Nanopores are promising structures for small-volume separations, but they often require complex top-down fabrication and are not easily (re)configurable to allow for dynamically tuned separations. Herein we report the first use of nanoporous gold (NPG) membranes as tunable, charge-based separation elements. NPG is fabricated into a robust network of interconnecting pores via simple solution de-alloying. We demonstrate that control over the selective permeation of small-molecule analytes can be controlled via three different methods: (1) anion adsorption at the Au surface; (2) pH-tuning of self-assembled monolayer (SAM) charge; and (3) electrochemical oxidation of a redox-active SAM. This simple and versatile membrane system is promising as a dynamically tunable element for small-volume separations.

Magnetic actuation has been introduced to an optical immunosensor technology resulting in improvements in both rapidity and limit of detection for an assay quantitating low concentrations of a representative protein biomarker. For purposes of demonstration, an assay was designed for monocyte chemotactic protein 1 (MCP-1), a small cytokine which regulates migration and infiltration of monocytes and macrophages, and is an emerging biomarker for several diseases. The immunosensor is based on arrays of highly multiplexed silicon photonic microring resonators. A one-step sandwich immunoassay was performed and the signal was further enhanced through a tertiary recognition event between biotinylated tracer antibodies and streptavidin-coated magnetic beads. By integrating a magnet under the sensor chip, magnetic beads were rapidly directed towards the sensor surface resulting in improved assay performance metrics. Notably, the time required in the bead binding step was reduced by a factor of 11 (4 vs. 45 min), leading to an overall decrease in assay time from 73 min to 32 min. The magnetically-actuated assay also lowered the limit of detection (LOD) for MCP-1 from 124 pg mL−1 down to 57 pg mL−1. In sum, the addition of magnetic actuation into bead-enhanced sandwich assays on a silicon photonic biosensor platform might facilitate improved detection of biomarkers in point-of-care diagnostics settings.

Extracellular signaling is commonly mediated through post-translational protein modifications that propagate messages from membrane-bound receptors to ultimately regulate gene expression. Signaling cascades are ubiquitously intertwined, and a full understanding of function can only be gleaned by observing dynamics across multiple key signaling nodes. Importantly, targets within signaling cascades often represent opportunities for therapeutic development or can serve as diagnostic biomarkers. Protein phosphorylation is a particularly important post-translational modification that controls many essential cellular signaling pathways. Not surprisingly, aberrant phosphorylation is found in many human diseases, including cancer, and phosphoprotein-based biomarker signatures hold unrealized promise for disease monitoring. Moreover, phosphoprotein analysis has wide-ranging applications across fundamental chemical biology, as many drug discovery efforts seek to target nodes within kinase signaling pathways. For both fundamental and translational applications, the analysis of phosphoprotein biomarker targets is limited by a reliance on labor-intensive and/or technically challenging methods, particularly when considering the simultaneous monitoring of multiplexed panels of phosphoprotein biomarkers. We have developed a technology based upon arrays of silicon photonic microring resonator sensors that fills this void, facilitating the rapid and automated analysis of multiple phosphoprotein levels from both cell lines and primary human tumor samples requiring only minimal sample preparation.

The incorporation of the photoreactive molecule benzophenone into polyacrylamide hydrogels, allowing for orthogonal control over spatial incorporation of biomolecules and selective modulation of matrix stiffness, is described. Adipose-derived mesenchymal stem cells were cultured on matrices whose elastic moduli are tuned to ∼5, ∼14, and ∼37 kPa combined with an immobilized growth factor, bone morphogenic protein 2 (BMP-2), and their lineage differentiation was determined. BMP-2 was observed to have the most pronounced effect at the intermediate stiffness, while the lowest and highest stiffness hydrogels are directed by elasticity alone. Together, this approach describes a facile platform for fundamental studies of cell fate decisions in the context of both mechanical and biochemical cues and may lead to improved insight and effectiveness of stem cell therapies.Keywords: biomaterials; hydrogels; matrix elasticity; mesenchymal stem cells; osteogenesis

Refractive index-based sensors offer attractive characteristics as nondestructive and universal detectors for liquid chromatographic separations, but a small dynamic range and sensitivity to minor thermal perturbations limit the utility of commercial RI detectors for many potential applications, especially those requiring the use of gradient elutions. As such, RI detectors find use almost exclusively in sample abundant, isocratic separations when interfaced with high-performance liquid chromatography. Silicon photonic microring resonators are refractive index-sensitive optical devices that feature good sensitivity and tremendous dynamic range. The large dynamic range of microring resonators allows the sensors to function across a wide spectrum of refractive indices, such as that encountered when moving from an aqueous to organic mobile phase during a gradient elution, a key analytical advantage not supported in commercial RI detectors. Microrings are easily configured into sensor arrays, and chip-integrated control microrings enable real-time corrections of thermal drift. Thermal controls allow for analyses at any temperature and, in the absence of rigorous temperature control, obviates extended detector equilibration wait times. Herein, proof of concept isocratic and gradient elution separations were performed using well-characterized model analytes (e.g., caffeine, ibuprofen) in both neat buffer and more complex sample matrices. These experiments demonstrate the ability of microring arrays to perform isocratic and gradient elutions under ambient conditions, avoiding two major limitations of commercial RI-based detectors and maintaining comparable bulk RI sensitivity. Further benefit may be realized in the future through selective surface functionalization to impart degrees of postcolumn (bio)molecular specificity at the detection phase of a separation. The chip-based and microscale nature of microring resonators also make it an attractive potential detection technology that could be integrated within lab-on-a-chip and microfluidic separation devices.

Ricin is a lethal protein toxin derived from the castor bean plant. Given its notorious history as a biowarfare agent and homicidal weapon, ricin has been classified as a category B bioterrorism agent. Current ricin detection methods based on immunoassays lack the required sensitivity and specificity for many homeland security surveillance applications. Importantly, many conventional antibody-based methodologies are unable to distinguish ricin from RCA 120, a nontoxic protein also found in the castor bean plant. Single domain antibodies (sdAbs), which are recombinantly derived from immunized llamas, are known to have high affinities for ricin A or B chains and low cross-reactivity with RCA 120. Herein, we demonstrate the use of silicon photonic microring resonators for antibody affinity profiling and one-step ricin detection at concentrations down to 300 pM using a 15 min, label-free assay format. These sdAbs were also simultaneously compared with a commercial anti-RCA IgG antibody in a multicapture agent, single target immunoassay using arrays of microrings, which allowed direct comparison of sensitivity and specificity. A selected sdAb was also found to exhibit outstanding specificity against another biotoxin, saporin, which has mechanism of action similar to ricin. Given the rapidity, scalability, and multiplexing capability of this silicon-based technology, this work represents a step toward using microring resonator arrays for the sensitive and specific detection of biowarfare agents.

Soluble proteins are key mediators of many biochemical signaling pathways via direct interaction with the lipid bilayer and via membrane-bound receptors. Components of the cell membrane are involved in many important biological processes, including viral infection, blood clotting, and signal transduction, and as such, they are common targets of therapeutic agents. Therefore, the development of analytical approaches to study interactions at the cell membrane is of critical importance. Herein, we integrate two key technologies, silicon photonic microring resonator arrays and phospholipid bilayer nanodiscs, which together allow multiplexed screening of soluble protein interactions with lipid and membrane-embedded targets. Microring resonator arrays are an intrinsically multiplexable, label-free analysis platform that has previously been applied to studying protein–protein, protein–nucleic acid, and nucleic acid–nucleic acid interactions. Nanodiscs are protein-stabilized lipid assemblies that represent a convenient construct to mimic the native phospholipid bilayer, investigate the effects of membrane composition, and solubilize membrane-embedded targets. Exploiting the natural affinity of nanodisc-supported lipid bilayers for oxide-passivated silicon, we assembled single and multiplex sensor arrays via direct physisorption, characterizing electrostatic effects on the nanodisc attachment. Using model systems, we demonstrate the applicability of this platform for the parallel screening of protein interactions with nanodisc-embedded lipids, glycolipids, and membrane proteins.

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.

Herein, we describe the utility of chaperone probes and a bead-based signal enhancement strategy for the analysis of full length mRNA transcripts using arrays of silicon photonic microring resonators. Changes in the local refractive index near microring sensors associated with biomolecular binding events are transduced as a shift in the resonant wavelength supported by the cavity, enabling the sensitive analysis of numerous analytes of interest. We employ the sensing platform for both the direct and bead-enhanced detection of three different mRNA transcripts, achieving a dynamic range spanning over 4 orders of magnitude and demonstrating expression profiling capabilities in total RNA extracts from the HL-60 cell line. Small, dual-use DNA chaperone molecules were developed and found to both enhance the binding kinetics of mRNA transcripts by disrupting complex secondary structure and serve as sequence-specific linkers for subsequent bead amplification. Importantly, this approach does not require amplification of the mRNA transcript, thereby allowing for simplified analyses that do not require expensive enzymatic reagents or temperature ramping capabilities associated with RT-PCR-based methods.

Viruses represent a continual threat to humans through a number of mechanisms, which include disease, bioterrorism, and destruction of both plant and animal food resources. Many contemporary techniques used for the detection of viruses and viral infections suffer from limitations such as the need for extensive sample preparation or the lengthy window between infection and measurable immune response, for serological methods. In order to develop a method that is fast, cost-effective, and features reduced sample preparation compared to many other virus detection methods, we report the application of silicon photonic microring resonators for the direct, label-free detection of intact viruses in both purified samples as well as in a complex, real-world analytical matrix. As a model system, we demonstrate the quantitative detection of Bean pod mottle virus, a pathogen of great agricultural importance, with a limit of detection of 10 ng/mL. By simply grinding a small amount of leaf sample in buffer with a mortar and pestle, infected leaves can be identified over a healthy control with a total analysis time of less than 45 min. Given the inherent scalability and multiplexing capability of the semiconductor-based technology, we feel that silicon photonic microring resonators are well-positioned as a promising analytical tool for a number of viral detection applications.

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 3.16×107 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.Highlights► tmRNAs are promising markers for distinguishing between pathogenic bacterial species. ► Quantitation of multiple tmRNAs is demonstrated using a silicon photonic detection platform. ► Scalable tmRNA detection is needed for biodefense, food monitoring, and health care, among others.

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.

We directly monitor in parallel and in real time the temporal profiles of polymer brushes simultaneously grown via multiple ATRP reaction conditions on a single substrate using arrays of silicon photonic microring resonators. In addition to probing relative polymerization rates, we show the ability to evaluate the dynamic properties of the in situ grown polymers. This presents a powerful new platform for studying modified interfaces that may allow for the combinatorial optimization of surface-initiated polymerization conditions.

Methods for the generation of substratespresenting biomolecules in a spatially controlled manner are enabling tools for applications in biosensor systems, microarray technologies, fundamental biological studies and biointerface science. We have implemented a method to create biomolecular patterns by using light to control the direct covalent immobilization of biomolecules onto benzophenone-modified glass substrates. We have generated substrates presenting up to three different biomolecules patterned in sequence, and demonstrate biomolecular photopatterning on corrugated substrates. The chemistry of the underlying monolayer was optimized to incorporate poly(ethylene glycol) to enable adhesive cell adhesion onto patterned extracellular matrix proteins. Substrates were characterized with contact angle goniometry, AFM, and immunofluorescence microscopy. Importantly, radioimmunoassays were performed to quantify the site density of immobilized biomolecules on photopatterned substrates. Retained function of photopatterned proteins was demonstrated both by native ligand recognition and cell adhesion to photopatterned substrates, revealing that substrates generated with this method are suitable for probing specific cell receptor–ligand interactions. This molecularly general photochemical patterning method is an enabling tool for the creation of substrates presenting both biochemical and topographical variation, which is an important feature of many native biointerfaces.Keywords: biointerfaces; cell patterning; model substrates; multicomponent; photopatterning;

High affinity capture agents against protein targets are essential components for immunoassays, regardless of specific analysis format. Here, we describe the use of DNA-encoded antibodies for rapidly screening the kinetic and equilibrium binding properties of twelve commercial antibodies in a parallel analysis format using a multiplexed array of microring optical resonators. We show that DNA-encoding offers advantages in terms of antigen binding capacity, compared to covalently tethered antibodies; we also demonstrate that this linkage modality facilitates the rapid self-assembly of multiplexed arrays on account of complementarity between the DNA sequences on the antibodies and sensor array, respectively. Furthermore, DNA-encoded antibodies also allow for sensor array regeneration and reprogramming, as chaotropic agents can be used to disrupt the DNA−DNA duplexes that link the capture agents to the sensor without harming the underlying DNA on the surface, which can subsequently be reloaded with antibodies either targeting the same or different antigens.

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.

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.

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.

By leveraging advances in semiconductor microfabrication technologies, chip-integrated optical biosensors are poised to make an impact as scalable and multiplexable bioanalytical measurement tools for lab-on-a-chip applications. In particular, waveguide-based optical sensing technology appears to be exceptionally amenable to chip integration and miniaturization, and, as a result, the recent literature is replete with examples of chip-integrated waveguide sensing platforms developed to address a wide range of contemporary analytical challenges. As an overview of the most recent advances within this dynamic field, this review highlights work from the last 2–3 years in the areas of grating-coupled, interferometric, photonic crystal, and microresonator waveguide sensors. With a focus towards device integration, particular emphasis is placed on demonstrations of biosensing using these technologies within microfluidically controlled environments. In addition, examples of multiplexed detection and sensing within complex matrices—important features for real-world applicability—are given special attention.

High affinity capture agents recognizing biomolecular targets are essential in the performance of many proteomic detection methods. Herein, we report the application of a label-free silicon photonic biomolecular analysis platform for simultaneously determining kinetic association and dissociation constants for two representative protein capture agents: a thrombin-binding DNA aptamer and an anti-thrombin monoclonal antibody. The scalability and inherent multiplexing capability of the technology make it an attractive platform for simultaneously evaluating the binding characteristics of multiple capture agents recognizing the same target antigen, and thus a tool complementary to emerging high-throughput capture agent generation strategies.

Model substrates presenting biochemical cues immobilized in a controlled and well-defined manner are of great interest for their applications in biointerface studies that elucidate the molecular basis of cell receptor–ligand interactions. Herein, we describe a direct, photochemical method to generate surface-immobilized biomolecular gradients that are applied to the study of selectin-mediated leukocyte rolling. The technique employs benzophenone-modified glass substrates, which upon controlled exposure to UV light (350–365 nm) in the presence of protein-containing solutions facilitate the generation of covalently immobilized protein gradients. Conditions were optimized to generate gradient substrates presenting P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1) immobilized at site densities over a 5- to 10-fold range (from as low as ∼200 molecules μm−2 to as high as 6000 molecules μm−2). The resulting substrates were quantitatively characterized via fluorescence analysis and radioimmunoassays before their use in the leukocyte rolling assays. HL-60 promyelocytes and Jurkat T lymphocytes were assessed for their ability to tether to and roll on substrates presenting immobilized P-selectin and PSGL-1 under conditions of physiologically relevant shear stress. The results of these flow assays reveal the combined effect of immobilized protein site density and applied wall shear stress on cell rolling behavior. Two-component substrates presenting P-selectin and ICAM-1 (intercellular adhesion molecule-1) were also generated to assess the interplay between these two proteins and their effect on cell rolling and adhesion. These proof-of-principle studies verify that the described gradient generation approach yields well-defined gradient substrates that present immobilized proteins over a large range of site densities that are applicable for investigation of cell-materials interactions, including multi-parameter leukocyte flow studies. Future applications of this enabling methodology may lead to new insights into the biophysical phenomena and molecular mechanism underlying complex biological processes such as leukocyte recruitment and the inflammatory response.

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 ∼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.

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), α-fetoprotein (AFP), carcinoembryonic antigen (CEA), tumor necrosis factor-α (TNF-α), 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.

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/mm2]/Δpm. By extrapolating to the instrument noise baseline, the mass/area limit of detection is found to be 1.5 ± 0.7 pg/mm2. 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.

In 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.

Aniline-catalyzed hydrazone ligation between surface-immobilized hydrazines and aldehyde-modified antibodies is shown to be an efficient method for attaching protein capture agents to model oxide-coated biosensor substrates. Silicon photonic microring resonators are used to directly evaluate the efficiency of this surface bioconjugate reaction at various pHs and in the presence or absence of aniline as a nucleophilic catalyst. It is found that aniline significantly increases the net antibody loading for surfaces functionalized over a pH range from 4.5 to 7.4, allowing derivatization of substrates with reduced incubation time and sample consumption. This increase in antibody loading directly results in more sensitive antigen detection when functionalized microrings are employed in a label-free immunoassay. Furthermore, these experiments also reveal an interesting pH-dependent noncovalent binding trend that plays an important role in dictating the amount of antibody attached onto the substrate, highlighting the competing contributions of the bioconjugate reaction rate and the dynamic interactions that control opportunities for a solution-phase biomolecule to react with a substrate-bound reagent.

Recent advances in label-free biosensing techniques have shown the potential to simplify clinical analyses. With this motivation in mind, this paper demonstrates for the first time the use of silicon-on-insulator microring optical resonator arrays for the robust and label-free detection of a clinically important protein biomarker in undiluted serum, using carcinoembryonic antigen (CEA) as the test case. We utilize an initial-slope-based quantitation method to sensitively detect CEA at clinically relevant levels and to determine the CEA concentrations of unknown samples in both buffer and undiluted fetal bovine serum. Comparison with a commercial enzyme-linked immunosorbent assay (ELISA) kit reveals that the label-free microring sensor platform has a comparable limit of detection (2 ng/mL) and superior accuracy in the measurement of CEA concentration across a 3 order of magnitude dynamic range. Notably, we report the lowest limit of detection to date for a microring resonator sensor applied to a clinically relevant cancer biomarker. Although this report describes the robust biosensing capabilities of silicon photonic microring resonator arrays for a single parameter assay, future work will focus on utilizing the platform for highly multiplexed, label-free bioanalysis.

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.

We describe an approach to generate multicomponent surface-immobilized patterns and gradients on the basis of the photochemically controlled covalent coupling of solution-phase biomolecules to benzophenone-modified substrates. Gradients were simply achieved by continuously varying the exposure to nondamaging UV light across the surface with the gradient profile controlled by biomolecule concentration and the spatial and temporal illumination of the surface. Sequential exposure of the same surface in the presence of different biomolecules resulted in overlapping patterns and gradients of proteins and carbohydrates. Finally, we preliminarily demonstrate that the resulting surfaces are suitable for generating model substrates to probe cell−substrate interactions.

We have developed a broadly applicable biosensing platform based upon chip-integrated silicon photonic microcavities. Large arrays of microring resonators are robustly fabricated using standard semiconductor processing methods on silicon-on-insulator wafers. Microring resonators are label-free sensors that support spectrally well-defined optical modes that are extremely sensitive to surface binding-induced changes in the local refractive index. Each microring can be uniquely functionalized with a biomolecularly-specific capture agent (antibody, cDNA, aptamer, etc.) allowing sensitive and multiplexed detection of a range of protein and nucleic acid targets at sub-pM levels and from within complex matrices, such as human serum. This paper describes recent advances in sensor design, characterization, and detection of biomolecules for applications in bioterrorism surveillance and medical diagnostics, with an added emphasis on the incorporation of emerging capture agents that give improved sensor performance.

Magnetic actuation has been introduced to an optical immunosensor technology resulting in improvements in both rapidity and limit of detection for an assay quantitating low concentrations of a representative protein biomarker. For purposes of demonstration, an assay was designed for monocyte chemotactic protein 1 (MCP-1), a small cytokine which regulates migration and infiltration of monocytes and macrophages, and is an emerging biomarker for several diseases. The immunosensor is based on arrays of highly multiplexed silicon photonic microring resonators. A one-step sandwich immunoassay was performed and the signal was further enhanced through a tertiary recognition event between biotinylated tracer antibodies and streptavidin-coated magnetic beads. By integrating a magnet under the sensor chip, magnetic beads were rapidly directed towards the sensor surface resulting in improved assay performance metrics. Notably, the time required in the bead binding step was reduced by a factor of 11 (4 vs. 45 min), leading to an overall decrease in assay time from 73 min to 32 min. The magnetically-actuated assay also lowered the limit of detection (LOD) for MCP-1 from 124 pg mL−1 down to 57 pg mL−1. In sum, the addition of magnetic actuation into bead-enhanced sandwich assays on a silicon photonic biosensor platform might facilitate improved detection of biomarkers in point-of-care diagnostics settings.