Enzyme detection has broad clinical and research applications. Both enzyme activity and concentration are important metrics, but direct comparison typically requires two separate assays. Previously, we demonstrated a multiplexed photonics-based diagnostic sensor that measured both cathepsin-L concentration and enzymatic activity in a single assay-the first such dual-measurement assay(1). The approach is generalizable to other enzymes and can be adapted during assay development. A single assay that distinguishes changes in enzyme concentration from reductions in activity enables new experimental and diagnostic applications. Here we present a general method for dual measurement of enzymes using multiplexed photonic integrated circuits and show preliminary data for collagenase detection. These results highlight the potential of photonic biosensors for multiplexed enzymology in translational and diagnostic contexts.

Von Willebrand Disease (VWD) is characterized by improper blood clotting, resulting from qualitative or quantitative changes in Von Willebrand factor (VWF). Diagnosis of VWD currently relies on measuring both the concentration of VWF and its activity by the binding ability to several different proteins, each of which are currently quantified separately. As such, the current diagnosis of VWD is complex and expensive, requiring multiple tests for a positive clinical determination. To address this challenge, we report a multiplexed biosensor that simultaneously measures VWF concentration and binding activity in plasma, enabling rapid diagnosis of VWD and discrimination among multiple subtypes. Using an 18-plex photonic ring resonator in a disposable, lateral flow assay-like format as the core technology, capture of VWF by an immobilized monoclonal antibody results in a red shift in resonance, which is referenced to a nonspecific binding control. Other ring resonators on the chip, functionalized with binding partners of VWF, allow simultaneous measurement of VWF binding to collagen, Factor VIII, and the GP1b receptor. Evaluation of a panel of 37 single-donor human plasma samples previously analyzed using FDA clinically approved assays demonstrated that the sensor has comparable concentration results and was able to accurately identify several categories of VWD (type 1, 2 A, and type 3).

Enzyme assays are a cornerstone of basic biology and clinical diagnosis. Typically, enzyme activity is measured, but concentration of the enzyme is also of interest, as are comparisons between concentration and activity. In these situations, separate concentration (i.e., ELISA) and activity (i.e., absorbance) assays are required to fully quantify. Here, we report a multiplex disposable photonic biosensor for simultaneous measurement of enzyme activity and concentration. Capture of the enzyme by a ring resonator-bound antibody produces a red shift in resonance, which can be referenced to a nonspecific binding control. At the same time, enzyme-mediated degradation of a ring-bound substrate produces a resonance blue shift, which can be referenced to a peptide inert to enzymatic cleavage. We tested the dual assay with human Cathepsin-L, dysfunction of which is a hallmark of several diseases, including COVID-19, kidney failure, and cancer. Both assays were found to be well-behaved analytically, with lower limits of detection of 2.0 ng·mL-1 (concentration) and 1.8 ng·mL-1 (activity), well within the range clinically relevant concentrations. Further assessment with a panel of 25 single-donor human serum samples confirmed utility of the assay in a complex, biologically relevant matrix. This approach therefore serves as a useful method for Cathepsin-L detection, and a prototype for other dual-mode photonic enzyme assays.

Enzyme assays are a cornerstone of basic biology and clinical diagnostics. While enzymatic assays are frequently used to indicate disease,[1] changes in enzyme activity can occur due to inhibitor or activator agents[2] without changing the concentration of the enzyme. ELISA assays can be used for quantification of concentration, but the technique must be performed separately and takes several hours[3]. To address this issue, we have developed the first multiplexed photonic biosensor able to measure enzyme activity and concentration simultaneously.

Since time immemorial, the value of rapidly diagnosing medical conditions in a broad range of environments, ranging from the doctor's office to the home, has been widely recognized. Rapid portable or handheld diagnostics have, however, remained in the realm of science fiction—like the Star Trek "tricorder"—until recently. Because of their utility in chemical and biological sensing, and their advantages of size, weight, and power (SWAP), photonic sensors have many characteristics that suggest they can fulfill the need for widely useful point-of-care (PoC) diagnostics. This chapter reviews the current state-of-the-art for PoC diagnostic technology, including commercially produced systems. We then introduce photonic biosensor platforms, which are being tested as potential PoC diagnostics, including both commercial systems and those currently the subject of academic research. Selected sensor platforms include ring resonators, Mach–Zehnder interferometers, and photonic crystals. We conclude with a discussion of sensor copackaging with microfluidics and possibilities for integration with light sources.

Beyond the optical and analytical performance of the sensor itself, the development of an optical detection tool in response to a pressing research or diagnostic need requires consideration of a host of additional factors. This talk will provide an overview of two photonic sensor systems developed for profiling the human immune response to COVID-19 infection and/or vaccination. One, focused on the design goal of high multiplexing (many targets per sensor), was built on the Arrayed Imaging Reflectometry (AIR) platform. AIR is a free-space optics technique that relies on the creation and target molecule binding-induced disruption of an antireflective coating on the surface of a silicon chip. The second method, focused on low cost and high speed, uses a small (1 x 4 mm) ring resonator photonic chip embedded in a plastic card able to provide passive transport of human samples. This "disposable photonics" platform is able to detect and quantify anti-COVID antibodies in a human sample in a minute, making it attractive for high-throughput testing applications.

Decades of research have shown that biosensors using photonic circuits fabricated using CMOS processes can be highly sensitive, selective, and quantitative. Unfortunately, the cost of these sensors combined with the complexity of sample handling systems has limited the use of such sensors in clinical diagnostics. We present a new "disposable photonics" sensor platform in which rice-sized (1 × 4 mm) silicon nitride ring resonator sensor chips are paired with plastic micropillar fluidic cards for sample handling and optical detection. We demonstrate the utility of the platform in the context of detecting human antibodies to SARS-CoV-2, both in convalescent COVID-19 patients and for subjects undergoing vaccination. Given its ability to provide quantitative data on human samples in a simple, low-cost single-use format, we anticipate that this platform will find broad utility in clinical diagnostics for a broad range of assays.

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. Array-based tools are desirable as methods for assessing broader patterns of antigen-specific responses, as well as providing information on SARS-CoV-2 immunity in the context of pre-existing immunity to other viruses. Also, methods that rapidly and quantitatively detect antibody responses to SARS-CoV-2 antigens using small (fingerstick) quantities of blood are essential for monitoring immunity at a global scale. This talk will describe the development of two optical sensor platforms (Arrayed Imaging Reflectometry, and an integrated photonics platform fabricated at AIM Photonics) for quantifying antibodies to SARS-CoV-2 and other upper respiratory pathogens, and oriented towards the needs of multiplex detection and speed.

Point-of-Care diagnostics are instrumental to patient care and are broadly applied in the clinical setting. The simplest such device is the lateral flow assay, which is used to influence clinical decisions ranging from pregnancy to malarial infection. Lateral flow assays are ubiquitous; however, they are semi-quantitative, require labeled reagents, and are often less sensitive than comparable clinical laboratory technology. We hypothesize that an attractive method to introduce label-free quantification to Point-of-Care diagnostics is to couple them to photonic sensors. Photonic sensors are attractive as biological measurement tools, as they have low size, weight, and power requirements while providing high sensitivity. In this manuscript we describe post-processing of foundry-prepared photonic sensor chips in preparation for integration with a lateral flow format.

Ring resonators fabricated in silicon or silicon nitride constitute one of the most versatile and widely studied platform photonic technologies for biosensing. As part of an effort by AIM Photonics to advance the photonics manufacturing infrastructure of the United States, we have designed, fabricated, and tested a series of silicon nitride ring resonators for biosensing. Optimized designs will be incorporated into the AIM Photonics photonic design kit (PDK) and made available to the broader community. This talk will describe the evolution of our designs and their performance, with a particular focus on the detection of cytokines under microfluidic flow.

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.

Cost-effective Point-of-Care (POC) diagnostics are of considerable interest to modern healthcare. Current POC devices are typically disposable, low-complexity, and qualitative, with quantitation only achievable at significant additional cost. Clinical diagnostic tools in centralized labs provide better quantitation, but are cumbersome, time-inefficient, expensive, and require trained operators. We hypothesized that Si3N4 and SU-8 photoresist ring resonators would allow for quantitative and inexpensive sensing of clinically relevant serum biomarkers. To test this hypothesis, we designed silicon nitride-based ring resonators that were then fabricated at the American Institute for Manufacturing Integrated Photonics (AIM Photonics). We also designed SU-8 polymer ring resonators, and fabricated those using in-house facilities. Single mode waveguides were designed for transverse electric and transverse magnetic polarizations at λ=1550 nm using COMSOL Multiphysics® and PhoeniX OptoDesigner. Devices were addressed by end-fire coupling and characterized by assessing spectral features including quality factor, finesse, and free spectral range. Bulk solution refractive index sensitivity was achieved using sucrose solutions. Specific interaction was shown by spiking C-Reactive Protein (CRP), an indicator of inflammatory response, into fetal bovine serum and identifying concentration dependent wavelength shift. This discussion will focus on device design, characterization, and the ability of silicon photonics to sense clinically relevant biomolecules in the label-free regime.

An effective response to human biowarfare agent exposure events requires the availability of simple, sensitive, reliable, and manufacturable sensing and diagnostic tools. While ring resonators fabricated on a silicon-on-insulator platform have found wide application as enabling components for biosensors, and have even been commercialized successfully, silicon nitride-based ring resonators have received less attention. We hypothesized that silicon nitride would provide both manufacturing and performance advantages over silicon in a biosensing context. To test that hypothesis, we designed a series of silicon nitride ring resonators. Designs were fabricated at the American Institute for Manufacturing Integrated Photonics (AIM Photonics) foundry. We will discuss the design process, optical performance of the manufactured devices, and their use in the label-free detection of biomedically relevant protein targets.