Multiplexed microRNA expression profiling by combined asymmetric PCR and label-free detection using silicon photonic sensor arrays
Analysis methods based upon the quantitative, real-time polymerase chain reaction are extremely powerful; however, they face intrinsic limitations in terms of target multiplexing. In contrast, silicon photonic microring resonators represent a modularly multiplexable sensor array technology that is well-suited to the analysis of targeted biomarker panels. In this manuscript we employ an asymmetric polymerase chain reaction approach to selectively amplify copies of cDNAs generated from targeted miRNAs before multiplexed, label-free quantitation through hybridization to microring resonator arrays pre-functionalized with capture sequences. This method, which shows applicability to low input amounts and a large dynamic range, was demonstrated for the simultaneous detection of eight microRNA targets from twenty primary brain tumor samples with expression profiles in good agreement with literature precedent. Click here to read the manuscript.
The group just got back from a breather down in Florida. When not giving talks or presenting their posters, everyone soaked up the sun and spent a night out at Universal Studios.
Combining asymmetric PCR-based enzymatic amplification with silicon photonic microring resonators for the detection of lncRNAs from low input human RNA samples
A method for quantifying biologically relevant long-non-coding RNAs by combining nucleic acid amplification via asymmetric polymerase chain reaction (PCR) with label-free PCR product detection using silicon photonic microring resonator arrays is described. This approach eliminates the need for fluorophores, which presents a limit for spectral multiplexing in conventional qPCR methods, and rather offers potential for much higher levels of plexity by spatially arraying capture probes. Here, we demonstrate the potential of this technique to detect two differentially expressed lncRNA transcripts and an internal control mRNA transcript in different commercial human tissue specimens, as well as in a glioblastoma cell line using only nanogram input amounts of total RNA. The obtained results were validated using single-plex RT-qPCR and found to be in good agreement, demonstrating the potential of this technique for lncRNA quantification applications. Click here to read the manuscript.
Dr. Heather Robison recently successfully defended her thesis titled “Multiplexed Immunoassay Development for Precision Medicine Diagnostics and Protein Characterization using Silicon Photonic Microring Resonators".
A Guide to Quantitative Biomarker Assay
Development using Whispering Gallery
Whispering gallery mode (WGM) sensors are a class of powerful analytical techniques defined by the measurement of changes in the local refractive index at or near the sensor surface. When functionalized with target-specific capture agents, analyte binding can be measured with very low limits of detection. There are many geometric manifestations of WGM sensors, with chip-integrated silicon photonic devices first commercialized because of the robust, wafer-scale device fabrication, facile optical interrogation, and amenability to the creation of multiplexed sensor arrays. Using these arrays, a number of biomolecular targets have been detected in both label-free and label-enhanced assay formats. For example, sub-picomolar detection limits for multiple cytokines were achieved using an enzymatically enhanced sandwich immunoassay that showed high analyte specificity suitable for detection in complex, clinical matrices. This protocol describes a generalizable approach for the development of quantitative, multiplexed immunoassays using silicon photonic microrings as an example WGM platform.
Multiplexed silicon photonic sensor arrays enable facile characterization of coagulation protein binding to Nanodiscs with variable lipid content
Interactions of soluble proteins with the cell membrane are critical within the blood coagulation cascade. Of particular interest are the interactions of γ-carboxyglutamic acid-rich (GLA) domain-containing clotting proteins with lipids. Variability among conventional analytical methods presents challenges for comparing clotting protein-lipid interactions. Most previous studies have investigated only a single clotting protein and lipid composition and have yielded widely different binding constants. Herein, we demonstrate that a combination of lipid bilayer Nanodiscs and a multiplexed silicon photonic analysis technology enables high-throughput probing of many protein-lipid interactions among blood-clotting proteins. This approach allowed direct comparison of the binding constants of prothrombin, factor X, activated factor VII, and activated protein C to seven different binary lipid compositions. In a single experiment, the binding constants of one protein interacting with all lipid compositions were simultaneously determined. A simple surface regeneration then facilitated similar binding measurements for three other coagulation proteins. As expected, our results indicated that all proteins exhibit tighter binding (lower Kd) as the proportion of anionic lipid increases. Interestingly, at high proportions of phosphatidylserine, the Kd values of all four proteins began to converge. We also found that although koff values for all four proteins followed trends similar to those observed for the Kd values, the variation among the proteins was much lower, indicating that much of the variation came from the kinetic binding (kon) of the proteins. These findings indicate that the combination of silicon photonic microring resonator arrays and Nanodiscs enables rapid interrogation of biomolecular binding interactions at model cell membrane interfaces.
The Bailey Lab recently got out into nature on their first kayaking trip since moving to the Wolverine State. While some suffered flip-flop and sunglasses casualties to the Huron River, Ryan managed to stay afloat this time.
Microfluidic Platform for Efficient Nanodisc Assembly, Membrane Protein Incorporation, and Purification
The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.