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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.

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.

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.

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.

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.

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.

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.

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.

Monitoring cytokine release by cells allows the investigation of cellular response to specific external stimuli, such as pathogens or candidate drugs. Unlike conventional colorimetric techniques, label-free detection of cytokines enables studying cellular secretions in real time by eliminating additional wash and labeling steps after the binding step. However, label-free techniques that are based on measuring mass accumulation on a sensor surface are challenging for measuring small cytokines binding to much larger capture agents (usually antibodies) because the relative signal change is small. This problem is exacerbated when the capturing antibodies desorb from the surface, a phenomenon that almost inevitably occurs in immunoassays but is rarely accounted for. Here, we demonstrate a quantitative dynamic detection of interleukine-6 (IL-6), a pro-inflammatory cytokine, using an interferometric reflectance imaging sensor (IRIS). We improved the accuracy of the quantitative analysis of this relatively small protein (21 kDa) by characterizing the antibody desorption rate and compensating for the antibody loss during the binding experiment. By correcting for protein desorption, we achieved an analytical limit of detection at 19 ng/mL IL-6 concentration. We enhanced the sensitivity by 7-fold by using detection antibodies that recognize a different epitope of the cytokine. We demonstrate that these detection antibodies, which we call "mass tags", can be used concurrently with the target analyte to eliminate an additional wash and binding step. Finally, we report successful label-free detection of IL-6 in cell culture medium (with 10% serum) with comparable signal to that obtained in PBS. This work is the first to report quantitative dynamic label-free detection of small protein in a complex biological fluid using IRIS.

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.

Rapid, chip-scale, and cost-effective single particle detection of biological agents is of great importance to human health and national security. We report real-time, high-throughput detection and sizing of individual, low-index polystyrene nanoparticles and H1N1 virus. Our widefield, common path interferometer detects nanoparticles and viruses over a very large sensing area, orders of magnitude larger than competing techniques. We demonstrate nanoparticle detection and sizing down to 70 nm in diameter. We clearly size discriminate nanoparticles with diameters of 70, 100, 150, and 200 nm. We also demonstrate detection and size characterization of hundreds of individual H1N1 viruses in a single experiment.