Sensitive Readout for Microfluidic High-Throughput Applications using Scanning SQUID Microscopy.

Microfluidic chips provide a powerful platform for high-throughput screening of diverse biophysical systems. The most prevalent detection methods are fluorescence based. Developing new readout techniques for microfluidics focusing on quantitative information in the low signal regime is desirable. In this work, we combine the well-established immunoassay approach, with magnetic nanoparticles, with a highly sensitive magnetic imaging technique. We offer to integrate a microfluidic array into a scanning superconducting quantum interference device (SQUID) microscope, to image nanoparticles that were moved through the microfluidic device. We demonstrate the technique on protein-protein interactions (PPI). We compare sensitivity to that of a conventional readout, quantify the amount of interactions, and demonstrate 0.1 atto-mole sensitivity. Our work serves as a proof of concept that will promote the development of a new set of eyes, a stable usable microfluidic-scanning SQUID microscopy.

An Integrated Microfluidics Approach for Personalized Cancer Drug Sensitivity and Resistance Assay.

Cancer is the second leading cause of death globally. Matching proper treatment and dosage is crucial for a positive outcome. Any given drug may affect patients with similar tumors differently. Personalized medicine aims to address this issue. Unfortunately, most cancer samples cannot be expanded in culture, limiting conventional cell-based testing. Herein, presented is a microfluidic device that combines a drug microarray with cell microscopy. The device can perform 512 experiments to test chemosensitivity and resistance to a drug array. MCF7 and 293T cells are cultured inside the device and their chemosensitivity and resistance to docetaxel, applied at various concentrations, are determined. Cell mortality is determined as a function of drug concentration and exposure time. It is found that both cell types form cluster morphology within the device, not evident in conventional tissue culture under similar conditions. Cells inside the clusters are less sensitive to drugs than dispersed cells. These findings support a heterogenous response of cancer cells to drugs. Then demonstrated is the principle of drug microarrays by testing cell response to four different drugs at four different concentrations. This approach may enable the personalization of treatment to the particular tumor and patient and may eventually improve final patient outcome.

Characterization of peptides self-assembly by low frequency Raman spectroscopy.

Low Frequency Vibrational (LFV) modes of peptides and proteins are attributed to the lattice vibrations and are dependent on their structural organization and self-assembly. Studies taken in order to assign specific absorption bands in the low frequency range to self-assembly behavior of peptides and proteins have been challenging. Here we used a single stage Low Frequency Raman (LF-Raman) spectrometer to study a series of diastereomeric analogue peptides to investigate the effect of peptides self-assembly on the LF-Raman modes. The structural variation of the diastereomeric analogues resulted in distinct self-assembly groups, as confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) data. Using LF-Raman spectroscopy, we consistently observed discrete peaks for each of the self-assembly groups. The correlation between the spectral features and structural morphologies was further supported by principal component analysis (PCA). The LFV modes provide further information on the degrees of freedom of the entire peptide within the higher order organization, reflecting the different arrangement of its hydrogen bonding and hydrophobic interactions. Thus, our approach provides a simple and robust complementary method to structural characterization of peptides assemblies.

New Method to Study the Vibrational Modes of Biomolecules in the Terahertz Range Based on a Single-Stage Raman Spectrometer.

The low-frequency vibrational (LFV) modes of biomolecules reflect specific intramolecular and intermolecular thermally induced fluctuations that are driven by external perturbations, such as ligand binding, protein interaction, electron transfer, and enzymatic activity. Large efforts have been invested over the years to develop methods to access the LFV modes due to their importance in the studies of the mechanisms and biological functions of biomolecules. Here, we present a method to measure the LFV modes of biomolecules based on Raman spectroscopy that combines volume holographic filters with a single-stage spectrometer, to obtain high signal-to-noise-ratio spectra in short acquisition times. We show that this method enables LFV mode characterization of biomolecules even in a hydrated environment. The measured spectra exhibit distinct features originating from intra- and/or intermolecular collective motion and lattice modes. The observed modes are highly sensitive to the overall structure, size, long-range order, and configuration of the molecules, as well as to their environment. Thus, the LFV Raman spectrum acts as a fingerprint of the molecular structure and conformational state of a biomolecule. The comprehensive method we present here is widely applicable, thus enabling high-throughput study of LFV modes of biomolecules.

An off-the-shelf integrated microfluidic device comprising self-assembled monolayers for protein array experiments.

Microfluidic-based protein arrays are promising tools for life sciences, with increased sensitivity and specificity. One of the drawbacks of this technology is the need to create fresh surface chemistry for protein immobilization at the beginning of each experiment. In this work, we attempted to include the process of surface functionalization as part of the fabrication of the device, which would substitute the time consuming step of surface functionalization at the beginning of each protein array experiment. To this end, we employed a novel surface modification using self-assembled monolayers (SAMs) to immobilize biomolecules within the channels of a polydimethylsiloxane (PDMS) integrated microfluidic device. As a model, we present a general method for depositing siloxane-anchored SAMs, with 1-undecyl-thioacetate-trichlorosilane (C11TA) on the silica surfaces. The process involved developing PDMS-compatible conditions for both SAM deposition and functional group activation. We successfully demonstrated the ability to produce, within an integrated microfluidic channel, a C11TA monolayer with a covalently conjugated antibody. The antibody could then bind its antigen with a high signal to background ratio. We further demonstrated that the antibody was still active after storage of the device for a week. Integration of the surface chemistry into the device as part of its fabrication process has potential to significantly simplify and shorten many experimental procedures involving microfluidic-based protein arrays. In turn, this will allow for broader dissemination of this important technology.

A sensitive microfluidic platform for a high throughput DNA methylation assay.

DNA methylation is an epigenetic modification essential for normal development and maintenance of somatic biological functions. DNA methylation provides heritable, long-term chromatin regulation and the aberrant methylation pattern is associated with complex diseases including cancer. Discovering novel therapeutic targets demands development of high-throughput, sensitive and inexpensive screening platforms for libraries of chemical or biological matter involved in DNA methylation establishment and maintenance. Here, we present a universal, high-throughput, microfluidic-based fluorometric assay for studying DNA methylation in vitro. The enzymatic activity of bacterial HPAII DNA methyltransferase and its kinetic properties are measured using the assay (K(m)(DNA) = 5.8 nM, K(m)(SAM) = 9.8 nM and Kcat = 0.04 s(-1)). Using the same platform, we then demonstrate a two-step approach for high-throughput in vitro identification and characterization of small molecule inhibitors of methylation. The approach is examined using known non-nucleoside inhibitors, SGI-1027 and RG108, for which we measured IC50 of 4.5 µM and 87.5 nM, respectively. The dual role of the microfluidic-based methylation assay both for the quantitative characterization of enzymatic activity and high-throughput screening of non-nucleoside inhibitors coupled with quantitative characterization of the inhibition potential highlights the advantages of our system for epigenetic studies.

Controlled formation of thiol and disulfide interfaces.

The work reported herein describes the controlled creation of uniform thiol-functionalized siloxane-anchored self-assembled monolayers (SAMs) and their selective transformation into intramonolayer (bridging) disulfides. These disulfides provide for the efficient immobilization of (bio)molecules bearing pendant thiols or disulfides, with no need for added oxidant. The unambiguous development of this surface chemistry required analytical methods that distinguish thiol and disulfide moieties on a surface. Physical properties such as wetting and monolayer thickness do not suffice nor do routine spectroscopic techniques (e.g., XPS, IR). Therefore, a method for distinguishing and quantifying thiol and disulfide surface functionality on a monolayer array based on the reaction with 2,4-dinitrofluorobenzene (DNFB, Sanger's reagent) is reported. DNFB readily reacts with thiol-SAMs (but not with disulfides) to form stable derivatives with distinctive IR, UV, and XPS signatures. Finally, the thiol-disulfide chemistry is applied to thiol-functionalized hybrid silica nanoparticles. These high-surface-area nanoparticles provide solid supports heavily loaded with thiol groups whose chemistry is also reported herein.