Frontiers in Biological Detection: From Nanosensors to Systems XIV

Recent advances in inkjet-printed optics have created a new class of lens fabrication technique. Dubbed DotLens, a single of which weighs less than 50 mg and occupies a volume less than 50 L. DotLens can be attached onto any smartphone camera akin to a contact lens, and turn the smartphones into a microscope. In this paper, we discuss various operational modes such as brightfield, darkfield, and fluorescence imaging, including counting and phenotyping nanoparticles by their distinct colors. As a portable microscope, this approach opens up opportunities in biomedical, environmental, and citizen science applications.

Whispering Gallery Mode (WGM) microresonators are a powerful class of optical devices in which light is confined within a small volume. These devices offer the advantages of high sensitivity, diversities in their geometries for different applications, and ease of integration with conventional electronic systems. In particular, microbubble resonators are a unique type of WGM device in which the optical and fluidic components are combined into a single system. We have developed a packaged silica microbubble resonator device for biosensing applications. HF etching is used to control the wall thickness and approach to the quasi-droplet regime in the packaged devices.

We experimentally demonstrate an EP-based sensor based on exceptional point(EP) of nanocylinders-loaded silicon microring for single particle detection. The EP is implemented by tailoring the spatial phase difference between the two nanocylinders placed close to the microring. When a nanoparticle is adsorbed onto the surface of the silicon microring, the degeneracy of two eigenvectors of the silicon microring is lifted, leading to mode splitting in the transmission spectrum. The wavelength difference of the split-mode is proportional to the square-root of the perturbation. To the best of our knowledge, this is the first sensor leveraging the EP of a silicon microring for single nanoparticle detection

Hydrogel microlaser utilizing a Fabry-Pérot cavity was demonstrated for biological fluid tracking and quantification. Taking advantage of the enhanced light-molecule interaction induced by the microcavity, subtle changes could be detected via stimulated emission at the nanoscale. By taking advantage of osmosis principle, here we present an optofluidic laser-based biosensor to detect the change in glucose concentration levels by analysing the amplified lasing emission. Both lasing spectra and spatial modes demonstrated the sensitivity and effectiveness of the microlaser. The results show the potential of the hydrogel microlaser array as a novel tool for biomedical analysis of body fluid in the near future.

Detection of biomarkers at low concentrations is essential for early diagnosis of numerous diseases. In many sensitive assays, the target molecules are tagged using fluorescently labeled probes and captured using magnetic beads. Current devices rely on quantifying the target molecules by detecting the fluorescent signal from individual beads. Here, we demonstrate a high-throughput optical modulation biosensing (ht-OMB) system Using the ht-OMB system to detect human Interleukin-8, we demonstrated a limit of detection of 0.14 ng/L and a 4-log dynamic range, values which are on par with the most sensitive devices, but are achieved without their bulk and laborious protocols.

Aflatoxin M1 (AFM1) is a carcinogenic compound routinely found in milk, especially in developing countries. This presentation will discuss the first application of wavelength-scanned cavity attenuated phase shift spectroscopy (WS-CAPS) in fiber cavities for AFM1 sensing. The sensor constitutes a fiber cavity along with a tapered fiber functionalized with DNA aptamers. We use the coupled-mode theory to arrive at mathematical equations for conducting the WS-CAPS measurements. Our demonstrated sensor can detect AFM1 as low as 20 ppt (20 ng/L) in an aqueous solution, which is better than the safety limits imposed by European and USA regulatory bodies.

In this presentation we will present two types of sensors for detecting SARS-CoV-2 symptoms. The first part of the presentation will address a contact-free sensor while its operation principle involves illuminating the inspected subject with a laser beam and analyzing with artificial intelligence (AI) based algorithms, the temporal-spatial changes occurring in the back scattered secondary 2D speckle patterns captured through properly defocused optics. The sensing is performed from a distance of several meters away and is applied to different regions of the subject’s body. We demonstrate measurements performed from the chest and then we extract various cardio-pulmonary bio-sign (several simultaneously) including the sounds of subject’s heart and lungs (like a remote stethoscope). We also perform measurements from the sclera and search for anomalies in the random eye movements. From those anomalies we estimate amount of saturated oxygen in the blood stream. All of the above-mentioned bio-parameters could be useful for remote early detection of SARS-CoV-2 symptoms. The AI algorithms are applied not only to extract the various bio-signs but also to perform the bio-medical diagnosis. In the second part of the presentation, we will present fiber based sensor that is incorporated into textile and clothing and make them a smart-clothing capable via a non-tight contact way to perform sensing of various vital bio-signs (several simultaneously). The bio-parameters to be sensed are related to cardio-pulmonary activity as well as blood-pressure and thus could be associated with early detection of SARS-CoV-2 symptoms. The fiber sensor is based on enhanced multi-mode fiber while at its output an artificial intelligence (AI) based algorithm analyses the temporal-spatial characterizations of the generated dynamic 2D speckle patterns. The fiber sensors are positioned in several locations in the clothing and can perform the bio-measurement from different organs of the wearer and thus allow a comparative measurement which could assist in obtaining more agnostic and more reliable bio-sensing. The AI algorithms are applied not only to extract the various bio-signs but also to perform the bio-medical diagnosis.

Here, we introduce a diagnostic platform for rapid and sensitive clinical diagnosis of COVID-19. Based on the biochemical principles of the RT-qPCR, it utilizes the end-point detection by the magnetic modulation biosensing (MMB) system, allowing the detection of two copies of SARS-CoV-2 in 30 minutes. Testing 309 RNA samples from clinical patients and healthy subjects resulted in 97.8% sensitivity, 100% selectivity, and 0% cross-reactivity. This is on par with the gold standard (RT-qPCR) but requires 1/3 of the time. The platform can be adapted to detect almost any other pathogen of choice.

The outbreak of the coronavirus disease emphasized the need for fast and sensitive inhibitor screening tools for new drug candidates’ identification. We demonstrate a rapid and quantitative method for the detection and classification of different types of molecules as inhibitors or non-inhibitors of the S1-ACE2 interaction using magnetically modulated biosensors (MMB). Inhibition of this interaction may limit the spread of the virus in the body. The MMB-based assay is much faster and has minimal non-specific binding than the commonly used ELISA. It can be adjusted to other interactions, and therefore can be utilized as a global tool for inhibitor screening.

Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in 1 h but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids in 40-60 min. Using SARS-CoV-2 and Epstein-Barr virus (EBV) as a proof-of-concept target, RADICA allows for absolute quantification with high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose a global public health threat. Effective and rapid serological assays are needed to provide valuable information about acute and past viral infections. Using the receptor-binding domain of the SARS-CoV-2 spike protein 1 antigen and a highly sensitive detection technology, termed magnetic modulation biosensing (MMB), we demonstrate a quantitative and rapid SARS-CoV-2 IgG antibody test with high sensitivity and specificity compared with the gold standard ELISA test. The improved analytical and clinical sensitivity of the MMB-based assay can help clinical laboratories provide critical information in a timely manner and monitor the spread of the disease.

Photonic crystals provide unique mechanisms to enhance light-matter interactions and have been widely adopted in optical biosensing. However, traditional photonic crystal biosensors still face significant challenges in cost, repeatability, and accuracy in quantification. Diatoms are unicellular algae in nature which have photonic crystal nanostructures with unique optical properties. In addition, they provide ultra-hydrophilic surface, excellent adsorption capabilities, microfluidic effect, and thin layer chromatography. This talk will provide an overview of our recent research progress in materials synthesis, various applications, and fusion with machine learning for better performance.

We report a system capable of classifying, quantifying and discriminating selected proteins using an array of porous silicon sensors with uniquely tuned properties and a combination of statistical and machine learning techniques. No capture agents or bioreceptors are utilized. The approach relies on differences in non-specific physisorption, and represents a step towards an unprecedented low cost, simple and robust sensor that can detect a vast range of biomolecules.

Infectious diseases are worldwide a major cause of morbidity and mortality. Here, we developed a set of near infrared (NIR) fluorescent nanosensors and used them for remote fingerprinting of clinically important bacteria. The nanosensors are based on single-walled carbon nanotubes (SWCNTs) that fluoresce in the NIR optical tissue transparency window, which offers ultra-low background and high tissue penetration. They are chemically tailored to detect released metabolites as well as specific virulence factors (lipopolysaccharides, siderophores, DNases, proteases) and integrated into functional hydrogel arrays with 9 different sensors. These hydrogels are exposed to clinical isolates of 6 important bacteria (Staphylococcus aureus, Escherichia coli, …) and remote (≥25 cm) NIR imaging allows to identify and distinguish bacteria. Sensors are also spectrally encoded (900 nm, 1000 nm, 1250 nm) to differentiate major pathogens and penetrate tissue (>5 mm).

Ultrabithorax (Ubx) is a recombinant protein derived from Drosophila melanogaster. Ubx self-assembles in aqueous buffers, forming thin surface film, from which microscale fibers and coatings are drawn. Ubx materials are elastic, bio- and cyto-compatible. They can also be functionalized pre- and post-formation by gene fusion and DNA binding to perform a range of functions, such as acting as a fluorescent beacon, binding analytes, or increasing cell proliferation. In this work, DNA aptamers bound to Ubx fibers were used to bind E.coli with a 57 % increase in binding. Upscaling process to create Ubx-based platform materials was assessed by photoluminescence studies.

We will present near-infrared fluorescent single wall carbon nanotube optical reporters that can measure spatially and temporally resolved dynamic behavior of neuromodulatory molecules on scales that elude conventional methods of inquiry. We will present work on the performance of such sensors on catecholinergic neuromodulators such as dopamine. We will additionally present new approaches for deploying such sensors in biological preparations of varying complexity that permit implementation of the sensors in ways that enable highly resolved imaging of synaptic neurochemical efflux.

GFR is till date the most used parameter for diagnosing kidney diseases. Nevertheless, tubular secretion and reabsorption play an equally important role in healthy functioning of the kidney. The primary focus of this study is to synthesise novel fluorescent markers for evaluating the three physiological processes of the kidney. This would help diagnosing function-/location-specific abnormality in kidney. Since the ideal goal would be to measure all the kidney parameters altogether using a transcutaneous device, the absorbance and emission wavelength for each marker is designed to be significantly different. This paves way for a wholesome, rapid, and non-invasive technique for kidney diagnosis.

Bonellin is a green pigment found in the female of echurian worm, Bonellia viridis, which typically lives in sediments or rock crevices at a depth of 3 to 10 m. Bonellin absorbs lights ~640 nm and has been utilized as a sensitizer for photodynamic therapy. Bonellin is a chlorin yet is distinct from the dominant naturally occurring chlorins, the chlorophylls. While historically known at depth in the Mediterranean Sea, at a wide shallow beach in Okinawa, Japan, samples of Bonellia viridis were acquired without diving equipment in the low water of a spring tide.

Near infrared photoimmunotherapy (NIR-PIT) is a new type of molecularly-targeted cancer photo-therapy, which employs a NIR silica-phthalocyanine, IR700, conjugated to a monoclonal antibody targeting cell-surface molecules. NIR-PIT targeting EGFR was approved for clinical use in Japan in September 2020 in late-stage recurrent head and neck squamous cell cancer patients. In this talk, cytotoxic mechanism of NIR-PIT is refocused. Based on chemical and biological mechanism, NIR-PIT could be performed safely under existence of L-NaAA without side-effects caused by unnecessary ROS production that induced harmful focal edema. This result indicates NIR-PIT cytotoxicity relies on photo-induced ligand release reaction rather than ROS.

Actin, as microfilaments in the cytoskeleton, is essential biological structure and mechanical properties for cell migration and division. These processes require new probes for visualization of actin. Fluorescent labeling as a traditional method accompanies photo bleaching and formation of free radicals that are harmful for live cells, resulting to hardly find a balance between more signals of observation and less light exposure. Here, we present new nanoparticle probes for continuous visualization of actin. We demonstrate continuous imaging of different cell division phases to reveal actin biological and mechanical properties.

Exosomes have received much attention as biomarkers for the diagnosis and treatment of various diseases such as diabetic cardiomyopathy and cancer. However, it remains an important challenge to quickly and precisely isolate and detect exosomes from various body fluids. Therefore, we selectively separated exosomes using anti-CD63 antibody-conjugated magnetic nanoparticles (anti-CD63 Ab-MNPs). And, to detect the exosomes optically, an aptamer-conjugated PDA liposome (Apt-PDA) was developed to target EpCAM overexpressed on the surface of cancer-derived exosome. The isolated exosomes were detected through the colorimetric change and the red fluorescence of PDA. We demonstrated simultaneous separation and detection of exosomes by anti-CD63 Ab-MNPs and anti-EpCAM Apt-PDA.

The fluorescence decay of two red fluorescent proteins (mNeptune, mCardinal) were measured as a function of pH. A change in the fluorescence lifetime of 0.5 ns was observed in mCardinal for a pH range of 5,0 to 8,0 while mNeptune exhibited no pH-sensitivity. Hydrogen bond analysis showed a lower occupancy in mCardinal, which results in increased conformational diversity and larger structural changes due to protonation. mCardinal was found to be a bright, pH-sensitive red fluorescent protein which may be used as a molecular probe in deep tissue imaging applications.

Red fluorescent proteins (RFPs) and biosensors built upon them provide an attractive advantage for two-photon laser microscopy because they can report from deeper layers of tissue compared to green fluorescent proteins. Using mCherry RFP we show that although the shorter wavelength excitation (740–800 nm) is several times more efficient compared to longer wavelength excitation (1000–1200 nm), the photobleaching of chromophore occurs much faster for the former. This can be explained by a different photobleaching mechanism: with higher energy-photons, 3-4 photons are sufficient to reach an energy threshold (ionization potential) and photodetach an electron from the chromophore.

Due to its accessibility, hemodynamics can be imaged in early stage quail embryo hearts longitudinally. Our group has linked abnormal shear stress patterns caused by regurgitant blood flow with resultant congenital heart defects (CHDs). To understand the mechanisms behind the development of these CHDs, it is imperative to image molecular expression at the sites of abnormal shear stress. However, molecular probes for the quail model are not extensive and 3D imaging is needed to accurately identify regions of interest in the looping heart. In this study, we present HCR FISH probes that target the shear stress responsive genes

Intracellular drug target validation and target engagement quantification have proven to be challenging, and all drugs have some degree of non-specific accumulation due to variable drug affinity, biodistribution, pharmacokinetics, and metabolism. Quantification of available drug targets necessitates accounting for both the drug that binds to its target as well as the drug that accumulates in the cells and tissues in a non-specific manner. We have developed a dynamic, fluorescence-based, three-compartment model termed intracellular paired-agent imaging that utilizes fluorophore labeled small molecule therapeutics as imaging agents to measure drug target availability in live cells and tissues.