Margo R. Monroe

Single nanoparticle detection for multiplexed protein diagnostics with attomolar sensitivity in serum and unprocessed whole blood.

Although biomarkers exist for a range of disease diagnostics, a single low-cost platform exhibiting the required sensitivity, a large dynamic-range and multiplexing capability, and zero sample preparation remains in high demand for a variety of clinical applications. The Interferometric Reflectance Imaging Sensor (IRIS) was utilized to digitally detect and size single gold nanoparticles to identify protein biomarkers in unprocessed serum and blood samples. IRIS is a simple, inexpensive, multiplexed, high-throughput, and label-free optical biosensor that was originally used to quantify biomass captured on a surface with moderate sensitivity. Here we demonstrate detection of β-lactoglobulin, a cows milk whey protein spiked in serum (>10 orders of magnitude) and whole blood (>5 orders of magnitude), at attomolar sensitivity. The clinical utility of IRIS was demonstrated by detecting allergen-specific IgE from microliters of characterized human serum and unprocessed whole blood samples by using secondary antibodies against human IgE labeled with 40 nm gold nanoparticles. To the best of our knowledge, this level of sensitivity over a large dynamic range has not been previously demonstrated. IRIS offers four main advantages compared to existing technologies: it (i) detects proteins from attomolar to nanomolar concentrations in unprocessed biological samples, (ii) unambiguously discriminates nanoparticles tags on a robust and physically large sensor area, (iii) detects protein targets with conjugated very small nanoparticle tags (~40 nm diameter), which minimally affect assay kinetics compared to conventional microparticle tagging methods, and (iv) utilizes components that make the instrument inexpensive, robust, and portable. These features make IRIS an ideal candidate for clinical and diagnostic applications.

Multiplexed Method to Calibrate and Quantitate Fluorescence Signal for Allergen-Specific IgE

Using a microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of serum. However, variation of probe immobilization on microarrays hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics. To address this problem, we have developed a calibrated, inexpensive, multiplexed, and rapid protein microarray method that directly correlates surface probe density to captured labeled secondary antibody in clinical samples. We have identified three major technological advantages of our calibrated fluorescence enhancement (CaFE) technique: (i) a significant increase in fluorescence emission over a broad range of fluorophores on a layered substrate optimized specifically for fluorescence; (ii) a method to perform label-free quantification of the probes in each spot while maintaining fluorescence enhancement for a particular fluorophore; and (iii) a calibrated, quantitative technique that combines fluorescence and label-free modalities to accurately measure probe density and bound target for a variety of antibody-antigen pairs. In this paper, we establish the effectiveness of the CaFE method by presenting the strong linear dependence of the amount of bound protein to the resulting fluorescence signal of secondary antibody for IgG, β-lactoglobulin, and allergen-specific IgEs to Ara h 1 (peanut major allergen) and Phl p 1 (timothy grass major allergen) in human serum.

Silicon biochips for dual label-free and fluorescence detection: Application to protein microarray development

A new silicon chip for protein microarray development, fabrication and validation is proposed. The chip is made of two areas with oxide layers of different thicknesses: an area with a 500 nm SiO2 layer dedicated to interferometric label-free detection and quantification of proteins and an area with 100 nm SiO2 providing enhanced fluorescence. The chip allows, within a single experiment performed on the same surface, label-free imaging of arrayed protein probes coupled with high sensitivity fluorescence detection of the molecular interaction counterparts. Such a combined chip is of high practical utility during assay development process to image arrays, check consistency and quality of the protein array, quantify the amount of immobilized probes and finally detect fluorescence of bioassays.

Interferometric silicon biochips for label and label‐free DNA and protein microarrays

Protein and DNA microarrays hold the promise to revolutionize the field of molecular diagnostics. Traditional microarray applications employ labeled detection strategies based on the use of fluorescent and chemiluminescent secondary antibodies. However, the development of high throughput, sensitive, label‐free detection techniques is attracting attention as they do not require labeled reactants and provide quantitative information on binding kinetics. In this article, we will provide an overview of the recent authors work in label and label‐free sensing platforms employing silicon/silicon oxide (Si/SiO2) substrates for interferometric and/or fluorescence detection of microarrays. The review will focus on applications of Si/SiO2 with controlled oxide layers to (i) enhance the fluorescence intensity by optical interferences, (ii) quantify with sub‐nanometer accuracy the axial locations of fluorophore‐labeled probes tethered to the surface, and (iii) detect protein–protein interactions label free. Different methods of biofunctionalization of the sensing surface will be discussed. In particular, organosilanization reactions for monodimensional coatings and polymeric coatings will be extensively reviewed. Finally, the importance of calibration of protein microarrays through the dual use of labeled and label‐free detection schemes on the same chip will be illustrated.

Precisely controlled smart polymer scaffold for nanoscale manipulation of biomolecules.

We demonstrate the application of a novel smart surface to modulate the orientation of immobilized double stranded DNA (dsDNA) and the conformation of a polymer scaffold through variation in buffer pH and ionic strength. An amphoteric poly(dimethylacrylamide) based coating containing weak acrylamido acids and bases, which are copolymerized together with the neutral monomer, is covalently bound to the surface. The coating can be made to contain any desired amount of buffering and titrant ionogenic monomers, allowing control of the surface charge when the surface is bathed in a given buffer pH. Spectral self-interference fluorescence microscopy (SSFM) is utilized to precisely quantify both the DNA orientation and the polymer conformation with subnanometer resolution. It is possible to utilize the polymer scaffold to functionalize a variety of common materials used in microfabrication, making it a general purpose building block for the next generation of nanomachines and biosensors.

Integrated imaging instrument for self-calibrated fluorescence protein microarrays

Protein microarrays, or multiplexed and high-throughput assays, monitor multiple protein binding events to facilitate the understanding of disease progression and cell physiology. Fluorescence imaging is a popular method to detect proteins captured by immobilized probes with high sensitivity and specificity. Reliability of fluorescence assays depends on achieving minimal inter- and intra-assay probe immobilization variation, an ongoing challenge for protein microarrays. Therefore, it is desirable to establish a label-free method to quantify the probe density prior to target incubation to calibrate the fluorescence readout. Previously, a silicon oxide on silicon chip design was introduced to enhance the fluorescence signal and enable interferometric imaging to self-calibrate the signal with the immobilized probe density. In this paper, an integrated interferometric reflectance imaging sensor and wide-field fluorescence instrument is introduced for sensitive and calibrated microarray measurements. This platform is able to analyze a 2.5 mm × 3.4 mm area, or 200 spots (100 μm diameter with 200 μm pitch), in a single field-of-view.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation. Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time. Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches. Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Precise quantification and control of surface immobilized DNA orientation

We utilize spectral self-interference fluorescent microscopy (SSFM) to measure fluorophore height with sub-nm precision to precisely quantify DNA orientation and conformation. A novel polymeric 3D scaffold is used to functionalize the sensor surface and permits controlled orientation of the surface anchored DNA.

Fluorescence enhancement on reflecting substrates for microarray applications

In this study, we report on the utilization of layered substrates for increased performance of fluorescent-based detection schemes. Through optimization of layer thicknesses, we demonstrate that enhancement with respect to commonly used microscope slides is achieved for the collected fluorescence signal.

Precise control of DNA orientation for improved functionlity in protein binding microarrays

We demonstrate controlled orientation of surface anchored DNA and utilize spectral self-interference fluorescent microscopy (SSFM) to measure fluorophore height with sub-nm precision to precisely quantify DNA orientation and conformation.