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.

Rapid, multiparameter profiling of cellular secretion using silicon photonic microring resonator arrays.

We have developed a silicon photonic biosensing chip capable of multiplexed protein measurements in a biomolecularly complex cell culture matrix. Using this multiplexed platform combined with fast one-step sandwich immunoassays, we perform a variety of T cell cytokine secretion studies with excellent time-to-result. Using 32-element arrays of silicon photonic microring resonators, the cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), and tumor necrosis factor alpha (TNFα) were simultaneously quantified with high accuracy in serum-containing cell media. Utilizing this cytokine panel, secretion profiles were obtained for primary human Th0, Th1, and Th2 subsets differentiated from naïve CD4+ T cells, and we show the ability to discriminate between lineage commitments at early stages of culture differentiation. We also utilize this approach to probe the temporal secretion patterns of each T cell type using real-time binding analyses for direct cytokine quantitation down to ∼100 pM with just a 5 min-analysis.

Crystallization and preliminary X-ray crystallographic analysis of ligand-free and arginine-bound forms of Thermotoga maritima arginine-binding protein.

The arginine-binding protein from Thermotoga maritima (TmArgBP) is an arginine-binding component of the ATP-binding cassette (ABC) transport system in this hyperthermophilic bacterium. This protein is endowed with an extraordinary stability towards thermal and chemical denaturation. Its structural characterization may provide useful insights for the clarification of structure-stability relationships and for the design of new biosensors. Crystallization trials were set up for both arginine-bound and ligand-free forms of TmArgBP and crystals suitable for crystallographic investigations were obtained for both forms. Ordered crystals of the arginine adduct of TmArgBP could only be obtained by using the detergent LDAO as an additive to the crystallization medium. These crystals were hexagonal, with unit-cell parameters a = 78.2, c = 434.7 Å, and diffracted to 2.7 Å resolution. The crystals of the ligand-free form were orthorhombic, with unit-cell parameters a = 51.8, b = 91.9, c = 117.9 Å, and diffracted to 2.25 Å resolution.

Sensitive on-chip detection of a protein biomarker in human serum and plasma over an extended dynamic range using silicon photonic microring resonators and sub-micron beads.

We demonstrate a three-step assay on a silicon photonic microring resonator-based detection platform that enables the quantitation of the cardiac biomarker C-reactive protein (CRP) over a dynamic range spanning six orders of magnitude. Using antibody-modified microrings, we sequentially monitor primary CRP binding, secondary recognition of bound CRP by a biotinylated antibody, and tertiary signal amplification using streptavidin-functionalized beads. This detection methodology is applied to CRP quantitation in human serum and plasma samples.

Characterization of the evanescent field profile and bound mass sensitivity of a label-free silicon photonic microring resonator biosensing platform.

Silicon photonic microring resonators have emerged as a sensitive and highly multiplexed platform for real-time biomolecule detection. Herein, we profile the evanescent decay of device sensitivity towards molecular binding as a function of distance from the microring surface. By growing multilayers of electrostatically bound polymers extending from the sensor surface, we are able to empirically determine that the evanescent field intensity is characterized by a 1/e response decay distance of 63 nm. We then applied this knowledge to study the growth of biomolecular assemblies consisting of alternating layers of biotinylated antibody and streptavidin, which follow a more complex growth pattern. Additionally, by monitoring the shift in microring resonance wavelength upon the deposition of a radioactively labeled protein, the mass sensitivity of the ring resonator platform was determined to be 14.7±6.7 [pg/mm(2)]/Δpm. By extrapolating to the instrument noise baseline, the mass/area limit of detection is found to be 1.5±0.7 pg/mm(2). Taking the small surface area of the microring sensor into consideration, this value corresponds to an absolute mass detection limit of 125 ag (i.e. 0.8 zmol of IgG), demonstrating the remarkable sensitivity of this promising label-free biomolecular sensing platform.

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima. 2. Molecular organization and structural stability.

ABC transport systems provide selective passage of metabolites across cell membranes and typically require the presence of a soluble binding protein with high specificity to a specific ligand. In addition to their primary role in nutrient gathering, the binding proteins associated with bacterial transport systems have been studied for their potential to serve as design scaffolds for the development of fluorescent protein biosensors. In this work, we used Fourier transform infrared spectroscopy and molecular dynamics simulations to investigate the physicochemical properties of a hyperthermophilic binding protein from Thermotoga maritima. We demonstrated preferential binding for the polar amino acid arginine and experimentally monitored the significant stabilization achieved upon binding of ligand to protein. The effect of temperature, pH, and detergent was also studied to provide a more complete picture of the protein dynamics. A protein structure model was obtained and molecular dynamic experiments were performed to investigate and couple the spectroscopic observations with specific secondary structural elements. The data determined the presence of a buried beta-sheet providing significant stability to the protein under all conditions investigated. The specific amino acid residues responsible for arginine binding were also identified. Our data on dynamics and stability will contribute to our understanding of bacterial binding protein family members and their potential biotechnological applications.

Silicon photonic microring resonators for quantitative cytokine detection and T-cell secretion analysis.

The ability to perform multiple simultaneous protein biomarker measurements in complex media with picomolar sensitivity presents a large challenge to disease diagnostics and fundamental biological studies. Silicon photonic microring resonators represent a promising platform for real-time detection of biomolecules on account of their spectral sensitivity toward surface binding events between a target and antibody-modified microrings. For all refractive index-based sensing schemes, the mass of bound analytes, in combination with other factors such as antibody affinity and surface density, contributes to the observed signal and measurement sensitivity. Therefore, proteins that are simultaneously low in abundance and have a lower molecular weight are often challenging to detect. By employing a more massive secondary antibody to amplify the signal arising from the initial binding event, it is possible to improve both the sensitivity and the specificity of protein assays, allowing for quantitative sensing in complex sample matrices. Herein, a sandwich assay is used to detect the 15.5 kDa human cytokine interleukin-2 (IL-2) at concentrations down to 100 pg/mL (6.5 pM) and to quantitate unknown solution concentrations over a dynamic range spanning 2.5 orders of magnitude. This same sandwich assay is then used to monitor the temporal secretion profile of IL-2 from Jurkat T lymphocytes in serum-containing cell culture media in the presence of the entire Jurkat secretome. The same temporal secretion analysis is performed in parallel using a commercial ELISA, revealing similar IL-2 concentration profiles but superior precision for the microring resonator sensing platform. Furthermore, we demonstrate the generality of the sandwich assay methodology on the microring resonator platform for the analysis of any biomolecular target for which two high-affinity antibodies exist by detecting the approximately 8 kDa cytokine interleukin-8 (IL-8) with a limit of detection and dynamic range similar to that of IL-2. This work demonstrates the first application of silicon photonic microring resonators for detecting cellular secretion of cytokines and represents an important advance for the detection of protein biomarkers on an emerging analytical platform.

Quantitative, label-free detection of five protein biomarkers using multiplexed arrays of silicon photonic microring resonators.

Because of the inherent complexity of biochemical pathways commonly altered in disease states, it has become accepted that multiplexed analyses can provide a more informative biomolecular understanding of disease onset and progression. Importantly, compared to conventional single-parameter assays, the detailed biomolecular insight gleaned from multiparameter measurements has the potential to greatly improve disease diagnostics, prognostics, and theragnostics. We have previously reported the utility of silicon photonic microring resonators for the sensitive quantitation of a single disease biomarker and herein demonstrate the first example of optical microcavity resonator arrays performing quantitative, label-free, multiplexed analyses of clinically relevant protein biomarkers. In this report, the concentrations of prostate specific antigen (PSA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), tumor necrosis factor-alpha (TNF-alpha), and interleukin-8 (IL-8) are simultaneously determined in three unknown protein cocktail solutions. This letter demonstrates that multiple immunoassays can be performed concurrently on a microresonator platform without any accompanying loss of sensitivity or measurement precision, and therefore, this report lays the groundwork for future applications involving multiplexed analysis of clinically relevant samples.

Amino acid transport in thermophiles: characterization of an arginine-binding protein in Thermotoga maritima.

Members of the periplasmic binding protein superfamily are involved in the selective passage of ligands through bacterial cell membranes. The hyperthermophilic eubacterium Thermotoga maritima was found to encode a highly stable and specific periplasmic arginine-binding protein (TM0593). Following signal sequence removal and overexpression in Escherichia coli, TM0593 was purified by thermoprecipitation and affinity chromatography. The ultra-stable protein with a monomeric molecular weight of 27.7 kDa was found to exist as both a homodimer and homotrimer at appreciable concentrations even under strongly denaturing conditions, with an estimated transition temperature of 116 degrees C. Its multimeric structure may provide further evidence of the importance of quaternary structure in the movement of nutrients across bacterial membranes. Purified and refolded TM0593 was further characterized by fluorescence spectroscopy, mass spectrometry, and circular dichroism to demonstrate the specificity of the protein for arginine and to elucidate structural changes associated with arginine binding. The protein binds arginine with a dissociation constant of 20 muM as determined by surface plasmon resonance measurements. Due to its high thermodynamic stability, TM0593 may serve as a scaffold for the creation of a robust fluorescent biosensor.