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Diagnostic approaches that combine the high sensitivity and specificity of laboratory-based digital detection with the ease of use and affordability of point-of-care (POC) technologies could revolutionize disease diagnostics. This is especially true in infectious disease diagnostics, where rapid and accurate pathogen detection is critical to curbing the spread of disease. We have pioneered an innovative label-free digital detection platform that utilizes Interferometric Reflectance Imaging Sensor (IRIS) technology. IRIS leverages light interference from an optically transparent thin film, eliminating the need for complex optical resonances to enhance the signal by harnessing light interference and the power of signal averaging in shot-noise-limited operation In our latest work, we have further improved our previous 'Single-Particle' IRIS (SP-IRIS) technology by allowing the construction of the optical signature of target nanoparticles (whole virus) from a single image. This new platform, 'Pixel-Diversity' IRIS (PD-IRIS), eliminated the need for z-scan acquisition, required in SP-IRIS, a time-consuming and expensive process, and made our technology more applicable to POC settings. Using PD-IRIS, we quantitatively detected the Monkeypox virus (MPXV), the etiological agent for Monkeypox (Mpox) infection. MPXV was captured by anti-A29 monoclonal antibody (mAb 69-126-3) on Protein G spots on the sensor chips and were detected at a limit-of-detection (LOD) - of 200 PFU/mL (∼3.3 aM). PD-IRIS was superior to the laboratory-based ELISA (LOD - 1800 PFU/mL) used as a comparator. The specificity of PD-IRIS in MPXV detection was demonstrated using Herpes simplex virus, type 1 (HSV-1), and Cowpox virus (CPXV). This work establishes the effectiveness of PD-IRIS and opens possibilities for its advancement in clinical diagnostics of Mpox at POC. Moreover, PD-IRIS is a modular technology that can be adapted for the multiplex detection of pathogens for which high-affinity ligands are available that can bind their surface antigens to capture them on the sensor surface., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Diagnostic approaches that combine the high sensitivity and specificity of laboratory-based digital detection with the ease of use and affordability of point-of-care (POC) technologies could revolutionize disease diagnostics. This is especially true in infectious disease diagnostics, where rapid and accurate pathogen detection is critical to curbing the spread of disease. We have pioneered an innovative label-free digital detection platform that utilizes Interferometric Reflectance Imaging Sensor (IRIS) technology. IRIS leverages light interference from an optically transparent thin film, eliminating the need for complex optical resonances to enhance the signal by harnessing light interference and the power of signal averaging in shot-noise-limited operation to achieve virtually unlimited sensitivity. In our latest work, we have further improved our previous 'Single-Particle' IRIS (SP-IRIS) technology by allowing the construction of the optical signature of target nanoparticles (whole virus) from a single image. This new platform, 'Pixel-Diversity' IRIS (PD-IRIS), eliminated the need for z-scan acquisition, required in SP-IRIS, a time-consuming and expensive process, and made our technology more applicable to POC settings. Using PD-IRIS, we quantitatively detected the Monkeypox virus (MPXV), the etiological agent for Monkeypox (Mpox) infection. MPXV was captured by anti-A29 monoclonal antibody (mAb 69-126-3) on Protein G spots on the sensor chips and were detected at a limit-of-detection (LOD) - of 200 PFU/ml (~3.3 attomolar). PD-IRIS was superior to the laboratory-based ELISA (LOD - 1800 PFU/mL) used as a comparator. The specificity of PD-IRIS in MPXV detection was demonstrated using Herpes simplex virus, type 1 (HSV-1), and Cowpox virus (CPXV). This work establishes the effectiveness of PD-IRIS and opens possibilities for its advancement in clinical diagnostics of Mpox at POC. Moreover, PD-IRIS is a modular technology that can be adapted for the multiplex detection of pathogens for which high-affinity ligands are available that can bind their surface antigens to capture them on the sensor surface.

Gold nanoparticles (AuNPs) play a vital role in biotechnology, medicine, and diagnostics due to their unique optical properties. Their conjugation with antibodies, antigens, proteins, or nucleic acids enables precise targeting and enhances biosensing capabilities. Functionalized AuNPs, however, may experience reduced stability, leading to aggregation or loss of functionality, especially in complex biological environments. Additionally, they can show non-specific binding to unintended targets, impairing assay specificity. Within this work, citrate-stabilized and silica-coated AuNPs (GNPs and SiGNPs, respectively) have been coated using N , N -dimethylacrylamide-based copolymers to increase their stability and enable their functionalization with biomolecules. AuNP stability after modification has been assessed by a combination of techniques including spectrophotometric characterization, nanoparticle tracking analysis, transmission electron microscopy and functional microarray tests. Two different copolymers were identified to provide a stable coating of AuNPs while enabling further modification through click chemistry reactions, due to the presence of azide groups in the polymers. Following this experimental design, AuNPs decorated with ssDNA and streptavidin were synthesized and successfully used in a biological assay. In conclusion, a functionalization scheme for AuNPs has been developed that offers ease of modification, often requiring single steps and short incubation time. The obtained functionalized AuNPs offer considerable flexibility, as the functionalization protocol can be personalized to match requirements of multiple assays.

Wet Age-related macular degeneration (AMD) is the leading cause of vision loss in industrialized nations, often resulting in blindness. Biologics, therapeutic agents derived from biological sources, have been effective in AMD, albeit at a high cost. Due to the high cost of AMD treatment, it is critical to determine the binding affinity of biologics to ensure their efficacy and make quantitative comparisons between different drugs. This study evaluates the in vitro VEGF binding affinity of two drugs used for treating wet AMD, monoclonal antibody-based bevacizumab and fusion protein-based aflibercept, performing quantitative binding measurements on an Interferometric Reflectance Imaging Sensor (IRIS) system. Both biologics can inhibit Vascular Endothelial Growth Factor (VEGF). For comparison, the therapeutic molecules were immobilized on to the same support in a microarray format, and their real-time binding interactions with recombinant human VEGF (rhVEGF) were measured using an IRIS. The results indicated that aflibercept exhibited a higher binding affinity to VEGF than bevacizumab, consistent with previous studies using ELISA and SPR. The IRIS system's innovative and cost-effective features, such as silicon-based semiconductor chips for enhanced signal detection and multiplexed analysis capability, offer new prospects in sensor technologies. These attributes make IRISs a promising tool for future applications in the development of therapeutic agents, specifically biologics.

Optical coherence tomography (OCT) is a label-free, non-invasive 3D imaging tool widely used in both biological research and clinical diagnosis. Conventional OCT modalities can only visualize specimen tomography without chemical information. Here, we report a bond-selective full-field OCT (BS-FF-OCT), in which a pulsed mid-infrared laser is used to modulate the OCT signal through the photothermal effect, achieving label-free bond-selective 3D sectioned imaging of highly scattering samples. We first demonstrate BS-FF-OCT imaging of 1 µm PMMA beads embedded in agarose gel. Next, we show 3D hyperspectral imaging of up to 75 µm of polypropylene fiber mattress from a standard surgical mask. We then demonstrate BS-FF-OCT imaging on biological samples, including cancer cell spheroids and C. elegans. Using an alternative pulse timing configuration, we finally demonstrate the capability of BS-FF-OCT on imaging a highly scattering myelinated axons region in a mouse brain tissue slice.

Clinical identification and fundamental study of viruses rely on the detection of viral proteins or viral nucleic acids. Yet, amplification-based and antigen-based methods are not able to provide precise compositional information of individual virions due to small particle size and low-abundance chemical contents (e.g., ~ 5000 proteins in a vesicular stomatitis virus). Here, we report a widefield interferometric defocus-enhanced mid-infrared photothermal (WIDE-MIP) microscope for high-throughput fingerprinting of single viruses. With the identification of feature absorption peaks, WIDE-MIP reveals the contents of viral proteins and nucleic acids in single DNA vaccinia viruses and RNA vesicular stomatitis viruses. Different nucleic acid signatures of thymine and uracil residue vibrations are obtained to differentiate DNA and RNA viruses. WIDE-MIP imaging further reveals an enriched β sheet components in DNA varicella-zoster virus proteins. Together, these advances open a new avenue for compositional analysis of viral vectors and elucidating protein function in an assembled virion., (© 2023. Springer Nature Limited.)

Illumination uniformity is a critical parameter for excitation and data extraction quality in widefield biological imaging applications. However, typical imaging systems suffer from spatial and spectral non-uniformity due to non-ideal optical elements, thus require complex solutions for illumination corrections. We present Effective Uniform Color-Light Integration Device (EUCLID), a simple and cost-effective illumination source for uniformity corrections. EUCLID employs a diffuse-reflective, adjustable hollow cavity that allows for uniform mixing of light from discrete light sources and modifies the source field distribution to compensate for spatial non-uniformity introduced by optical components in the imaging system. In this study, we characterize the light coupling efficiency of the proposed design and compare the uniformity performance with the conventional method. EUCLID demonstrates a remarkable illumination improvement for multi-spectral imaging in both Nelsonian and Koehler alignment with a maximum spatial deviation of ≈ 1% across a wide field-of-view., Competing Interests: The authors have read the journal’s policy and have the following competing interests: MSU is the founder and CEO at iRiS Kinetics. There are no patents, products in development or marketed products under iRiS Kinetics associated with this research to declare. Boston University filed a patent application regarding this study: patent application no. 63/392,639. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Çelebi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Viral infections can pose a major threat to public health by causing serious illness, leading to pandemics, and burdening healthcare systems. The global spread of such infections causes disruptions to every aspect of life including business, education, and social life. Fast and accurate diagnosis of viral infections has significant implications for saving lives, preventing the spread of the diseases, and minimizing social and economic damages. Polymerase chain reaction (PCR)-based techniques are commonly used to detect viruses in the clinic. However, PCR has several drawbacks, as highlighted during the recent COVID-19 pandemic, such as long processing times and the requirement for sophisticated laboratory instruments. Therefore, there is an urgent need for fast and accurate techniques for virus detection. For this purpose, a variety of biosensor systems are being developed to provide rapid, sensitive, and high-throughput viral diagnostic platforms, enabling quick diagnosis and efficient control of the virus's spread. Optical devices, in particular, are of great interest due to their advantages such as high sensitivity and direct readout. The current review discusses solid-phase optical sensing techniques for virus detection, including fluorescence-based sensors, surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), optical resonators, and interferometry-based platforms. Then, we focus on an interferometric biosensor developed by our group, the single-particle interferometric reflectance imaging sensor (SP-IRIS), which has the capability to visualize single nanoparticles, to demonstrate its application for digital virus detection.

Pathogenic microorganisms and viruses can easily transfer from one host to another and cause disease in humans. The determination of these pathogens in a time- and cost-effective way is an extreme challenge for researchers. Rapid and label-free detection of pathogenic microorganisms and viruses is critical in ensuring rapid and appropriate treatment. Sensor technologies have shown considerable advancements in viral diagnostics, demonstrating their great potential for being fast and sensitive detection platforms. In this review, we present a summary of the use of an interferometric reflectance imaging sensor (IRIS) for the detection of microorganisms. We highlight low magnification modality of IRIS as an ensemble biomolecular mass measurement technique and high magnification modality for the digital detection of individual nanoparticles and viruses. We discuss the two different modalities of IRIS and their applications in the sensitive detection of microorganisms and viruses.

Monoclonal antibodies (mAbs) play an important role in diagnostics and therapy of infectious diseases. Here we utilize a single-particle interferometric reflectance imaging sensor (SP-IRIS) for screening 30 mAbs against Ebola, Sudan, and Lassa viruses (EBOV, SUDV, and LASV) to find out the ideal capture antibodies for whole virus detection using recombinant vesicular stomatitis virus (rVSV) models expressing surface glycoproteins (GPs) of EBOV, SUDV, and LASV. We also make use of the binding properties on SP-IRIS to develop a model for mapping the antibody epitopes on the GP structure. mAbs that bind to mucin-like domain or glycan cap of the EBOV surface GP show the highest signal on SP-IRIS, followed by mAbs that target the GP1-GP2 interface at the base domain. These antibodies were shown to be highly efficacious against EBOV infection in non-human primates in previous studies. For LASV detection, 8.9F antibody showed the best performance on SP-IRIS. This antibody binds to a unique region on the surface GP compared to other 15 mAbs tested. In addition, we demonstrate a novel antibody competition assay using SP-IRIS and rVSV-EBOV models to reveal the competition between mAbs in three successful therapeutic mAb cocktails against EBOV infection. We provide an explanation as to why ZMapp cocktail has higher efficacy compared to the other two cocktails by showing that three mAbs in this cocktail (13C6, 2G4, 4G7) do not compete with each other for binding to EBOV GP. In fact, the binding of 13C6 enhances the binding of 2G4 and 4G7 antibodies. Our results establish SP-IRIS as a versatile tool that can provide high-throughput screening of mAbs, multiplexed and sensitive detection of viruses, and evaluation of therapeutic antibody cocktails., (© 2023. The Author(s).)

MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA-451a (miR-451) and miRNA-223-3p (miR-223). We demonstrated improvements in sensitivity in comparison to traditional end-point assays that employ capture on solid phase support, by implementing real-time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low-abundance biomarkers in the presence of low-affinity but high-abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to [Formula: see text] 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min., (© 2022. The Author(s).)

Considering the limitations of well-known traditional detection techniques, innovative research studies have focused on the development of new sensors to offer label-free, highly sensitive, real-time, low-cost, and rapid detection for biomolecular interactions. In this study, we demonstrate immunoglobulin G (IgG) detection in aqueous solutions by using real-time and label-free kinetic measurements of the Interferometric Reflectance Imaging Sensor (IRIS) system. By performing kinetic characterization experiments, the sensor's performance is comprehensively evaluated and a high correlation coefficient value (>0.94) is obtained in the IgG concentration range of 1-50 μg/mL with a low detection limit (0.25 μg/mL or 1.67 nM). Moreover, the highly sensitive imaging system ensures accurate quantification and reliable validation of recorded binding events, offering new perspectives in terms of direct biomarker detection for clinical applications., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The field of biosensing is in constant evolution, propelled by the need for sensitive, reliable platforms that provide consistent results, especially in the drug development industry, where small molecule characterization is of uttermost relevance. Kinetic characterization of small biochemicals is particularly challenging, and has required sensor developers to find solutions to compensate for the lack of sensitivity of their instruments. In this regard, surface chemistry plays a crucial role. The ligands need to be efficiently immobilized on the sensor surface, and probe distribution, maintenance of their native structure and efficient diffusion of the analyte to the surface need to be optimized. In order to enhance the signal generated by low molecular weight targets, surface plasmon resonance sensors utilize a high density of probes on the surface by employing a thick dextran matrix, resulting in a three-dimensional, multilayer distribution of molecules. Despite increasing the binding signal, this method can generate artifacts, due to the diffusion dependence of surface binding, affecting the accuracy of measured affinity constants. On the other hand, when working with planar surface chemistries, an incredibly high sensitivity is required for low molecular weight analytes, and furthermore the standard method for immobilizing single layers of molecules based on self-assembled monolayers (SAM) of epoxysilane has been demonstrated to promote protein denaturation, thus being far from ideal. Here, we will give a concise overview of the impact of tridimensional immobilization of ligands on label-free biosensors, mostly focusing on the effect of diffusion on binding affinity constants measurements. We will comment on how multilayering of probes is certainly useful in terms of increasing the sensitivity of the sensor, but can cause steric hindrance, mass transport and other diffusion effects. On the other hand, probe monolayers on epoxysilane chemistries do not undergo diffusion effect but rather other artifacts can occur due to probe distortion. Finally, a combination of tridimensional polymeric chemistry and probe monolayer is presented and reviewed, showing advantages and disadvantages over the other two approaches.

Anthrax lethal factor (LF) is one of the enzymatic components of the anthrax toxin responsible for the pathogenic responses of the anthrax disease. The ability to screen multiplexed ligands against LF and subsequently estimate the effective kinetic rates (kon and koff) and complementary binding behavior provides critical information useful in diagnostic and therapeutic development for anthrax. Tools such as biolayer interferometry (BLI) and surface plasmon resonance imaging (SPRi) have been developed for this purpose; however, these tools suffer from limitations such as signal jumps when the solution in the chamber is switched or low sensitivity. Here, we present multiplexed antibody affinity measurements obtained by the interferometric reflectance imaging sensor (IRIS), a highly sensitive, label-free optical biosensor, whose stability, simplicity, and imaging modality overcomes many of the limitations of other multiplexed methods. We compare the multiplexed binding results obtained with the IRIS system using two ligands targeting the anthrax lethal factor (LF) against previously published results obtained with more traditional surface plasmon resonance (SPR), which showed consistent results, as well as kinetic information previously unattainable with SPR. Additional exemplary data demonstrating multiplexed binding and the corresponding complementary binding to sequentially injected ligands provides an additional layer of information immediately useful to the researcher.

Mid-infrared photothermal (MIP) microscopy has been a promising label-free chemical imaging technique for functional characterization of specimens owing to its enhanced spatial resolution and high specificity. Recently developed wide-field MIP imaging modalities have drastically improved speed and enabled high-throughput imaging of micron-scale subjects. However, the weakly scattered signal from subwavelength particles becomes indistinguishable from the shot-noise as a consequence of the strong background light, leading to limited sensitivity. Here, we demonstrate background-suppressed chemical fingerprinting at a single nanoparticle level by selectively attenuating the reflected light through pupil engineering in the collection path. Our technique provides over 3 orders of magnitude background suppression by quasi-darkfield illumination in the epi-configuration without sacrificing lateral resolution. We demonstrate 6-fold signal-to-background noise ratio improvement, allowing for simultaneous detection and discrimination of hundreds of nanoparticles across a field of view of 70 μ m × 70 μ m. A comprehensive theoretical framework for photothermal image formation is provided and experimentally validated with 300 and 500 nm PMMA beads. The versatility and utility of our technique are demonstrated via hyperspectral dark-field MIP imaging of S. aureus and E. coli bacteria and MIP imaging of subcellular lipid droplets inside C. albicans and cancer cells., Competing Interests: Notes The authors declare no competing financial interest.

Extracellular vesicles (EVs) have attracted significant attention as impactful diagnostic biomarkers, since their properties are closely related to specific clinical conditions. However, designing experiments that involve EVs phenotyping is usually highly challenging and time-consuming, due to laborious optimization steps that require very long or even overnight incubation durations. In this work, we demonstrate label-free, real-time detection, and phenotyping of extracellular vesicles binding to a multiplexed surface. With the ability for label-free kinetic binding measurements using the Interferometric Reflectance Imaging Sensor (IRIS) in a microfluidic chamber, we successfully optimize the capture reaction by tuning various assay conditions (incubation time, flow conditions, surface probe density, and specificity). A single (less than 1 h) experiment allows for characterization of binding affinities of the EVs to multiplexed probes. We demonstrate kinetic characterization of 18 different probe conditions, namely three different antibodies, each spotted at six different concentrations, simultaneously. The affinity characterization is then analyzed through a model that considers the complexity of multivalent binding of large structures to a carpet of probes and therefore introduces a combination of fast and slow association and dissociation parameters. Additionally, our results confirm higher affinity of EVs to aCD81 with respect to aCD9 and aCD63. Single-vesicle imaging measurements corroborate our findings, as well as confirming the EVs nature of the captured particles through fluorescence staining of the EVs membrane and cargo.

The importance of microarrays in diagnostics and medicine has drastically increased in the last few years. Nevertheless, the efficiency of a microarray-based assay intrinsically depends on the density and functionality of the biorecognition elements immobilized onto each sensor spot. Recently, researchers have put effort into developing new functionalization strategies and technologies which provide efficient immobilization and stability of any sort of molecule. Here, we present an overview of the most widely used methods of surface functionalization of microarray substrates, as well as the most recent advances in the field, and compare their performance in terms of optimal immobilization of the bioreceptor molecules. We focus on label-free microarrays and, in particular, we aim to describe the impact of surface chemistry on two types of microarray-based sensors: microarrays for single particle imaging and for label-free measurements of binding kinetics. Both protein and DNA microarrays are taken into consideration, and the effect of different polymeric coatings on the molecules' functionalities is critically analyzed.

Optical technologies for label-free detection are an attractive solution for monitoring molecular binding kinetics; however, these techniques measure the changes in the refractive index, making it difficult to distinguish surface binding from a change in the refractive index of the analyte solution in the proximity of the sensor surface. The solution refractive index changes, due to solvents, temperature changes, or pH variations, can create an unwanted background signal known as the bulk effect. Technologies such as biolayer interferometry and surface plasmon resonance offer no bulk-effect compensation, or they alternatively offer a reference channel to correct in postprocessing. Here, we present a virtually bulk-effect-free method, without a reference channel or any computational correction, for measuring kinetic binding using the interferometric reflectance imaging sensor (IRIS), an optical label-free biomolecular interaction analysis tool. Dynamic spectral illumination engineering, through tailored LED contributions, is combined with the IRIS technology to minimize the bulk effect, with the potential to enable kinetic measurements of a broader range of analytes. We demonstrate that the deviation in the reflectivity signal is reduced to ∼8 × 10 -6 for a solution change from phosphate-buffered saline (PBS) ( n = 1.335) to 1% dimethyl sulfoxide (DMSO) in PBS ( n = 1.336). As a proof of concept, we applied the method to a biotin-streptavidin interaction, where biotin (MW = 244.3 Da) was dissolved at a final concentration of 1 μM in a 1% solution of DMSO in PBS and flowed over immobilized streptavidin. Clear binding results were obtained without a reference channel or any computational correction., Competing Interests: The authors declare the following competing financial interest(s): M. Selim Unlu is the founder the startup iRiS Kinetics, Inc., which is working towards the commercialization of the IRIS technique., (© 2021 The Authors. Published by American Chemical Society.)

We report a confocal interferometric mid-infrared photothermal (MIP) microscope for ultra-sensitive and spatially resolved chemical imaging of individual viruses. The interferometric scattering principle is applied to detect the very weak photothermal signal induced by infrared absorption of chemical bonds. Spectroscopic MIP detection of single vesicular stomatitis viruses (VSVs) and poxviruses is demonstrated. The single virus spectra show high consistency within the same virus type. The dominant spectral peaks are contributed by the amide I and amide II vibrations attributed to the viral proteins. The ratio of these two peaks is significantly different between VSVs and poxviruses, highlighting the potential of using interferometric MIP microscopy for label-free differentiation of viral particles. This all-optical chemical imaging method opens a new way for spectroscopic detection of biological nanoparticles in a label-free manner and may facilitate in predicting and controlling the outbreaks of emerging virus strains.

Here, we demonstrate real-time multiplexed virus detection by applying a DNA-directed antibody immobilization technique in a single-particle interferometric reflectance imaging sensor (SP-IRIS). In this technique, the biosensor chip surface spotted with different DNA sequences is converted to a multiplexed antibody array by flowing antibody-DNA conjugates and allowing for specific DNA-DNA hybridization. The resulting antibody array is shown to detect three different recombinant vesicular stomatitis viruses (rVSVs), which are genetically engineered to express surface glycoproteins of Ebola, Marburg, and Lassa viruses in real time in a disposable microfluidic cartridge. We also show that this method can be modified to produce a single-step, homogeneous assay format by mixing the antibody-DNA conjugates with the virus sample in the solution phase prior to incubation in the microfluidic cartridge, eliminating the antibody immobilization step. This homogenous approach achieved detection of the model Ebola virus, rVSV-EBOV, at a concentration of 100 PFU/mL in 1 h. Finally, we demonstrate the feasibility of this homogeneous technique as a rapid test using a passive microfluidic cartridge. A concentration of 10 4 PFU/mL was detectable under 10 min for the rVSV-Ebola virus. Utilizing DNA microarrays for antibody-based diagnostics is an alternative approach to antibody microarrays and offers advantages such as configurable sensor surface, long-term storage ability, and decreased antibody use. We believe that these properties will make SP-IRIS a versatile and robust platform for point-of-care diagnostics applications.

The manufacture of a very high-quality microarray support is essential for the adoption of this assay format in clinical routine. In fact, poorly surface-bound probes can affect the diagnostic sensitivity or, in worst cases, lead to false negative results. Here we report on a reliable and easy quality control method for the evaluation of spotted probe properties in a microarray test, based on the Interferometric Reflectance Imaging Sensor (IRIS) system, a high-resolution label free technique able to evaluate the variation of the mass bound to a surface. In particular, we demonstrated that the IRIS analysis of microarray chips immediately after probe immobilization can detect the absence of probes, which recognizably causes a lack of signal when performing a test, with clinical relevance, using fluorescence detection. Moreover, the use of the IRIS technique allowed also to determine the optimal concentration of the probe, that has to be immobilized on the surface, to maximize the target recognition, thus the signal, but to avoid crowding effects. Finally, through this preliminary quality inspection it is possible to highlight differences in the immobilization chemistries. In particular, we have compared NHS ester versus click chemistry reactions using two different surface coatings, demonstrating that, in the diagnostic case used as an example (colorectal cancer) a higher probe density does not reflect a higher binding signal, probably because of a crowding effect.

Single particle interferometric reflectance (SPIR) microscopy has been studied as a powerful imaging platform for label-free and highly sensitive biological nanoparticle detection and characterization. SPIR's interferometric nature yields a unique 3D defocus intensity profile of the nanoparticles over a large field of view. Here, we utilize this defocus information to recover high signal-to-noise ratio nanoparticle images with a computationally and memory efficient reconstruction framework. Our direct inversion approach recovers this image from a 3D defocus intensity stack using the vectorial-optics-based forward model developed for sub-diffraction-limited dielectric nanoparticles captured on a layered substrate. We demonstrate proof-of-concept experiments on silica beads with a 50 nm nominal diameter.

Investigating the binding interaction of small molecules to large ligands is a compelling task for the field of drug development, as well as agro-biotechnology, since a common trait of drugs and toxins is often a low molecular weight (MW). Here, we improve the limit of detection of the Interferometric Reflectance Imaging Sensor (IRIS), a label-free, highly multiplexed biosensor, to perform small-molecule screening. In this work, characterization of small molecules binding to immobilized probes in a microarray format is demonstrated, with a limit of detection of 1 pg/mm 2 in mass density. First, as a proof of concept to show the impact of spatial and temporal averaging on the system noise, detection of biotin (MW = 244.3 Da) binding to a streptavidin-functionalized chip is performed and the parameters are tuned to achieve maximum signal-to-noise ratio (SNR ≈ 34). The optimized system is then applied to the screening of a 20-multiplexed antibody chip against fumonisin B1 (MW = 721.8 Da), a mycotoxin found in cereal grains. The simultaneously recorded binding curves yield an SNR ≈ 8. Five out of twenty antibodies are also screened against the toxin in a lateral flow assay, obtaining consistent results. With the demonstrated noise characteristics, further sensitivity improvements are expected with the advancement of camera sensor technology., Competing Interests: The authors declare the following competing financial interest(s): Prof. M. Selim Ünlü is the principal investigator of these technology translation grants. He is the founder of a start up company (iRiS Kinetics, Inc.) for the commercialization of the multiplexed affinity technique.

Bacterial infectious diseases are a major threat to human health. Timely and sensitive pathogenic bacteria detection is crucial in bacterial contaminations identification and preventing the spread of infectious diseases. Due to limitations of conventional bacteria detection techniques there have been concerted research efforts towards developing new biosensors. Biosensors offering label-free, whole bacteria detection are highly desirable over those relying on label-based or pathogenic molecular components detection. The major advantage is eliminating the additional time and cost required for labeling or extracting the desired bacterial components. Here, we demonstrate rapid, sensitive and label-free Escherichia coli (E. coli) detection utilizing interferometric reflectance imaging enhancement allowing visualizing individual pathogens captured on the surface. Enabled by our ability to count individual bacteria on a large sensor surface, we demonstrate an extrapolated limit of detection of 2.2 CFU/ml from experimental data in buffer solution with no sample preparation. To the best of our knowledge, this level of sensitivity for whole E. coli detection is unprecedented in label-free biosensing. The specificity of our biosensor is validated by comparing the response to target bacteria E. coli and non-target bacteria S. aureus, K. pneumonia and P. aeruginosa. The biosensor's performance in tap water proves that its detection capability is unaffected by the sample complexity. Furthermore, our sensor platform provides high optical magnification imaging and thus validation of recorded detection events as the target bacteria based on morphological characterization. Therefore, our sensitive and label-free detection method offers new perspectives for direct bacterial detection in real matrices and clinical samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Surface chemistry is a crucial aspect for microarray modality biosensor development. The immobilization capability of the functionalized surface is indeed a limiting factor for the final yield of the binding reaction. In this work, we were able to simultaneously compare the functionality of protein ligands that were locally immobilized on different polymers, while on the same solid support, therefore demonstrating a new way of multiplexing. Our goal was to investigate, in a single experiment, both the immobilization efficiency of a group of reactive polymers and the resulting affinity of the tethered molecules. This idea was demonstrated by spotting many reactive polymers on a Si/SiO 2 chip and depositing the molecular probes on the spots immediately after. As a proof of concept, we focused on which polymers would better immobilize a model protein (α-Lactalbumin) and a peptide (LAC-1). We successfully showed that this protocol is applicable to proteins and peptides with a good efficiency. By means of real-time binding measurements performed with the interferometric reflectance imaging sensor (IRIS), local functionalization proved to be comparable to the classical flat coating solution. The final outcome highlights the multiplexing power of this method: first, it allows to characterize dozens of polymers at once. Secondly, it removes the limitation, related to coated surfaces, that only molecules with the same functional groups can be tethered to the same solid support. By applying this protocol, many types of molecules can be studied simultaneously and immobilization for each probe can be individually optimized.

Label-free, visible light microscopy is an indispensable tool for studying biological nanoparticles (BNPs). However, conventional imaging techniques have two major challenges: (i) weak contrast due to low-refractive-index difference with the surrounding medium and exceptionally small size and (ii) limited spatial resolution. Advances in interferometric microscopy have overcome the weak contrast limitation and enabled direct detection of BNPs, yet lateral resolution remains as a challenge in studying BNP morphology. Here, we introduce a wide-field interferometric microscopy technique augmented by computational imaging to demonstrate a 2-fold lateral resolution improvement over a large field-of-view (>100 × 100 μm 2 ), enabling simultaneous imaging of more than 10 4 BNPs at a resolution of ∼150 nm without any labels or sample preparation. We present a rigorous vectorial-optics-based forward model establishing the relationship between the intensity images captured under partially coherent asymmetric illumination and the complex permittivity distribution of nanoparticles. We demonstrate high-throughput morphological visualization of a diverse population of Ebola virus-like particles and a structurally distinct Ebola vaccine candidate. Our approach offers a low-cost and robust label-free imaging platform for high-throughput and high-resolution characterization of a broad size range of BNPs.

We review some emerging trends in transduction, connectivity and data analytics for Point-of-Care Testing (POCT) of infectious and non-communicable diseases. The patient need for POCT is described along with developments in portable diagnostics, specifically in respect of Lab-on-chip and microfluidic systems. We describe some novel electrochemical and photonic systems and the use of mobile phones in terms of hardware components and device connectivity for POCT. Developments in data analytics that are applicable for POCT are described with an overview of data structures and recent AI/Machine learning trends. The most important methodologies of machine learning, including deep learning methods, are summarised. The potential value of trends within POCT systems for clinical diagnostics within Lower Middle Income Countries (LMICs) and the Least Developed Countries (LDCs) are highlighted.

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format., Competing Interests: The authors declare no conflict of interest.

The determination of kinetic information and appropriate binding pairs is fundamental to the proper optimization and selection of ligands used in immunoassays, diagnostics, and therapeutics. However, the ability to estimate such parameters in a multiplexed and inexpensive format remains difficult and modification of the ligand is often necessary. Here, we detail the methods and materials necessary to evaluate hundreds of unlabeled ligands simultaneously using the interferometric reflectance imaging sensor (IRIS). The incorporation of a low-cost fluidic cartridge that integrates on the top of the sensor simplifies reagent handling considerably.

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology's Achilles' heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ("digital") regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique's simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.

Fluorescence based microarray detection systems provide sensitive measurements; however, variation of probe immobilization and poor repeatability negatively affect the final readout, and thus quantification capability of these systems. Here, we demonstrate a label-free and high-throughput optical biosensor that can be utilized for calibration of fluorescence microarrays. The sensor employs a commercial flatbed scanner, and we demonstrate transformation of this low cost (∼100 USD) system into an Interferometric Reflectance Imaging Sensor through hardware and software modifications. Using this sensor, we report detection of DNA hybridization and DNA directed antibody immobilization on label-free microarrays with a noise floor of ∼30 pg/mm 2 , and a scan speed of 5 s (50 s for 10 frames averaged) for a 2 mm × 2 mm area. This novel system may be used as a standalone label-free sensor especially in low-resource settings, as well as for quality control and calibration of microarrays in existing fluorescence-based DNA and protein detection platforms.

Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.

The sensitive detection and quantitative measurement of biological nanoparticles such as viruses or exosomes is of growing importance in biology and medicine since these structures are implicated in many biological processes and diseases. Interferometric reflectance imaging is a label-free optical biosensing method which can directly detect individual biological nanoparticles when they are immobilized onto a protein microarray. Previous efforts to infer bio-nanoparticle size and shape have relied on empirical calibration using a 'ruler' of particle samples of known size, which was inconsistent and qualitative. Here, we present a mechanistic physical explanation and experimental approach by which interferometric reflectance imaging may be used to not only detect but also quantitatively measure bio-nanoparticle size and shape. We introduce a comprehensive optical model that can quantitatively simulate the scattering of arbitrarily-shaped nanoparticles such as rod-shaped or filamentous virions. Finally, we optimize the optical design for the detection and quantitative measurement of small and low-index bio-nanoparticles immersed in water.

Single-molecule and single-nanoparticle biosensors are a growing frontier in diagnostics. Digital biosensors are those which enumerate all specifically immobilized biomolecules or biological nanoparticles, and thereby achieve limits of detection usually beyond the reach of ensemble measurements. Here we review modern optical techniques for single nanoparticle detection and describe the single-particle interferometric reflectance imaging sensor (SP-IRIS). We present challenges associated with reliably detecting faint nanoparticles with SP-IRIS, and describe image acquisition processes and software modifications to address them. Specifically, we describe a image acquisition processing method for the discrimination and accurate counting of nanoparticles that greatly reduces both the number of false positives and false negatives. These engineering improvements are critical steps in the translation of SP-IRIS towards applications in medical diagnostics.

Light microscopy is a straightforward and highly portable imaging approach that is used for the detection of parasites, fungi, and bacteria. The detection of individual virus particles has historically not been possible through this approach. Thus, characterization of virus particles is typically performed using high-energy approaches such as electron microscopy. These approaches require purification of virions away from its normal milieu, significant levels of expertise, and only count a small number of particles at a time. To correct these deficiencies we created a platform that allows label-free, point-of-need virus imaging and counting. We adapted a multiplex-capable, interferometric imaging technique to a closed-system that allows real-time particle detection in complex mixtures. To maximize virus particle binding we constructed a disposable device with a constant flow rate of ∼3 μl min -1 . Biosafety was achieved by having a sealable sample addition port. Using this platform we were able to readily identify virus binding in a 20 minute experiment. Sensitivity was comparable to laboratory-based assays such as ELISA and plaque assay, and showed equal or better sensitivity against paper-based assays designed for point-of-need use. Our results demonstrate a platform that can be used for rapid multiplexed detection and visualization of whole virus particles. We envision this technology as a sample-to-answer platform for detection and visualization of viruses without the need for prior labeling. This would enable both research investigation of virus particle behavior and morphology and have the potential to be used in a diagnostic context, where direct imaging from samples such as blood and urine would be valuable.

Protein microarrays are emerging tools which have become very powerful in multiplexed detection technologies. A variety of proteins can be immobilized on a sensor chip allowing for multiplexed diagnostics. Therefore, various types of analyte in a small volume of sample can be detected simultaneously. Protein immobilization is a crucial step for creating a robust and sensitive protein microarray-based detection system. In order to achieve a successful protein immobilization and preserve the activity of the proteins after immobilization, DNA-directed immobilization is a promising technique. Here, we present the design and the use of DNA-directed immobilized (DDI) antibodies in fabrication of robust protein microarrays. We focus on application of protein microarrays for capturing and detecting nanoparticles such as intact viruses. Experimental results on Single-particle interferometric reflectance imaging sensor (SP-IRIS) are used to validate the advantages of the DDI method.

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30-100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders., Competing Interests: The authors declare the following competing financial interest(s): George G. Daaboul is the chief scientific officer of Nexgenarrays LLC. David Freedman is the chief executive officer of NexgenArrays LLC. All remaining contributing authors declare no competing financial interests.

A critical requirement for controlling outbreaks of viral infection is sensitive and accurate diagnostics, which can be expensive and are frequently located in resource-intensive clinical laboratories. Outbreaks of many viral infections occur in countries where healthcare resources are limited and clinical laboratories scarce. This creates a fulfillment gap, one that could be filled through the development of inexpensive, sensitive, easy to use, and portable diagnostics. Here we describe our efforts to develop a diagnostic technology that detects viruses without needing to label the particle directly. Our approach has the advantage of speed and assay simplicity while maintaining high sensitivity. Essential in this approach has been the assembly of an integrated, diverse, and interdisciplinary team that worked together to evaluate technologies, spin-out a company, and produce a product for infectious disease diagnostics. The synergy of different individuals with complementary skills has been critical for the development of our transformative technology.

Interferometric imaging schemes have gained significant interest due to their superior sensitivity over imaging techniques that are solely based on scattered signal. In this study, we outline the theoretical foundations of imaging and characterization of single nanoparticles in an interferometric microscopy scheme, examine key parameters that influence the signal, and benchmark the model against experimental findings.

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.

Label-free imaging of individual viruses and nanoparticles directly in complex solutions is important for virology research and biosensing applications. A successful visualization technique should be rapid, sensitive, and inexpensive, while needing minimal sample preparation or user expertise. Current approaches typically require fluorescent labeling or the use of an electron microscope, which are expensive and time-consuming to use. We have developed an imaging technique for real-time, sensitive, and label-free visualization of viruses and nanoparticles directly in complex solutions such as serum. By combining the advantages of a single-particle reflectance imaging sensor, with microfluidics, we perform real-time digital detection of individual 100 nm vesicular stomatitis viruses as they bind to an antibody microarray. Using this approach, we have shown capture and visualization of a recombinant vesicular stomatitis virus Ebola model (rVSV-ZEBOV) at 100 PFU/mL in undiluted fetal bovine serum in less than 30 min.

Extracellular neural recording, with multi-electrode arrays (MEAs), is a powerful method used to study neural function at the network level. However, in a high density array, it can be costly and time consuming to integrate the active circuit with the expensive electrodes. In this paper, we present a 4 mm × 4 mm neural recording integrated circuit (IC) chip, utilizing IBM C4 bumps as recording electrodes, which enable a seamless active chip and electrode integration. The IC chip was designed and fabricated in a 0.13 μm BiCMOS process for both in vitro and in vivo applications. It has an input-referred noise of 4.6 μV rms for the bandwidth of 10 Hz to 10 kHz and a power dissipation of 11.25 mW at 2.5 V, or 43.9 μW per input channel. This prototype is scalable for implementing larger number and higher density electrode arrays. To validate the functionality of the chip, electrical testing results and acute in vivo recordings from a rat barrel cortex are presented.

Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications.

Over the last decade, the growing need in disease diagnostics has stimulated rapid development of new technologies with unprecedented capabilities. Recent emerging infectious diseases and epidemics have revealed the shortcomings of existing diagnostics tools, and the necessity for further improvements. Optical biosensors can lay the foundations for future generation diagnostics by providing means to detect biomarkers in a highly sensitive, specific, quantitative and multiplexed fashion. Here, we review an optical sensing technology, Interferometric Reflectance Imaging Sensor (IRIS), and the relevant features of this multifunctional platform for quantitative, label-free and dynamic detection. We discuss two distinct modalities for IRIS: (i) low-magnification (ensemble biomolecular mass measurements) and (ii) high-magnification (digital detection of individual nanoparticles) along with their applications, including label-free detection of multiplexed protein chips, measurement of single nucleotide polymorphism, quantification of transcription factor DNA binding, and high sensitivity digital sensing and characterization of nanoparticles and viruses.

Resolution improvement through signal processing techniques for integrated circuit imaging is becoming more crucial as the rapid decrease in integrated circuit dimensions continues. Although there is a significant effort to push the limits of optical resolution for backside fault analysis through the use of solid immersion lenses, higher order laser beams, and beam apodization, signal processing techniques are required for additional improvement. In this work, we propose a sparse image reconstruction framework which couples overcomplete dictionary-based representation with a physics-based forward model to improve resolution and localization accuracy in high numerical aperture confocal microscopy systems for backside optical integrated circuit analysis. The effectiveness of the framework is demonstrated on experimental data.

Single-molecule detection and counting is the new frontier in biomarker analysis. Here, we report on recent techniques for the digital detection of biomolecules for clinical application. First we highlight methods based on the immunocapture of proteins onto microparticles, followed by isolation of individual particles in microenvironments so that a sufficient signal is acquired for each binding event to make a binary decision, thus dramatically enhancing the signal:noise ratio. Various approaches are categorized based on the method used for particle confinement in an isolated microenvironment. We go on to describe methods for the detection of individual biological nanoparticles as well as the digital detection of proteins by artificial nanoparticle labels. The discussion of the methods emphasizes the practical considerations and their clinical applicability., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Efficient graph-cut methods have been used with great success for labeling and denoising problems occurring in computer vision. Unfortunately, the presence of linear image mappings has prevented the use of these techniques in most discrete-amplitude image reconstruction problems. In this paper, we develop a graph-cut based framework for the direct solution of discrete amplitude linear image reconstruction problems cast as regularized energy function minimizations. We first analyze the structure of discrete linear inverse problem cost functions to show that the obstacle to the application of graph-cut methods to their solution is the variable mixing caused by the presence of the linear sensing operator. We then propose to use a surrogate energy functional that overcomes the challenges imposed by the sensing operator yet can be utilized efficiently in existing graph-cut frameworks. We use this surrogate energy functional to devise a monotonic iterative algorithm for the solution of discrete valued inverse problems. We first provide experiments using local convolutional operators and show the robustness of the proposed technique to noise and stability to changes in regularization parameter. Then we focus on nonlocal, tomographic examples where we consider limited-angle data problems. We compare our technique with state-of-the-art discrete and continuous image reconstruction techniques. Experiments show that the proposed method outperforms state-of-the-art techniques in challenging scenarios involving discrete valued unknowns.

Quantitative determination of the density and conformation of DNA molecules tethered to the surface can help optimize and understand DNA nanosensors and nanodevices, which use conformational or motional changes of surface-immobilized DNA for detection or actuation. We present an interferometric sensing platform that combines (i) dual-color fluorescence spectroscopy for precise axial co-localization of two fluorophores attached at different nucleotides of surface-immobilized DNA molecules and (ii) independent label-free quantification of biomolecule surface density at the same site. Using this platform, we examined the conformation of DNA molecules immobilized on a three-dimensional polymeric surface and demonstrated simultaneous detection of DNA conformational change and binding in real-time. These results demonstrate that independent quantification of both surface density and molecular nanoscale conformation constitutes a versatile approach for nanoscale solid-biochemical interface investigations and molecular binding assays.

Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.