Abraham J. Qavi

Multiplexed Detection and Label-Free Quantitation of microRNAs using Arrays of Silicon Photonic Microring Resonators

MicroRNAs (miRNAs) are short (19 to 24 nucleotides), single-stranded, non-protein-coding RNAs that are powerful transcriptional and post-transcriptional regulators of gene expression. Unlike small interfering RNAs (siRNAs), miRNAs are genomically encoded and play key roles in a range of normal cellular processes, including proliferation, apoptosis, and development.[1-4] Not surprisingly, miRNAs have also been implicated in a number of diseases, including cancer,[5-8] neurodegenerative disorders,[9-11] and diabetes,[12-14] and represent promising biomarker candidates for informative diagnostics. Despite their increasingly well-understood importance in gene regulation, the development of sensitive analytical techniques for the quantitation of multiple miRNAs has lagged behind. Furthermore, current methodologies for miRNA expression analysis are not applicable to a clinical setting where sample sizes are limited and assay cost and time-to-result is of tremendous importance.

Label-free technologies for quantitative multiparameter biological analysis

In the postgenomic era, information is king and information-rich technologies are critically important drivers in both fundamental biology and medicine. It is now known that single-parameter measurements provide only limited detail and that quantitation of multiple biomolecular signatures can more fully illuminate complex biological function. Label-free technologies have recently attracted significant interest for sensitive and quantitative multiparameter analysis of biological systems. There are several different classes of label-free sensors that are currently being developed both in academia and in industry. In this critical review, we highlight, compare, and contrast some of the more promising approaches. We describe the fundamental principles of these different methods and discuss advantages and disadvantages that might potentially help one in selecting the appropriate technology for a given bioanalytical application.

Anti-DNA:RNA Antibodies and Silicon Photonic Microring Resonators: Increased Sensitivity for Multiplexed microRNA Detection

In this paper, we present a method for the sensitive detection of microRNAs (miRNAs) utilizing an antibody that specifically recognizes DNA:RNA heteroduplexes and a silicon photonic microring resonator array transduction platform. Microring resonator arrays are covalently functionalized with DNA capture probes that are complementary to solution phase miRNA targets. Following hybridization on the sensor, the anti-DNA:RNA antibody is introduced and binds selectively to the heteroduplexes, giving a larger signal than the original miRNA hybridization due to the increased mass of the antibody, as compared to the 22-mer oligoribonucleotide. Furthermore, the secondary recognition step is performed in neat buffer solution and at relatively higher antibody concentrations, facilitating the detection of miRNAs of interest. The intrinsic sensitivity of the microring resonator platform coupled with the amplification provided by the anti-DNA:RNA antibodies allows for the detection of microRNAs at concentrations as low as 10 pM (350 amol). The simplicity and sequence generality of this amplification method position it as a promising tool for high-throughput, multiplexed miRNA analysis as well as a range of other RNA based detection applications.

Isothermal Discrimination of Single-Nucleotide Polymorphisms via Real-Time Kinetic Desorption and Label-Free Detection of DNA Using Silicon Photonic Microring Resonator Arrays

We report a sensitive, label-free method for detecting single-stranded DNA and discriminating between single nucleotide polymorphisms (SNPs) using arrays of silicon photonic microring resonators. In only a 10 min assay, DNA is detected at subpicomole levels with a dynamic range of 3 orders of magnitude. Following quantitation, sequence discrimination with single nucleotide resolution is achieved isothermally by monitoring the dissociation kinetics of the duplex in real-time using an array of SNP-specific capture probes. By leveraging the capabilities of the microring resonator platform, we successfully generate multiplexed arrays to quickly screen for the presence and identity of SNPs and show the robustness of this methodology by analyzing multiple target sequences of varying GC content. Furthermore, we show that this technique can be used to distinguish both homozygote and heterozygote alleles.

Sizing up the future of microRNA analysis

AbstractIn less than 20 years, our appreciation for micro-RNA molecules (miRNAs) has grown from an original, curious observation in worms to their current status as incredibly important global regulators of gene expression that play key roles in many transformative biological processes. As our understanding of these small, non-coding transcripts continues to evolve, new approaches for their analysis are emerging. In this critical review we describe recent improvements to classical methods of detection as well as innovative new technologies that are poised to help shape the future landscape of miRNA analysis. FigureDriven by the ever increasing appreciation of the critical biological roles played by microRNAs, new technologies are continually reshaping the landscape of microRNA analysis. This review highlights existing and emerging technologies for the detection of microRNAs

Label-free, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators

A label-free biosensing method for the sensitive detection and identification of bacterial transfer-messenger RNA (tmRNA) is presented employing arrays of silicon photonic microring resonators. Species specific tmRNA molecules are targeted by complementary DNA capture probes that are covalently attached to the sensor surface. Specific hybridization is monitored in near real-time by observing the resonance wavelength shift of each individual microring. The sensitivity of the biosensing platform allowed for detection down to 53 fmol of Streptococcus pneumoniae tmRNA, equivalent to approximately 3.16×10(7) CFU of bacteria. The simplicity and scalability of this biosensing approach makes it a promising tool for the rapid identification of different bacteria via tmRNA profiling.

Subpicogram per milliliter detection of interleukins using silicon photonic microring resonators and an enzymatic signal enhancement strategy

The detection of biomolecules at ultralow (low to subpicogram per milliliter) concentrations and within complex, clinically relevant matrices is a formidable challenge that is complicated by limitations imposed by the Langmuir binding isotherm and mass transport, for surface-based affinity biosensors. Here we report the integration of an enzymatic signal enhancement scheme onto a multiplexable silicon photonic microring resonator detection platform. To demonstrate the analytical value of this combination, we simultaneously quantitated levels of the interleukins IL-2, IL-6, and IL-8 in undiluted cerebrospinal fluid in an assay format that is multiplexable, relatively rapid (90 min), and features a 3 order of magnitude dynamic range and a limit of detection ≤1 pg/mL. The modular nature of this assay and technology should lend itself broadly amenable to different analyte classes, making it a versatile tool for biomarker analysis in clinically relevant settings.

A robust silicon photonic platform for multiparameter biological analysis

Silicon photonic technology has incredible potential to transform multiplexed bioanalysis on account of the scalability of device fabrication, which maps favorably to a myriad of medical diagnostic applications. The optical properties of CMOS-fabricated microring resonators are incredibly responsive to changes in the local dielectric environment accompanying a biological binding event near the ring surface. Arrays of high-Q microrings were designed to be individually addressable both in surface derivitization, using well-established microarraying technologies, and in optical evaluation. The optical response of each ring can be determined in near real time allowing multiple biomolecular interactions to be simultaneously monitored. We describe a stable and robust measurement platform that allows sensitive visualization of small molecule surface chemical derivitization as well as monitoring of biological interactions, including the detection of proteins and nucleic acids. We also present recent results demonstrating multiplexed measurement of cancer markers. These demonstrations establish a pathway to higher level multiparameter analysis from real-world patient samples; a development that will enable individualized disease diagnostics and personalized medicine.

Silicon photonic microring resonator arrays for scalable and multiplexable bioanalysis

Silicon photonic devices, such as SOI microring resonators, have optical properties that are incredibly responsive to changes in the local dielectric environment accompanying a biological binding event at or near the ring surface. We have developed a platform in which arrays of uniquely addressable microrings are functionalized with target-specific capture agents so as to create a powerful tool for multiplexed biomolecular detection. We will discuss applications of this technology to the multiplexed detection of DNA, RNA, and proteins in clinically-relevant matrices, and will also describe the rigorous empirical determination of key sensitivity metrics.

Applications of Silicon Photonic Technology for Clinical Diagnostics and Highly Multiplexed Biomolecular Detection

Silicon photonic structures are incredibly responsive to binding-induced changes in the refractive index environment surrounding the device. We are developing microring resonator arrays as a robust platform for sensitive, label-free, and highly multiplexed biomolecular detection.