A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

A rapid and sensitive aptamer-based biosensor for beta-lactoglobulin in milk.

Beta-lactoglobulin (ß-Lg), a prominent milk protein, is a major contributor to milk allergies. The quantitative assessment of ß-Lg is a valuable method for assessing the allergenic potential of dairy products. In this study, a specific aptamer, ß-Lg-01, with an affinity constant (KD) of 28.6 nM for ß-Lg was screened through seven rounds of magnetic bead SELEX (MB-SELEX). A novel bio-layer interferometry (BLI)-based aptasensor was developed, which had a limit of detection (LOD) of 0.3 ng mL-1, a linear range of 1.5 ng mL-1-15 µg mL-1, and a recovery rate of 102-116% among the milk samples. This aptasensor provides a potential tool for the detection and risk assessment of ß-Lg within 10 min.

Influence of Aberration-Free, Narrowband Light on the Choroidal Thickness and Eye Length.

Purpose: To determine whether light chromaticity without defocus induced by longitudinal chromatic aberration (LCA) is sufficient to regulate eye growth. Methods: An interferometric setup based on a spatial light modulator was used to illuminate the dominant eyes of 23 participants for 30 minutes with three aberration-free stimulation conditions: (1) short wavelength (450 nm), (2) long wavelength (638 nm), and (3) broadband light (450-700 nm), covering a retinal area of 12°. The non-dominant eye was occluded and remained as the control eye. Axial length and choroidal thickness were measured before and after the illumination period. Results: Axial length increased significantly from baseline for short-wavelength (P < 0.01, 7.4 ± 2.2 µm) and long-wavelength (P = 0.01, 4.8 ± 1.7 µm) light. The broadband condition also showed an increase in axial length with no significance (P = 0.08, 5.1 ± 3.5 µm). The choroidal thickness significantly decreased in the case of long-wavelength light (P < 0.01, -5.7 ± 2.2 µm), but there was no significant change after short-wavelength and broadband illumination. The axial length and choroidal thickness did not differ significantly between the test and control eyes or between the illumination conditions (all P > 0.05). Also, the illuminated versus non-illuminated choroidal zone did not show a significant difference (all P > 0.05). Conclusions: All stimulation conditions with short- and long-wavelength light and broadband light led to axial elongation and choroidal thinning. Therefore, light chromaticity without defocus induced by LCA is suggested to be insufficient to regulate eye growth. Translational Relevance: This study helps in understanding if light chromaticity alone is a sufficient regulator of eye growth.

Effect of sound-induced vibrations of the pinna on head-related transfer functions: Experimental and numerical investigations.

Numerical simulations of head-related transfer functions (HRTFs) conventionally assume a rigid boundary condition for the pinna. The human pinna, however, is an elastic deformable body that can vibrate due to incident acoustic waves. This work investigates how sound-induced vibrations of the pinna can affect simulated HRTF magnitudes. The work will motivate the research question by measuring the sound-induced vibrational patterns of an artificial pinna with a high-speed holographic interferometric system. Then, finite element simulations are used to determine HRTFs for a tabletop model of the B&K 5128 head and torso simulator for a number of directions. Two scenarios are explored: one where the pinna is modeled as perfectly rigid, and another where the pinna is modeled as linear elastic with material properties close to that of auricular cartilage. The findings suggest that pinna vibrations have negligible effects on HRTF magnitudes up to 5 kHz. The same conclusion, albeit with less certainty, is drawn for higher frequencies. Finally, the importance of the elastic domain's material properties is emphasized and possible implications for validation studies on dummy heads 1as well as the limitations of the present work are discussed in detail.

Discovery of drug targets based on traditional Chinese medicine microspheres (TCM-MPs) fishing strategy combined with bio-layer interferometry (BLI) technology.

Target discovery of natural products is a key step in the development of new drugs, and it is also a difficult speed-limiting step. In this study, a traditional Chinese medicine microspheres (TCM-MPs) target fishing strategy was developed to discover the key drug targets from complex system. The microspheres are composed of Fe3O4 magnetic nanolayer, oleic acid modified layer, the photoaffinity group (4- [3-(Trifluoromethyl)-3H-diazirin-3-yl] benzoic acid, TAD) layer and active small molecule layer from inside to outside. TAD produces highly reactive carbene under ultraviolet light, which can realize the self-assembly and fixation of drug active small molecules with non-selective properties. Here, taking Shenqi Jiangtang Granules (SJG) as an example, the constructed TCM-MPs was used to fish the related proteins of human glomerular mesangial cells (HMCs) lysate. 28 differential proteins were screened. According to the target analysis based on bioinformatics, GNAS was selected as the key target, which participated in insulin secretion and cAMP signaling pathway. To further verify the interaction effect of GNAS and small molecules, a reverse fishing technique was established based on bio-layer interferometry (BLI) coupled with UHPLC-Q/TOF-MS/MS. The results displayed that 26 small molecules may potentially interact with GNAS, and 7 of them were found to have strong binding activity. In vitro experiments for HMCs have shown that 7 active compounds can significantly activate the cAMP pathway by binding to GNAS. The developed TCM-MPs target fishing strategy combined with BLI reverse fishing technology to screen out key proteins that directly interact with active ingredients from complex target protein systems is significant for the discovery of drug targets for complex systems of TCM.

Protein-Protein Binding Kinetics by Biolayer Interferometry.

The specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, immobilization, the amount of sample required, the number of measurements that can be accomplished simultaneously, or the cost. Bio-Layer Interferometry (BLI) is a label-free biophysical method to measure the kinetics of protein-protein interactions. Label-free interaction assays are a broad family of methods that do not require protein modifications (other than immobilization) or labels such as fusions with fluorescent proteins or transactivating domains or chemical modifications like biotinylation or reaction with radionuclides. Besides BLI, other label-free techniques that are widely used for determining protein-protein interactions include surface plasmon resonance (SPR), thermophoresis, and isothermal titration calorimetry (ITC), among others.

Advancing the study of protein-G4 interactions in DNA repair: Insights from biolayer interferometry.

Biolayer interferometry (BLI) is a powerful tool that enables direct observations of protein-G4 interactions in real-time. In this article, we discuss the crucial aspects in conducting a BLI experiment by using the TAR DNA-binding protein (TDP43) and a G4 DNA formed by (GGGGCC)4 as a sample application. We also describe the necessary precautions in designing the DNA substrate and evaluating the signal contributions arising from nonspecific binding interactions. A comprehensive guide is included that details the necessary materials and reagents, experimental procedures, and data analysis methods for researchers who are interested in using BLI for similar studies. The insights provided in this article will allow researchers to harness the potential of BLI and unravel the complexities of protein-G4 interactions with precision and confidence.

Analyzer-free hard x-ray interferometry.

Objective. To enable practical interferometry-based phase contrast CT using standard incoherent x-ray sources, we propose an imaging system where the analyzer grating is replaced by a high-resolution detector. Since there is no need to perform multiple exposures (with the analyzer grating at different positions) at each scan angle, this scheme is compatible with continuous-rotation CT apparatus, and has the potential to reduce patient radiation dose and patient motion artifacts.Approach. Grating-based x-ray interferometry is a well-studied technique for imaging soft tissues and highly scattering objects embedded in such tissues. In addition to the traditional x-ray absorption-based image, this technique allows reconstruction of the object phase and small-angle scattering information. When using conventional incoherent, polychromatic, hard x-ray tubes as sources, three gratings are usually employed. To sufficiently resolve the pattern generated in these interferometers with contemporary x-ray detectors, an analyzer grating is used, and consequently multiple images need to be acquired for each view angle. This adds complexity to the imaging system, slows image acquisition and thus increases sensitivity to patient motion, and is not dose efficient. By simulating image formation based on wave propagation, and proposing a novel phase retrieval algorithm based on a virtual grating, we assess the potential of a analyzer-grating-free system to overcome these limitations.Main results. We demonstrate that the removal of the analyzer-grating can produce equal image contrast-to-noise ratio at reduced dose (by a factor of 5), without prolonging scan duration.Significance.By demonstrating that an analyzer-free CT system, in conjuction with an efficient phase retrieval algorithm, can overcome the prohibitive dose and workflow penalties associated grating-stepping, an alternative path towards realizing clinical inteferometric CT appears possible.

Tapered Fiber Bioprobe Based on U-Shaped Fiber Transmission for Immunoassay.

In this paper, a tapered fiber bioprobe based on Mach-Zehnder interference (MZI) is proposed. To retain the highly sensitive straight-tapered fiber MZI sensing structure, we designed a U-shaped transmission fiber structure for the collection of optical sensing signals to achieve a miniature-insert-probe design. The spectrum responses from the conventional straight-tapered fiber MZI sensor and our proposed sensor were compared and analyzed, and experimental results showed that our proposed sensor not only has the same sensing capability as the straight-tapered fiber sensor, but also has the advantages of being flexible, convenient, and less liquid-consuming, which are attributed to the inserted probe design. The tapered fiber bioprobe obtained a sensitivity of 1611.27 nm/RIU in the refractive index detection range of 1.3326-1.3414. Finally, immunoassays for different concentrations of human immunoglobulin G were achieved with the tapered fiber bioprobe through surface functionalization, and the detection limit was 45 ng/mL. Our tapered fiber bioprobe has the insert-probe advantages of simpleness, convenience, and fast operation. Simultaneously, it is low-cost, highly sensitive, and has a low detection limit, which means it has potential applications in immunoassays and early medical diagnosis.

High energy x-ray Talbot-Lau interferometer employing a microarray anode-structured target source to extend the field of view.

Objective. High energy and large field of view (FOV) phase contrast imaging is crucial for biological and even medical applications. Although some works have devoted to achieving a large FOV at high energy through bending gratings and so on, which would be extremely challenging in medical high energy imaging.Approach.We analyze the angular shadowing effect of planar gratings in high-energy x-ray Talbot-Lau interferometer (XTLI). Then we design and develop an inverse XTLI coupled with a microarray anode-structured target source to extend the FOV at high energy.Main results.Our experimental results demonstrate the benefit of the source in the inverse XTLI and a large FOV of 106.6 mm in the horizontal direction is achieved at 40 keV. Based on this system, experiments of a mouse demonstrate the potential advantage of phase contrast mode in imaging lung tissue.Significance.We extend the FOV in a compact XTLI using a microarray anode-structured target source coupled with an inverse geometry, which eliminates grating G0 and relaxes the fabrication difficulty of G2. We believe the established design idea and imaging system would facilitate the wide applications of XTLI in high energy phase contrast imaging.

Comparison of the ocular ultrasonic and optical biometry devices in the different quality measurements.

PURPOSE: To compare the reliability and agreement of axial length (AL), anterior chamber depth (ACD), and lens thickness (LT) measurements obtained with optical biometry based on swept-source optical coherence tomography (IOLMaster 700; Carl Zeiss, Germany) and an ultrasound biometry device (Nidek; US-4000 Echoscan, Japan) in different qualities of AL measurement. METHODS: A total of 239 consecutive eyes of 239 cataract surgery candidates with a mean age of 56 ± 14 years were included. The quality measurements were grouped according to the quartiles of SD of the measured AL by IOLMaster 700. The first and fourth quartile's SD are defined as high and low-quality measurement, respectively, and the second and third quartiles' SD is defined as moderate-quality. RESULTS: The reliability of AL and ACD between the two devices in all patients and in different quality measurement groups was excellent with highly statistically significant (AL: all ICC=0.999 and P<0.001, ACD: all ICC>0.920 and P<0.001). AL and ACD in all quality measurements showed a very strong correlation between devices with highly statistically significant. However, there was poor (ICC=0.305), moderate (ICC=0.742), and good (ICC=0.843) reliability in measuring LT in low-, moderate-, and high-quality measurements, respectively. LT showed a very strong correlation (r = 0.854) with highly statistically significant (P<0.001) between devices only in patients with high-quality measurements. CONCLUSIONS: AL and ACD of the IOLMaster700 had outstanding agreements with the US-4000 ultrasound in different quality measurements of AL and can be used interchangeably. But LT should be used interchangeably cautiously only in the high-quality measurements group.

Atom Interferometric Imaging of Differential Potentials Using an Atom Laser.

Interferometry is a prime technique for modern precision measurements. Atoms, unlike light, have significant interactions with electric, magnetic, and gravitational fields, making their use in interferometric applications particularly versatile. Here, we demonstrate atom interferometry to image optical and magnetic potential landscapes over an area exceeding 240 µm×600 µm. The differential potentials employed in our experiments generate phase imprints in an atom laser that are made visible through a Ramsey pulse sequence. We further demonstrate how advanced pulse sequences can enhance desired imaging features, e.g., to image steep potential gradients. A theoretical discussion is presented that provides a semiclassical analysis and matching numerics.

Towards virtual histology with X-ray grating interferometry.

Breast cancer is the most common type of cancer worldwide. Diagnosing breast cancer relies on clinical examination, imaging and biopsy. A core-needle biopsy enables a morphological and biochemical characterization of the cancer and is considered the gold standard for breast cancer diagnosis. A histopathological examination uses high-resolution microscopes with outstanding contrast in the 2D plane, but the spatial resolution in the third, Z-direction, is reduced. In the present paper, we propose two high-resolution table-top systems for phase-contrast X-ray tomography of soft-tissue samples. The first system implements a classical Talbot-Lau interferometer and allows to perform ex-vivo imaging of human breast samples with a voxel size of 5.57 µm. The second system with a comparable voxel size relies on a Sigray MAAST X-ray source with structured anode. For the first time, we demonstrate the applicability of the latter to perform X-ray imaging of human breast specimens with ductal carcinoma in-situ. We assessed image quality of both setups and compared it to histology. We showed that both setups made it possible to target internal features of breast specimens with better resolution and contrast than previously achieved, demonstrating that grating-based phase-contrast X-ray CT could be a complementary tool for clinical histopathology.

Modified Mach-Zehnder interferometer for spatial coherence measurement.

Spatial coherence of light sources is usually obtained by using the classical Young's interferometer. Although the original experiment was improved upon in successive works, some drawbacks still remain. For example, several pairs of points must be used to obtain the complex coherence degree (normalized first-order correlation function) of the source. In this work, a modified Mach-Zehnder interferometer which includes a pair of lenses and is able to measure the spatial coherence degree is presented. With this modified Mach-Zehnder interferometer, it is possible to measure the full 4D spatial coherence function by displacing the incoming beam laterally. To test it, we have measured only a 2D projection (zero shear) of the 4D spatial coherence, which is enough to characterize some types of sources. The setup has no movable parts, making it robust and portable. To test it, the two-dimensional spatial coherence of a high-speed laser with two cavities was measured for different pulse energy values. We observe from the experimental measurements that the complex degree of coherence changes with the selected output energy. Both laser cavities seem to have similar complex coherence degrees for the maximum energy, although it is not symmetrical. Thus, this analysis will allow us to determine the best configuration of the double-cavity laser for interferometric applications. Furthermore, the proposed approach can be applied to any other light sources.

Epitope Binning of Monoclonal and Polyclonal Antibodies by Biolayer Interferometry.

Understanding the epitopes of antibodies elicited by infection and vaccination is often useful in immunogen design. In this chapter, we describe biolayer interferometry (BLI)-based methods to evaluate such epitopes and permit simultaneous analysis of antibodies from several sources, including monoclonal antibodies (mAbs) and polyclonal serum antibodies (pAbs). Using previously characterized antibodies with known epitopes as controls, the distribution of epitopes for the influenza hemagglutinin (HA) is shown for isolated human mAbs and pooled serum from HA-immunized mice. This method is versatile, high-throughput, and can be adapted to several antigens.

Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer interferometry.

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free expression as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

Spectrally Selective Time-Resolved Emission through Fourier-Filtering (STEF).

We demonstrate a method for separating and resolving the dynamics of multiple emitters without the use of conventional filters. By directing the photon emission through a fixed path-length imbalanced Mach-Zehnder interferometer, we interferometrically cancel (or enhance) certain spectral signatures corresponding to one emissive species. Our approach, Spectrally selective Time-resolved Emission through Fourier-filtering (STEF), leverages the detection and subtraction of both outputs of a tuned Mach-Zehnder interferometer, which can be combined with time-correlated single photon counting (TCSPC) or confocal imaging to demix multiple emitter signatures. We develop a procedure to calibrate out imperfections in Mach-Zehnder interferometry schemes. Additionally, we demonstrate the range and utility of STEF by performing the following procedures with one measurement: (1) filtering out laser scatter from a sample, (2) separating and measuring a fluorescence lifetime from a binary chromophore mixture with overlapped emission spectra, (3) confocally imaging and separately resolving the standard fluorescent stains in bovine pulmonary endothelial cells and nearly overlapping fluorescent stains on RAW 264.7 cells. This form of spectral balancing can allow for robust and tunable signal sorting.

Modeling Vibrations of a Tiled Talbot-Lau Interferometer on a Clinical CT.

X-ray computed tomography (CT) is an invaluable imaging technique for non-invasive medical diagnosis. However, for soft tissue in the human body the difference in attenuation is inherently small. Grating-based X-ray phase-contrast is a relatively novel imaging method which detects additional interaction mechanisms between photons and matter, namely refraction and small-angle scattering, to generate additional images with different contrast. The experimental setup involves a Talbot-Lau interferometer whose susceptibility to mechanical vibrations hindered acquisition schemes suitable for clinical routine in the past. We present a processing pipeline to identify spatially and temporally variable fluctuations occurring in an interferometer installed on a continuously rotating clinical CT gantry. The correlations of the vibrations in the modular grating setup are exploited to identify a small number of relevant fluctuation modes, allowing for a sample reconstruction free of vibration artifacts.

Ultrasensitive deafness gene DNA hybridization detection employing a fiber optic Mach-Zehnder interferometer: Enabled by a black phosphorus nanointerface.

The rapid and efficient detection of deafness gene DNA plays an important role in the clinical diagnosis of deafness diseases. This study demonstrates the ultrasensitive detection of complementary DNA (cDNA) by employing a nanointerface-sensitized fiber optic biosensor. The sensor consists of SMF-TNCF-MMF-SMF (abbreviated as STMS) structure with lateral offset. Besides, it is functionalized with a nanointerface of black phosphorus (BP) to enhance the light-matter interaction and eventually improve the sensing performances. Relying on this nanointerface-sensitized sensor, we successfully realize the in-situ detection of cDNA at concentrations ranging from 1 pM to 1 µM, with a sensitivity of 0.719 nm/lgM. The limit of detection (LOD) is as low as 0.24 pM, which is at least two orders of magnitude lower than those of existing methods. The sensor exhibits the advantages of simple operation, fast response, label-free measurement, excellent repeatability, and high selectivity. Our contribution suggests a convenient approach for deafness gene DNA detection and can be extended for general ultra-low concentration DNA detection applications.

Technical Design Considerations of a Human-Scale Talbot-Lau Interferometer for Dark-Field CT.

Computed tomography (CT) as an important clinical diagnostics method can profit from extension with dark-field imaging, as it is currently restricted to X-rays' attenuation contrast only. Dark-field imaging allows access to more tissue properties, such as micro-structural texture or porosity. The up-scaling process to clinical scale is complex because several design constraints must be considered. The two most important ones are that the finest grating is limited by current manufacturing technology to a [Formula: see text] period and that the interferometer should fit into the CT gantry with minimal modifications only. In this work we discuss why an inverse interferometer and a triangular G1 profile are advantageous and make a compact and sensitive interferometer implementation feasible. Our evaluation of the triangular grating profile reveals a deviation in the interference pattern compared to standard grating profiles, which must be considered in the subsequent data processing. An analysis of the grating orientation demonstrates that currently only a vertical layout can be combined with cylindrical bending of the gratings. We also provide an in-depth discussion, including a new simulation approach, of the impact of the extended X-ray source spot which can lead to large performance loss and present supporting experimental results. This analysis reveals a vastly increased sensitivity to geometry and grating period deviations, which must be considered early in the system design process.