The seasonal outbreaks of respiratory diseases and increasing awareness of personal healthcare drive the development of advanced in vitro diagnostic methods, in order to combat the hospitalization burden and improve patient lives. Respiratory Syncytial Virus (RSV) is historically the major respiratory pathogen in infants and children worldwide. It accounts for ~200,000 deaths per year globally in children below age 5 and is the second-highest cause of death in infants under one year. Current viral assay methods, falling into the broad categories of molecular tests such as polymerase chain reaction, and antigen tests, such as lateral flow assay, either involve delayed testing in a laboratory environment or lack sensitivity.
In the last decades, we have witnessed digital immunoassays as a remarkable conceptual advance that allows us to detect single-molecular profiles. Of a standard operation, they utilize microbeads to capture a single molecule of interest, partition the sample in a microwells array or droplet flow, add substrates for signal amplification, and count the events digitally. While sensitive, it has been largely limited by heavy instrumentations, well-trained medical professionals, and a lengthy assay protocol. Despite a burst of novel paradigms as technology progressed, there remains a substantial unmet need for digital immunoassays paired with rapid turnaround and easy-to-use platforms.
In our group at the University of Texas at Dallas and collaborators at the University of Texas Southwestern Medical Center, we have developed digital plasmonic nanobubble detection, or DIAMOND, to better deliver diagnostic strategies for infectious disease diagnosis. The study has been recently published in Nature Communications.
How does DIAMOND work?
We focus on laser-plasmonic nanoparticle interactions that enable single-nanoparticle detection. Particularly, the laser pulses actuate gold nanoparticles (AuNPs) that can “boil” the surrounding water and nucleate vapor bubbles (termed plasmonic nanobubbles, PNBs) that rapidly grow and collapse. PNBs used to be a surgical technology for in vivo cancer theranostics, as they allow for optical or acoustical detection and mechanical destruction of cancer cells. Recent efforts demonstrated that the PNBs can also be used for in vitro diagnosis and provide amplified signals for ensemble measurements. Inspired by those frontier works, we aim to further improve the technology with the capability of digital counting. We first designed an optofluidic setup that is similar to a flow cytometry system, where two aligned laser beams synchronically activate the NPs flowing through and detect the as-generated PNBs. At this early stage, unfortunately, we knew little about how to statistically analyze the PNB signals and failed the digital counting.
Figure 1. Schematic illustration of the generation and optofluidic detection of a plasmonic nanobubble from a gold nanoparticle (AuNP).
With a deeper understanding of the Poisson statistics, we started making good progress on our DIAMOND technology. We first found the characteristic pattern of digital counting ꟷ the sequential “on” and “off” PNB signals in a diluted AuNP suspension ꟷ and confirmed the capability of single-nanoparticle detection of DIAMOND. We also noticed that the PNBs are transient events and have no crosstalk between laser pulses, which allows DIAMOND to create “virtual detection zones” in a flow without the need for droplets and thus counts the signals in a compartment-free manner. A standard method for data analysis was developed utilizing MATLAB code. We then tested complex systems involving mixtures of different-sized AuNPs and silica beads. The Poisson-probability-corrected results validated the successful implementation of DIAMOND on a homogeneous assay. Such a combination leads to rapid detection results and excellent sensitivity with minimal hands-on time (< 1 min). Using RSV as the model, we demonstrated that DIAMOND allows for rapid and single-virion detection in spiked nasal swab samples and its detection limit rivals the digital loop-mediated isothermal amplification at a single RNA copy.
Figure 2. Schematic illustration of the digital counting of plasmonic nanobubble (PNB) signals from nanoparticles and nanoparticle-conjugated viruses.
Undoubtfully, digital immunoassays create high standards as next-generation diagnostic platforms, yet the major barriers to its widespread use are the time-consuming protocols and laboratory infrastructures. Our DIAMOND technology overcomes some of these bottlenecks, such as bypassing the steps of sample washing, separation, and signal amplification steps. On the other hand, DIAMOND can detect intact viruses without additional liquid handling (i.e., virus extraction, thermal incubation, and chip loading), offering a simplified diagnostic approach at room temperature. Our future improvements are focused on the fabrication of prototype devices, including device miniaturization, laser modulation, and detection scheme, as we wish to bring this technology to a broad range of labs and practical applications.