Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor.

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.

Nanoscale characterization of DNA conformation using dual-color fluorescence axial localization and label-free biosensing.

Quantitative determination of the density and conformation of DNA molecules tethered to the surface can help optimize and understand DNA nanosensors and nanodevices, which use conformational or motional changes of surface-immobilized DNA for detection or actuation. We present an interferometric sensing platform that combines (i) dual-color fluorescence spectroscopy for precise axial co-localization of two fluorophores attached at different nucleotides of surface-immobilized DNA molecules and (ii) independent label-free quantification of biomolecule surface density at the same site. Using this platform, we examined the conformation of DNA molecules immobilized on a three-dimensional polymeric surface and demonstrated simultaneous detection of DNA conformational change and binding in real-time. These results demonstrate that independent quantification of both surface density and molecular nanoscale conformation constitutes a versatile approach for nanoscale solid-biochemical interface investigations and molecular binding assays.

Microfluidic, marker-free isolation of circulating tumor cells from blood samples.

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen-independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2-5 d.

Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing-enhanced microfluidic CTC capture platform, termed "CTC-iChip," that is capable of sorting rare CTCs from whole blood at 10(7) cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.

Precisely controlled smart polymer scaffold for nanoscale manipulation of biomolecules.

We demonstrate the application of a novel smart surface to modulate the orientation of immobilized double stranded DNA (dsDNA) and the conformation of a polymer scaffold through variation in buffer pH and ionic strength. An amphoteric poly(dimethylacrylamide) based coating containing weak acrylamido acids and bases, which are copolymerized together with the neutral monomer, is covalently bound to the surface. The coating can be made to contain any desired amount of buffering and titrant ionogenic monomers, allowing control of the surface charge when the surface is bathed in a given buffer pH. Spectral self-interference fluorescence microscopy (SSFM) is utilized to precisely quantify both the DNA orientation and the polymer conformation with subnanometer resolution. It is possible to utilize the polymer scaffold to functionalize a variety of common materials used in microfabrication, making it a general purpose building block for the next generation of nanomachines and biosensors.

Quantification of surface etching by common buffers and implications on the accuracy of label-free biological assays.

High throughput analyses in biochemical assays are gaining popularity in the post-genomic era. Multiple label-free detection methods are especially of interest, as they allow quantitative monitoring of biomolecular interactions. It is assumed that the sensor surface is stable to the surrounding medium while the biochemical processes are taking place. Using the Interferometric Reflectance Imaging Sensor (IRIS), we found that buffers commonly used in biochemical reactions can remove silicon dioxide, a material frequently used as the solid support in the microarray industry. Here, we report 53 pm to 731 pm etching of the surface silicon oxide over a 12-h period for several different buffers, including various concentrations of SSC, SSPE, PBS, TRIS, MES, sodium phosphate, and potassium phosphate buffers, and found that PBS and MES buffers are much more benign than the others. We observe a linear dependence of the etch depth over time, and we find the etch rate of silicon dioxide in different buffers that ranges from 2.73±0.76 pm/h in 1M NaCl to 43.54±2.95 pm/h in 6×SSC. The protective effects by chemical modifications of the surface are explored. We demonstrate unaccounted glass etching leading to erroneous results with label-free detection of DNA microarrays, and offer remedies to increase the accuracy of quantitative analysis.

Floating light-activated microelectrical stimulators tested in the rat spinal cord.

Microelectrodes of neural stimulation utilize fine wires for electrical connections to driving electronics. Breakage of these wires and the neural tissue response due to their tethering forces are major problems encountered with long-term implantation of microelectrodes. The lifetime of an implant for neural stimulation can be substantially improved if the wire interconnects are eliminated. Thus, we proposed a floating light-activated microelectrical stimulator (FLAMES) for wireless neural stimulation. In this paradigm, a laser beam at near infrared (NIR) wavelengths will be used as a means of energy transfer to the device. In this study, microstimulators of various sizes were fabricated, with two cascaded GaAs p-i-n photodiodes, and tested in the rat spinal cord. A train of NIR pulses (0.2 ms, 50 Hz) was sent through the tissue to wirelessly activate the devices and generate the stimulus current. The forces elicited by intraspinal stimulation were measured from the ipsilateral forelimb with a force transducer. The largest forces were around 1.08 N, a significant level of force for the rat forelimb motor function. These in vivo tests suggest that the FLAMES can be used for intraspinal microstimulation even for the deepest implant locations in the rat spinal cord. The power required to generate a threshold arm movement was investigated as the laser source was moved away from the microstimulator. The results indicate that the photon density does not decrease substantially for horizontal displacements of the source that are in the same order as the beam radius. This gives confidence that the stimulation threshold may not be very sensitive to small displacement of the spinal cord relative to the spine-mounted optical power source.

A nanoelectromechanical biosensor based on precise quantification and control of DNA orientation.

We utilize spectral self-interference fluorescent microscopy (SSFM) to measure fluorophore height with sub-nm precision to precisely quantify DNA orientation. A novel polymeric 3D scaffold is used to functionalize the sensor surface and to control orientation of the surface anchored DNA.

Platform for in situ real-time measurement of protein-induced conformational changes of DNA.

A platform for in situ and real-time measurement of protein-induced conformational changes in dsDNA is presented. We combine electrical orientation of surface-bound dsDNA probes with an optical technique to measure the kinetics of DNA conformational changes. The sequence-specific Escherichia coli integration host factor is utilized to demonstrate protein-induced bending upon binding of integration host factor to dsDNA probes. The effects of probe surface density on binding/bending kinetics are investigated. The platform can accommodate individual spots of microarrayed dsDNA on individually controlled, lithographically designed electrodes, making it amenable for use as a high throughput assay.

In vitro testing of floating light activated micro-electrical stimulators.

Chronic tissue response to microelectrode implants stands in the way as a major challenge to development of many neural prosthetic applications. The long term tissue response is mostly due to the movement of interconnects and the resulting mechanical stress between the electrode and the surrounding neural tissue. Remotely activated floating micro-stimulators are one possible method of eliminating the interconnects. As a method of energy transfer to the micro-stimulator, we proposed to use a laser beam at near infrared (NIR) wavelengths. FLAMES of various sizes were fabricated with integrated silicon PIN photodiodes. Sizes varied from 120 (Width) x 300 (Length) x 100 (Height) microm to 200 x 500 x 100microm. Devices were bench tested using 850nm excitation from a Ti:Sapphire laser. To test this method, the voltage field of the FLAMES was experimentally tested in saline solution pulsed with a NIR laser beam. The voltage generated is around 196mV in peak at the cathodic contact as a response to a single pulse. When a train of laser pulses was applied at 100Hz, the peak voltage at the cathodic contact remained around 141mV suggesting the feasibility of this approach for applications with pulse frequencies up to 100Hz.