Using Temporally and Spatially Resolved Measurements to Improve the Sensitivity of Fluorescence-Based Immunoassays.

Detecting low concentrations of biomarkers is essential in clinical laboratories. To improve analytical sensitivity, especially in identifying fluorescently labeled molecules, typical optical detection systems, consisting of a photodetector or camera, utilize time-resolved measurements. Taking a different approach, magnetic modulation biosensing (MMB) is a novel technology that combines fluorescently labeled probes and magnetic particles to create a sandwich assay with the target molecules.

Recent Advances in Rapid and Highly Sensitive Detection of Proteins and Specific DNA Sequences Using a Magnetic Modulation Biosensing System.

In early disease stages, biomolecules of interest exist in very low concentrations, presenting a significant challenge for analytical devices and methods. Here, we provide a comprehensive overview of an innovative optical biosensing technology, termed magnetic modulation biosensing (MMB), its biomedical applications, and its ongoing development. In MMB, magnetic beads are attached to fluorescently labeled target molecules.

High throughput optical modulation biosensing for highly sensitive and rapid detection of biomarkers.

Rapid, highly sensitive, and high-throughput detection of biomarkers at low concentrations is invaluable for early diagnosis of various diseases. In many highly sensitive immunoassays, magnetic beads are used to capture fluorescently labeled target molecules. The target molecules are then quantified by detecting the fluorescent signal from individual beads, which is time consuming and requires a complicated and expensive detection system.

PrrT/A, a Pseudomonas aeruginosa Bacterial Encoded Toxin-Antitoxin System Involved in Prophage Regulation and Biofilm Formation.

Toxin-antitoxin (TA) systems are genetic modules that consist of a stable protein-toxin and an unstable antitoxin that neutralizes the toxic effect. In type II TA systems, the antitoxin is a protein that inhibits the toxin by direct binding. Type II TA systems, whose roles and functions are under intensive study, are highly distributed among bacterial chromosomes.

Highly Sensitive and Specific SARS-CoV-2 Serological Assay Using a Magnetic Modulation Biosensing System.

Sensitive serological assays are needed to provide valuable information about acute and past viral infections. For example, detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies could serve as the basis for an "immunity passport" that would enable individuals to travel internationally. Here, utilizing a novel Magnetic Modulation Biosensing (MMB) system and the receptor-binding domain of the SARS-CoV-2 spike protein, we demonstrate a highly sensitive and specific anti-SARS-CoV-2 IgG serological assay.

Rapid Biosensing Method for Detecting Protein-DNA Interactions.

Identifying and investigating protein-DNA interactions, which play significant roles in many biological processes, is essential for basic and clinical research. Current techniques for identification of protein-DNA interactions are laborious, time-consuming, and suffer from nonspecific binding and limited sensitivity. To overcome these challenges and assess protein-DNA interactions, we use a magnetic modulation biosensing (MMB) system.

Optical modulation biosensing system for rapid detection of biological targets at low concentrations.

In many sensitive assays, target molecules are tagged using fluorescently labeled probes and captured using magnetic beads. Here, we introduce an optical modulation biosensing (OMB) system, which aggregates the beads into a small detection area and separates the signal from the background noise by manipulating the laser beam in and out of the cluster of beads. Using the OMB system to detect human interleukin-8, we demonstrated a limit of detection of 0.

A Magnetic Modulation Biosensing-Based Molecular Assay for Rapid and Highly Sensitive Clinical Diagnosis of Coronavirus Disease 2019 (COVID-19).

Rapid and sensitive detection of human pathogens, such as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is an urgent and challenging task for clinical laboratories. Currently, the gold standard for SARS-CoV-2-specific RNA is based on quantitative RT-PCR (RT-qPCR), which relies on target amplification by Taq polymerase and uses a fluorescent resonance energy transfer-based hydrolysis probe. Although this method is accurate and specific, it is also time consuming.

Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors.

Inhibitor screening is an important tool for drug development, especially during the COVID-19 pandemic. The most used in vitro inhibitor screening tool is an enzyme-linked immunosorbent assay (ELISA). However, ELISA-based inhibitor screening is time consuming and has a limited dynamic range.

Characterization of PfiT/PfiA toxin-antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction.

Toxin-antitoxin (TA) systems are small genetic modules usually consisting of two elements-a toxin and an antitoxin. The abundance of TA systems among various bacterial strains may indicate an important evolutionary role. Pseudomonas aeruginosa, which can be found in a variety of niches in nature, is an opportunistic pathogen for various hosts.

Configuration and Design of Electromagnets for Rapid and Precise Manipulation of Magnetic Beads in Biosensing Applications.

Rapid and precise manipulation of magnetic beads on the nano and micro scales is essential in many biosensing applications, such as separating target molecules from background molecules and detecting specific proteins and DNA sequences in plasma. Accurately moving magnetic beads back and forth requires at least two adjustable magnetic field gradients. Unlike permanent magnets, electromagnets are easy to design and can produce strong and adjustable magnetic field gradients without mechanical motion, making them desirable for use in robust and safe medical devices.

Rapid and Sensitive Detection of Repetitive Nucleic Acid Sequences Using Magnetically Modulated Biosensors.

Repetitive DNA sequences are abundant in the genome of most biological species. These sequences are naturally "preamplified", which makes them a preferential target for a variety of biological assays. Current methods to detect specific DNA sequences are based on the quantitative polymerase chain reaction (PCR), which relies on target amplification by polymerase and uses a fluorescent resonance energy transfer (FRET)-based probe.

Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay.

We present a novel assay for rapid and highly sensitive detection of specific nucleic acid fragments in human serum. In a magnetic modulation biosensing (MMB) system, magnetic beads and fluorescently labeled probes are attached to the target analyte and form a "sandwich" complex. An alternating external magnetic field gradient condenses the magnetic beads (and hence the target molecules with the fluorescently labeled probes) to the detection volume and sets them in a periodic motion, in and out of a laser beam.

Improving the Sensitivity of Fluorescence-Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads.

In fluorescence-based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay.

Highly Sensitive and Specific Zika Virus Serological Assays Using a Magnetic Modulation Biosensing System.

Background: Zika virus has created global alarm because it has been associated with catastrophic fetal abnormalities, including microcephaly, spontaneous abortion, and intrauterine growth restriction. Current serological assays that detect antiviral antibodies suffer from low sensitivity and high cross-reactivity among different flaviviruses.

Methods: In this study, utilizing a novel magnetic modulation biosensing (MMB) system and the Zika nonstructural 1 protein, we show highly sensitive and specific Zika serological assays.

Nonlinear photoacoustic spectroscopy of hemoglobin.

As light intensity increases in photoacoustic imaging, the saturation of optical absorption and the temperature dependence of the thermal expansion coefficient result in a measurable nonlinear dependence of the photoacoustic (PA) signal on the excitation pulse fluence. Here, under controlled conditions, we investigate the intensity-dependent photoacoustic signals from oxygenated and deoxygenated hemoglobin at varied optical wavelengths and molecular concentrations. The wavelength and concentration dependencies of the nonlinear PA spectrum are found to be significantly greater in oxygenated hemoglobin than in deoxygenated hemoglobin.

Optical-resolution photoacoustic endomicroscopy in vivo.

Optical-resolution photoacoustic microscopy (OR-PAM) has become a major experimental tool of photoacoustic tomography, with unique imaging capabilities for various biological applications. However, conventional imaging systems are all table-top embodiments, which preclude their use in internal organs. In this study, by applying the OR-PAM concept to our recently developed endoscopic technique, called photoacoustic endoscopy (PAE), we created an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system, which enables internal organ imaging with a much finer resolution than conventional acoustic-resolution PAE systems.

Label-free photoacoustic nanoscopy.

Super-resolution microscopy techniques - capable of overcoming the diffraction limit of light - have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents.

Calibration-free quantification of absolute oxygen saturation based on the dynamics of photoacoustic signals.

Photoacoustic tomography (PAT) is a hybrid imaging technique that has broad preclinical and clinical applications. Based on the photoacoustic effect, PAT directly measures specific optical absorption, which is the product of the tissue-intrinsic optical absorption coefficient and the local optical fluence. Therefore, quantitative PAT, such as absolute oxygen saturation (sO₂) quantification, requires knowledge of the local optical fluence, which can only be estimated through invasive measurements or sophisticated modeling of light transportation.

Functional photoacoustic microscopy of pH.

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, an imbalance of pH regulation may result from or result in serious illness. In this paper, we report photoacoustic microscopy (PAM) of a commercially available pH-sensitive fluorescent dye (SNARF-5F carboxylic acid) in tissue phantoms.

Real-time four-dimensional optical-resolution photoacoustic microscopy with Au nanoparticle-assisted subdiffraction-limit resolution.

Photoacoustic microscopy (PAM) offers label-free, optical absorption contrast. A high-speed, high-resolution PAM system in an inverted microscope configuration with a laser pulse repetition rate of 100,000 Hz and a stationary ultrasonic transducer was built. Four-dimensional in vivo imaging of microcirculation in mouse skin was achieved at 18 three-dimensional volumes per second with repeated two-dimensional (2D) raster scans of 100 by 50 points.

Single-wavelength functional photoacoustic microscopy in biological tissue.

Recently, we developed a reflection-mode relaxation photoacoustic microscope, based on saturation intensity, to measure picosecond relaxation times using a nanosecond laser. Here, using the different relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, the oxygen saturation was quantified in vivo with single-wavelength photoacoustic microscopy. All previous functional photoacoustic microscopy measurements required imaging with multiple-laser-wavelength measurements to quantify oxygen saturation.

Picosecond absorption relaxation measured with nanosecond laser photoacoustics.

Picosecond absorption relaxation-central to many disciplines-is typically measured by ultrafast (femtosecond or picosecond) pump-probe techniques, which however are restricted to optically thin and weakly scattering materials or require artificial sample preparation. Here, we developed a reflection-mode relaxation photoacoustic microscope based on a nanosecond laser and measured picosecond absorption relaxation times. The relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, were measured at 576 nm.