StaphAIR: A Label-Free Antigen Microarray Approach to Detecting Anti-Staphylococcus aureus Antibody Responses in Orthopedic Infections.

Arrayed imaging reflectometry (AIR) is an optical biosensor platform for simple, multiplex measurement of antigen-specific antibody responses in patient blood samples. Here, we report the development of StaphAIR, an 8-plex Staphylococcus aureus antigen array on the AIR platform for profiling antigen-specific anti-S. aureus humoral immune responses. Initial validation experiments with mouse and humanized monoclonal antibodies against the S. aureus autolysin glucosaminidase (Gmd) domain, and subsequent testing with dilution series of pooled positive human serum confirmed analytically robust behavior of the array, with all antigens displaying Langmuir-type dose-response curves. Testing a cohort of 82 patients with S. aureus musculoskeletal infections (MSKI) and 30 healthy individuals enabled discrimination of individual patient responses to different S. aureus antigens, with statistical significance between osteomyelitis patients and controls obtained overall for four individual antigens (IsdA, IsdB, Gmd, and SCIN). Multivariate analyses of the antibody titers obtained from StaphAIR revealed its utility as a potential diagnostic tool for detecting S. aureus MSKI (area under the receiver operating characteristic curve (AUC) > 0.85). We conclude that StaphAIR has utility as a high-throughput immunoassay for studying and diagnosing osteomyelitis in patients.

High-performance, low-voltage electroosmotic pumps with molecularly thin silicon nanomembranes.

We have developed electroosmotic pumps (EOPs) fabricated from 15-nm-thick porous nanocrystalline silicon (pnc-Si) membranes. Ultrathin pnc-Si membranes enable high electroosmotic flow per unit voltage. We demonstrate that electroosmosis theory compares well with the observed pnc-Si flow rates. We attribute the high flow rates to high electrical fields present across the 15-nm span of the membrane. Surface modifications, such as plasma oxidation or silanization, can influence the electroosmotic flow rates through pnc-Si membranes by alteration of the zeta potential of the material. A prototype EOP that uses pnc-Si membranes and Ag/AgCl electrodes was shown to pump microliter per minute-range flow through a 0.5-mm-diameter capillary tubing with as low as 250 mV of applied voltage. This silicon-based platform enables straightforward integration of low-voltage, on-chip EOPs into portable microfluidic devices with low back pressures.

Influence of silicon dioxide capping layers on pore characteristics in nanocrystalline silicon membranes.

Porous nanocrystalline silicon (pnc-Si) membranes are a new class of membrane material with promising applications in biological separations. Pores are formed in a silicon film sandwiched between nm thick silicon dioxide layers during rapid thermal annealing. Controlling pore size is critical in the size-dependent separation applications. In this work, we systematically studied the influence of the silicon dioxide capping layers on pnc-Si membranes. Even a single nm thick top oxide layer is enough to switch from agglomeration to pore formation after annealing. Both the pore size and porosity increase with the thickness of the top oxide, but quickly reach a plateau after 10 nm of oxide. The bottom oxide layer acts as a barrier layer to prevent the a-Si film from undergoing homo-epitaxial growth during annealing. Both the pore size and porosity decrease as the thickness of the bottom oxide layer increases to 100 nm. The decrease of the pore size and porosity is correlated with the increased roughness of the bottom oxide layer, which hinders nanocrystal nucleation and nanopore formation.

Highly porous silicon membranes fabricated from silicon nitride/silicon stacks.

Nanopore formation in silicon films has previously been demonstrated using rapid thermal crystallization of ultrathin (15 nm) amorphous Si films sandwiched between nm-thick SiO2 layers. In this work, the silicon dioxide barrier layers are replaced with silicon nitride, resulting in nanoporous silicon films with unprecedented pore density and novel morphology. Four different thin film stack systems including silicon nitride/silicon/silicon nitride (NSN), silicon dioxide/silicon/silicon nitride (OSN), silicon nitride/silicon/silicon dioxide (NSO), and silicon dioxide/silicon/silicon dioxide (OSO) are tested under different annealing temperatures. Generally the pore size, pore density, and porosity positively correlate with the annealing temperature for all four systems. The NSN system yields substantially higher porosity and pore density than the OSO system, with the OSN and NSO stack characteristics fallings between these extremes. The higher porosity of the Si membrane in the NSN stack is primarily due to the pore formation enhancement in the Si film. It is hypothesized that this could result from the interfacial energy difference between the silicon/silicon nitride and silicon/silicon dioxide, which influences the Si crystallization process.

Charge- and size-based separation of macromolecules using ultrathin silicon membranes.

Commercial ultrafiltration and dialysis membranes have broad pore size distributions and are over 1,000 times thicker than the molecules they are designed to separate, leading to poor size cut-off properties, filtrate loss within the membranes, and low transport rates. Nanofabricated membranes have great potential in molecular separation applications by offering more precise structural control, yet transport is also limited by micrometre-scale thicknesses. This limitation can be addressed by a new class of ultrathin nanostructured membranes where the membrane is roughly as thick (approximately 10 nm) as the molecules being separated, but membrane fragility and complex fabrication have prevented the use of ultrathin membranes for molecular separations. Here we report the development of an ultrathin porous nanocrystalline silicon (pnc-Si) membrane using straightforward silicon fabrication techniques that provide control over average pore sizes from approximately 5 nm to 25 nm. Our pnc-Si membranes can retain proteins while permitting the transport of small molecules at rates an order of magnitude faster than existing materials, separate differently sized proteins under physiological conditions, and separate similarly sized molecules carrying different charges. Despite being only 15 nm thick, pnc-Si membranes that are free-standing over 40,000 microm2 can support a full atmosphere of differential pressure without plastic deformation or fracture. By providing efficient, low-loss macromolecule separations, pnc-Si membranes are expected to enable a variety of new devices, including membrane-based chromatography systems and both analytical and preparative microfluidic systems that require highly efficient separations.

Pore size control of ultrathin silicon membranes by rapid thermal carbonization.

Rapid thermal carbonization in a dilute acetylene (C(2)H(2)) atmosphere has been used to chemically modify and precisely tune the pore size of ultrathin porous nanocrystalline silicon (pnc-Si). The magnitude of size reduction was controlled by varying the process temperature and time. Under certain conditions, the carbon coating displayed atomic ordering indicative of graphene layer formation conformal to the pore walls. Initial experiments show that carbonized membranes follow theoretical predictions for hydraulic permeability and retain the precise separation capabilities of untreated membranes.

Ion-selective permeability of an ultrathin nanoporous silicon membrane as probed by scanning electrochemical microscopy using micropipet-supported ITIES tips.

We report on the application of scanning electrochemical microscopy (SECM) to the measurement of the ion-selective permeability of porous nanocrystalline silicon membrane as a new type of nanoporous material with potential applications in analytical, biomedical, and biotechnology device development. The reliable measurement of high permeability in the molecularly thin nanoporous membrane to various ions is important for greater understanding of its structure-permeability relationship and also for its successful applications. In this work, this challenging measurement is enabled by introducing two novel features into amperometric SECM tips based on the micropipet-supported interface between two immiscible electrolyte solutions (ITIES) to reveal the important ion-transport properties of the ultrathin nanopore membrane. The tip of a conventional heat-pulled micropipet is milled using the focused ion beam (FIB) technique to be smoother, better aligned, and subsequently, approach closer to the membrane surface, which allows for more precise and accurate permeability measurement. The high membrane permeability to small monovalent ions is determined using FIB-milled micropipet tips to establish a theoretical formula for the membrane permeability that is controlled by free ion diffusion across water-filled nanopores. Moreover, the ITIES tips are rendered selective for larger polyions with biomedical importance, i.e., polyanionic pentasaccharide Arixtra and polycationic peptide protamine, to yield the membrane permeability that is lower than the corresponding diffusion-limited permeability. The hindered transport of the respective polyions is unequivocally ascribed to electrostatic and steric repulsions from the wall of the nanopores, i.e., the charge and size effects.

A structure-permeability relationship of ultrathin nanoporous silicon membrane: a comparison with the nuclear envelope.

We report on a simple, quantitative relationship between structure and permeability of a novel ultrathin nanoporous membrane based on nanocrystalline silicon. Large permeability of the free-standing nanomembrane to Ru(NH3)63+, O2, or 1,1'-ferrocenedimethanol, which was able to be measured for the first time by employing scanning electrochemical microscopy, is proportional to the density (67 mum-2) and average radius (5.6 nm) of nanopores. As solution electrolyte concentration decreases down to 0.01 M, the nanopores are selectively "closed" against Fe(CN)64- because of electrostatic repulsion against negative charges at the pore wall. Permeability of the silicon nanomembrane was compared to permeability of the nuclear envelope to find that the channel diameter of the nuclear pore complex that perforates the nuclear envelope is much larger than the average diameter of the silicon nanopores and concomitantly a hypothetical diameter of 10 nm.

Detection of human proteins using arrayed imaging reflectometry.

Assays built upon protein arrays are critical tools in determining the basic nature of biology, and have considerable promise in diagnosing human disease. These protein arrays aid in the elucidation of mapping pathway interactions, disease biomarker discovery, and regulatory processes. The solutions used in these experiments, including cellular lysate and serum, are inherently complex mixtures and are high in total protein content. Therefore, array-based assays must be robust and maintain a high level of selectivity and sensitivity. We report herein that arrayed imaging reflectometry (AIR), a label-free biosensing platform we have previously disclosed, is highly suitable for the detection of human proteins in complex solutions. In particular, we demonstrate array-based detection of cytokines in buffered solutions, and in undiluted human serum.

Label-free microarray-based detection of autoantibodies in human serum.

Multiplex assays for autoantibodies have shown utility both in research towards understanding the basic biology of autoimmune disease, and as tools for clinical diagnosis. New label-free multiplex analysis methods have the potential to streamline both the process of assay development and assay workflow. We report fabrication and testing of a 5-plex autoantigen microarray using the Arrayed Imaging Reflectometry (AIR) platform. This label-free technology provides rapid, sensitive, and quantitative detection of an arbitrary number of analytes in a standard multiwell format. In this work, we demonstrate that AIR is able to detect antibodies to Ro60, La/SSB, Scl-70, BicD2, and Ro52 in single-donor human serum samples with multiplex results comparable to singleplex ELISA or Luminex assays.

A label-free, multiplex competitive assay for small molecule pollutants.

Understanding the amount of exposure individuals have had to common chemical pollutants critically requires the ability to detect those compounds in a simple, sensitive, and specific manner. Doing so using label-free biosensor technology has proven challenging, however, given the small molecular weight of many pollutants of interest. To address this issue, we report the development of a pollutant microarray based on the label-free arrayed imaging reflectometry (AIR) detection platform. The sensor is able to detect three common environmental contaminants (benzo[a]pyrene, bisphenol A, and acrolein) in human serum via a competitive binding scheme.

Ultrathin silicon membranes for wearable dialysis.

The development of wearable or implantable technologies that replace center-based hemodialysis (HD) hold promise to improve outcomes and quality of life for patients with ESRD. A prerequisite for these technologies is the development of highly efficient membranes that can achieve high toxin clearance in small-device formats. Here we examine the application of the porous nanocrystalline silicon (pnc-Si) to HD. pnc-Si is a molecularly thin nanoporous membrane material that is orders of magnitude more permeable than conventional HD membranes. Material developments have allowed us to dramatically increase the amount of active membrane available for dialysis on pnc-Si chips. By controlling pore sizes during manufacturing, pnc-Si membranes can be engineered to pass middle-molecular-weight protein toxins while retaining albumin, mimicking the healthy kidney. A microfluidic dialysis device developed with pnc-Si achieves urea clearance rates that confirm that the membrane offers no resistance to urea passage. Finally, surface modifications with thin hydrophilic coatings are shown to block cell and protein adhesion.

Analysis of inflammatory biomarkers by Arrayed Imaging Reflectometry.

Levels of serum cytokines are important markers for a broad range of human health conditions, ranging from infectious disease and cancer, to pollutant exposure and stress. In the interest of developing new rapid label-free methods for profiling serum cytokines, we have examined the utility of Arrayed Imaging Reflectometry (AIR) microarrays for this application. We find that AIR is readily able to profile 14 cytokines and other inflammatory biomarker proteins in a background of buffered bovine serum albumin or 1% bovine serum with performance metrics comparable to singleplex ELISA, but in a multiplex, chip-based, reagentless format. Further experiments with interferon-gamma (IFN-γ) demonstrated that the concentration of bovine serum could be increased to at least 20% without changing the overall analytical profile or limit of detection (<10 pg/mL).

High-performance separation of nanoparticles with ultrathin porous nanocrystalline silicon membranes.

Porous nanocrystalline silicon (pnc-Si) is a 15 nm thin free-standing membrane material with applications in small-scale separations, biosensors, cell culture, and lab-on-a-chip devices. Pnc-Si has already been shown to exhibit high permeability to diffusing species and selectivity based on molecular size or charge. In this report, we characterize properties of pnc-Si in pressurized flows. We compare results to long-standing theories for transport through short pores using actual pore distributions obtained directly from electron micrographs. The measured water permeability is in agreement with theory over a wide range of pore sizes and porosities and orders of magnitude higher than those exhibited by commercial ultrafiltration and experimental carbon nanotube membranes. We also show that pnc-Si membranes can be used in dead-end filtration to fractionate gold nanoparticles and protein size ladders with better than 5 nm resolution, insignificant sample loss, and little dilution of the filtrate. These performance characteristics, combined with scalable manufacturing, make pnc-Si filtration a straightforward solution to many nanoparticle and biological separation problems.

Theoretical and experimental analysis of arrayed imaging reflectometry as a sensitive proteomics technique.

Arrayed imaging reflectometry (AIR) is a newly developed label-free optical biosensing technique based on the creation and perturbation of a condition of zero reflectance on a silicon substrate. The antireflective coating is formed by covalently immobilizing arrayed probes on a silicon dioxide film. Probe-target complex formation causes a localized increase in optical thickness and a measurable reflectance change. To evaluate the performance of AIR, we have employed two proteins, intimin and tir, from enteropathogenic E. coli that are critical to the bacterium's mechanism of host infection. Using substrates functionalized with the intimin-binding domain of tir, we demonstrate detection of the extracellular domain of intimin at concentrations as low as 10 pM. Through the use of a diffusion-limited model for the intimin-tir binding interaction at this concentration, we estimate the detected intimin surface concentration to be 0.33 pg/mm2.