Towards Early Diagnosis and Screening of Alzheimer's Disease Using Frequency Locked Whispering Gallery Mode Microtoroid Biosensors.

Alzheimer's disease (AD) is a progressive form of dementia affecting almost 55 million people worldwide. It is characterized by the abnormal deposition of amyloid plaques and neurofibrillary tangles within the brain, leading to a pathological cascade of neuron degeneration and death as well as memory loss and cognitive decline. Amyloid beta (Aß) is an AD biomarker present in cerebrospinal fluid and blood serum and correlates with the presence of amyloid plaques and tau tangles in the brain. Measuring the levels of Aß can help with early diagnosis of AD, which is key for studying novel AD drugs and delaying the symptoms of dementia. However, this goal is difficult to achieve due to the low levels of AD biomarkers in biofluids. Here we demonstrate for the first time the use of FLOWER (frequency locked optical whispering evanescent resonator) for quantifying the levels of post-mortem cerebrospinal fluid (CSF) Aß42 in clinicopathologically classified control, mild cognitive impairment (MCI), and AD participants. FLOWER is capable of measuring CSF Aß42 (area under curve, AUC = 0.92) with higher diagnostic performance than standard ELISA (AUC = 0.82) and was also able to distinguish between control and MCI samples. Our results demonstrate the capability of FLOWER for screening CSF samples for early diagnosis of Alzheimer's pathology.

Methotrexate Inhibits the Binding of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Receptor Binding Domain to the Host-Cell Angiotensin-Converting Enzyme-2 (ACE-2) Receptor.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus mutates, finding effective drugs becomes more challenging. In this study, we use ultrasensitive frequency locked microtoroid optical resonators in combination with in silico screening to search for COVID-19 drugs that can stop the virus from attaching to the human angiotensin-converting enzyme 2 (hACE2) receptor in the lungs. We found 29 promising candidates that could block the binding site and selected four of them that were likely to bind very strongly. We tested three of these candidates using frequency locked optical whispering evanescent resonator (FLOWER), a label-free sensing method based on microtoroid resonators. FLOWER has previously been used for sensing single macromolecules. Here we show, for the first time, that FLOWER can provide accurate binding affinities and sense the inhibition effect of small molecule drug candidates without labels, which can be prohibitive in drug discovery. One of the candidates, methotrexate, showed binding to the spike protein 1.8 million times greater than that to the receptor binding domain (RBD) binding to hACE2, making it difficult for the virus to enter cells. We tested methotrexate against different variants of the SARS-CoV-2 virus and found that it is effective against all four of the tested variants. People taking methotrexate for other conditions have also shown protection against the original SARS-CoV-2 virus. Normally, it is assumed that methotrexate inhibits the replication and release of the virus. However, our findings suggest that it may also block the virus from entering cells. These studies additionally demonstrate the possibility of extracting candidate ligands from large databases, followed by direct receptor-ligand binding experiments on the best candidates using microtoroid resonators, thus creating a workflow that enables the rapid discovery of new drug candidates for a variety of applications.

Label-free, real-time monitoring of membrane binding events at zeptomolar concentrations using frequency-locked optical microresonators.

Binding events to elements of the cell membrane act as receptors which regulate cellular function and communication and are the targets of many small molecule drug discovery efforts for agonists and antagonists. Conventional techniques to probe these interactions generally require labels and large amounts of receptor to achieve satisfactory sensitivity. Whispering gallery mode microtoroid optical resonators have demonstrated sensitivity to detect single-molecule binding events. Here, we demonstrate the use of frequency-locked optical microtoroids for characterization of membrane interactions in vitro at zeptomolar concentrations using a supported biomimetic membrane. Arrays of microtoroids were produced using photolithography and subsequently modified with a biomimetic membrane, providing high quality (Q) factors (>106) in aqueous environments. Fluorescent recovery after photobleaching (FRAP) experiments confirmed the retained fluidity of the microtoroid supported-lipid membrane with a diffusion coefficient of 3.38±0.26 µm2⋅s-1. Utilizing this frequency-locked membrane-on-a-chip model combined with auto-balanced detection and non-linear post-processing techniques, we demonstrate zeptomolar detection levels The binding of Cholera Toxin B- monosialotetrahexosyl ganglioside (GM1) was monitored in real-time, with an apparent equilibrium dissociation constant kd=1.53 nM. The measured affiny of the agonist dynorphin A 1-13 to the κ-opioid receptor revealed a kd=3.1 nM using the same approach. Radioligand binding competition with dynorphin A 1-13 revealed a kd in agreement (1.1 nM) with the unlabeled method. The biosensing platform reported herein provides a highly sensitive real-time characterization of membrane embedded protein binding kinetics, that is rapid and label-free, for toxin screening and drug discovery, among other applications.

Optical frequency combs in aqueous and air environments at visible to near-IR wavelengths.

The ability to detect and identify molecules at high sensitivity without the use of labels or capture agents is important for medical diagnostics, threat identification, environmental monitoring, and basic science. Microtoroid optical resonators, when combined with noise reduction techniques, have been shown capable of label-free single molecule detection; however, they still require a capture agent and prior knowledge of the target molecule. Optical frequency combs can potentially provide high precision spectroscopic information on molecules within the evanescent field of the microresonator; however, this has not yet been demonstrated in air or aqueous biological sensing. For aqueous solutions in particular, impediments include coupling and thermal instabilities, reduced Q factor, and changes to the mode spectrum. Here we overcome a key challenge toward single-molecule spectroscopy using optical microresonators: the generation of a frequency comb at visible to near-IR wavelengths when immersed in either air or aqueous solution. The required dispersion is achieved via intermodal coupling, which we show is attainable using larger microtoroids, but with the same shape and material that has previously been shown ideal for ultra-high sensitivity biosensing. We believe that the continuous evolution of this platform will allow us in the future to simultaneously detect and identify single molecules in both gas and liquid at any wavelength without the use of labels.

How Fast It Can Stick: Visualizing Flow Delivery to Microtoroid Biosensors.

Sensitive and rapid biosensors are of critical importance for a variety of applications including infectious disease detection and monitoring as well as medical diagnostics and drug discovery. Whispering gallery mode microtoroid optical resonators are among the most sensitive biochemical sensors in existence. When combined with frequency-locking and data-processing techniques, these sensors have been shown to be capable of single-molecule detection in under 30 s. The sensitivity of these sensors is affected by how a concentration of analyte molecules is transported to the surface of the sensors and the average time it takes the molecules to bind at that concentration. Currently, one question in the field is that at these low concentrations, how these microsensors achieve such rapid response times. Here, we reconcile theory and experiment and demonstrate through flow visualization experiments and finite-element simulations that the total analyte arrival and binding time can be on the order of seconds. This fast response time provides an advantage over nanoscale sensors such as nanowires or nanorods. We anticipate that these results can help us to control, with confidence, when and how many molecules bind to these sensors, thus enabling the building of faster and more sensitive sensors.

Wide-angle MEMS-based imaging lidar by decoupled scan axes.

We present an optical architecture for a scanning lidar in which a digital micromirror device (DMD) is placed at an intermediate image plane in a receiver to decouple the trade-offs between scan angle, scan speed, and aperture size of the lidar's transmitter and receiver. In the architecture, the transmitter with a galvo mirror and the receiver with a DMD scan the horizontal and vertical fields of view, respectively, to enable an increased field of view of 50°, centimeter transmitter beam diameter, and video frame rate range finding captures. We present our optimized system and discuss the adjustable parameter trade-offs.

Ultrasensitive Detection of Human Chorionic Gonadotropin Using Frequency Locked Microtoroid Optical Resonators.

Clean sport competition is of significant concern to many governments and sporting organizations. Highly sensitive and rapid sensors are needed to improve the detection of performance enhancing drugs in sports as athletes take diuretics to dilute the concentration of drugs in their urine and microdose under the detectable limits of current sensors. Here we demonstrate, using frequency locked microtoroid optical resonators, a 3 orders of magnitude improvement in detection limit over the current gold standard, mass spectrometry, for the common performance enhancing drug, human chorionic gonadotropin (hCG). hCG, also known as the pregnancy hormone, was detected both in simulated urine and in the urine of pregnant donors at a concentration of 1 and 3 femtomolar, respectively. We anticipate that the sensitivity provided by frequency locked optical microcavities can enable a new standard in antidoping research.

Single chip lidar with discrete beam steering by digital micromirror device.

A novel method of beam steering enables a large field of view and reliable single chip light detection and ranging (lidar) by utilizing a mass-produced digital micromirror device (DMD). Using a short pulsed laser, the micromirrors' rotation is frozen in mid-transition, which forms a programmable blazed grating. The blazed grating efficiently redistributes the light to a single diffraction order, among several. We demonstrated time of flight measurements for five discrete angles using this beam steering method with a nano second 905nm laser and Si avalanche diode. A distance accuracy of < 1 cm over a 1 m distance range, a 48° full field of view, and a measurement rate of 3.34k points/s is demonstrated.