functionalization procedures have been developed to meet the challenges of routinely coupling such membrane proteins to nanotubes, while preserving the function of the protein. We have successfully isolated olfactory proteins and attached them to carbon nanotube transistors, which provide fast, all-electronic readout of analyte binding by the olfactory receptor. Several different olfactory proteins have been tested, each showing a different sensing response. This work opens the way for future coupling of biology to nanoelectronics and improved biomimetic chemical sensing. This work is supported by the DARPA RealNose Project and the Nano/Bio Interface Center   

Flow Cytometry- Current Detection Limits and Future Prospects

Flow Cytometry measures optical signals from particles, usually biological cells, flowing through intense illumination. Typical illumination time of each particle is 1 to 100 microseconds. Detection of dim fluorescence signals is limited by multiple factors including the properties of the fluorescent molecules used to stain the cells. Fluorescence intensity from a fluorophore bound to a probe molecule is affected by environment and rarely the same as from the fluorophore in solution [1]. For any particular fluorophore, there is a maximum optimal excitation intensity above which signal to noise will decrease [2]. Detection of the emitted fluorescence is limited by the overall detection efficiency of the optical system, background light and electronic noise in the data acquisition system. The ultimate limitation is due to the photon statistics of fluorescence from the stained and unstained populations of cells. A practical approach to quantitative assessment of fluorescence detection capability based on physical factors has been developed and implemented in routine testing of commercial flow cytometers [3]. When a photomultiplier is used to detect fluorescence, the overall detection efficiency, Q, is the effective number of photoelectrons per equivalent fluorescence emitter. Contributions to background light, B, include Raman scatter of water, unbound fluorescent probe and spectral overlap from different fluorophores in multicolor applications. Knowledge of Q and B and basic information about the sample allow prediction of the fluorescence population distributions of cells. New photon counting detectors, signal analysis methods and luminescent nanoparticles may provide increased detection sensitivity in the future. \\[4pt] [1] J Res Natl Inst Stand Technol. 107:83--91 (2002) \\[0pt] [2] Cytometry 29:204--214 (1997) \\[0pt] [3] Cytometry 33:267--279 (1998)   

Hand-held flow cytometer for point of care CD4 testing

Commercial flow cytometers are sophisticated analytical instruments extensively used in research and clinical laboratories. However, they do not meet the challenging practical requirements for point-of-care (POC) testing. PARC has demonstrated a new optical detection technique termed `spatially modulated emission' that delivers high signal-to-noise discrimination without precision optics to enable a flow cytometer that can combine high performance, robustness, compactness, low cost, and ease of use. The detection technique has been extensive evaluated with measurements of absolute CD4+ and percentage CD4 counts in human blood. To benchmark our system we performed a direct one-to-one comparison of measurements on the same samples with a commercial instrument (BD FACSCount) and obtained excellent agreement for both absolute CD4 and percentage CD4. We have assembled the first generation of a compact ($\sim $5x3x2 inch), single-parameter, flow cytometer based on the spatial modulation technique which uses a pin photodiode for detection rather than a PMT or APD. Measurements of the sensitivity and dynamic range of the prototype were conducted with 3.8-um ultra-rainbow calibration beads (Spherotech) and yielded a detection limit of $\sim $1000 MEPE, which meets the needs for a wide range of bio-particle-detection applications.   

A Hybrid Integrated-Circuit/Microfluidic Device for Positioning, Porating and Fusing Individual Cells

Here we report a hybrid integrated-circuit/microfluidic device which can position, porate and fuse individual cells. Existing electroporation and fusion devices can only act on cells in bulk. Our device consists of a microarray of electrode pixels$^{1}$ and a grounded conducting plate. Cells were positioned with dielectrophoretic forces induced by the pixels and porated or fused with voltage pulses which caused a dielectric breakdown of the cell membrane. The device positioned cells with 10$\mu $m precision and porated or fused them with high yields. It is programmable and mass-parallelization on a single device enables bulk applications. $^{1}$ T. Hunt, D. Issadore, R. Westervelt, \textit{Lab on a Chip}, 2008, \textbf{8}, 81-87.   

Phenotypic variability and selection of lipid-producing microalgae in a microfluidic centrifuge

Isogenic cells are known to display various expression levels that may result in different phenotypes within a population. Here we focus on the phenotypic variability of a species of unicellular algae that produce neutral lipids. Lipid-producing algae are one of the most promising sources of biofuel. We have implemented a simple microfluidic method to assess lipid-production variability in a population of algae that relays on density differences. We will discuss the reasons of this variability and address the promising avenues of this technique for directing the evolution of algae towards high lipid productivity.   

Resolving sub-cellular force dynamics using arrays of magnetic microposts

The biological response of cells to mechanical forces is integral to both normal cell function and the progression of many diseases, such as hypertensive vascular wall thickening. This likely results from the fact that mechanical stresses can directly affect many cellular processes, including signal transduction, gene expression, growth, differentiation, and survival. The need to understand the relationship between applied forces and the mechanical response of cells as a critical step towards understanding mechanotransduction calls for tools that can apply forces to cells while measuring their contractile response. This talk will describe an approach that simultaneously allows local mechanical stimulation of the adherent surface of a cell and spatially resolved measurement of the local force fields generated throughout the cell in response to this stimulation. Cells are cultured on the top surfaces of arrays of micrometer-scale posts made from a flexible elastomer (PDMS), and the contractile forces generated by an adherent cell bend the posts. Measurements of the displacement of each post allow the contractile force field of the cell to be mapped out with sub-cellular precision. To apply forces to cells, rod- shaped magnetic nanoparticles are embedded in some of the posts so that externally applied magnetic fields selectively deform these ``magnetic posts,'' thereby exerting tunable local, mechanical stresses to the adherent surface of attached cells. Alternatively, magnetic particles bound to or internalized by the cell may be employed to apply forces and torques to the cell. With either approach, measuring the deflection of the surrounding non-magnetic posts probes the full mechanical response of the cell to these stresses. Results that illustrate the temporal dynamics and spatial distribution of the non-local response of fibroblasts and smooth muscle cells to local stresses will be discussed.   

Limits of Bioparticle Detection in NanoLaser Microfluidic Chips and Application to Cancer Detection in Single Cells and Mitochondria

BioChips comprising light-emitting semiconductors can be configured as microfluidic laser cavities used for ultrafast analysis of bioparticles such as whole cells, organelles, virons, and macromolecules (protein,DNA,RNA). Three regimes of operation include: 1.Geometrical limit (particle radius a$>>\lambda $ the laser wavelength), laser exhibits multimode spectra useful to study particle morphology, shape, and composition. 2. Mie regime (a$\approx \lambda )$ laser exhibits nano-squeezed light with single mode operation to study particle size and composition. 3.Rayleigh limit (a$<<\lambda )$ laser exhibits cavity mode fluctuations to study nanoparticle mass and motion. We have recently used these biochips to study the nanolaser spectra of submicron mitochondrial bioparticles as a new probe of cancer in single cells. These high-speed, nanophotonic tools may play an important role in advancing early detection of cancer and offer improvements over conventional tumor pathology that relies on labor-intensive microscopic examination and/or older cell-staining methods that can be time-consuming and may give false readings.   

Microwave dielectric heating of drops in microfluidic devices

A technique is presented to locally and rapidly heat water drops in microfluidic devices using microwave dielectric heating. Water absorbs microwave power more efficiently than polymers, glass, and oils of microfluidic devices due to its permanent molecular dipole moment that has large dielectric loss at GHz frequencies. The relevant heat capacity of the system is that of a single thermally isolated picolitre-scale drop of water, enabling very fast thermal cycling. Microwave dielectric heating is demonstrated in a microfluidic device that integrates a flow-focusing drop maker, drop splitters, and metal electrodes to locally deliver microwave power from an inexpensive, commercially available 3.0 GHz source and amplifier. The temperature change of the drops is measured by observing the temperature dependent fluorescence intensity of cadmium selenide nanocrystals suspended in the water drops. Characteristic heating times as short as 15 ms to steady-state temperature changes as large as 30 \r{ }C above the base temperature of the microfluidic device are demonstrated. Many common biological and chemical applications require rapid and local control of temperature and can benefit from this new technique.   

Immunomagnetic Nano-Screening Chip for Circulating Tumor Cells Detection in Blood

We present a novel method towards diagnose cancer at an early stage via a blood test. Early diagnosis is high on the future agenda of oncologists because of significant evidence that it will result in a higher cure rate. Capture of circulating tumor cells (CTCs) which are known to escape from carcinomas at an early stage offers such an opportunity. We design, fabricate and optimize the nanomagnetic-screening chip that captures the CTCs in microfluid, and further integrate the nano-chip with the new multispectral imaging system so that it can quantify different tumor markers and automate the entire instrument. Specifically, hybrid plasmonic (Fe$_{2}$O$_{3}$-core Au shell) nanoparticles, conjugated a collection of antibodies especially chosen to target breast cancer CTCs, with high magnetic susceptibility will be used for effective immunomagnetic CTC isolation. Greatly increased sensitivity over previous attempts is demonstrated by decreasing the length scale for interactions between the magnetic-nanoparticle-tagged CTCs and the isolative magnetic field, while increasing the effective cross-sectional area over which this interaction takes place. The screening chip is integrated with a novel hyperspectral microscopic imaging (HMI) platform capable of recording the entire emission spectra in a single pass evaluation. The combined system will precisely quantify up to 10 tumor markers on CTCs.