A Target Recycling Amplification Process for the Digital Detection of Exosomal MicroRNAs through Photonic Resonator Absorption Microscopy.

Exosomal microRNAs (miRNAs) have considerable potential as pivotal biomarkers to monitor cancer development, dis-ease progression, treatment effects and prognosis. Here, we report an efficient target recycling amplification process (TRAP) for the digital detection of miRNAs using photonic resonator absorption microscopy. We achieve multiplex digital detection with sub-attomolar sensitivity in 20 minutes, robust selectivity for single nucleotide variants, and a broad dynamic range from 1 aM to 1 pM. Compared with traditional qRT-PCR, TRAP showed similar accuracy in profiling exosomal miRNAs derived from cancer cells, but also exhibited at least 31-fold and 61-fold enhancement in the limits of miRNA-375 and miRNA-21 detection, respectively. The TRAP approach is ideal for exosomal or circulating miRNA biomarker quantification, where the miRNAs are present in low concentrations or sample volume, with potentials for frequent, low-cost, and minimally invasive point-of-care testing.

Photonic crystal enhanced fluorescence emission and blinking suppression for single quantum dot digital resolution biosensing.

While nanoscale quantum emitters are effective tags for measuring biomolecular interactions, their utilities for applications that demand single-unit observations are limited by the requirements for large numerical aperture (NA) objectives, fluorescence intermittency, and poor photon collection efficiency resulted from omnidirectional emission. Here, we report a nearly 3000-fold signal enhancement achieved through multiplicative effects of enhanced excitation, highly directional extraction, quantum efficiency improvement, and blinking suppression through a photonic crystal (PC) surface. The approach achieves single quantum dot (QD) sensitivity with high signal-to-noise ratio, even when using a low-NA lens and an inexpensive optical setup. The blinking suppression capability of the PC improves the QDs on-time from 15% to 85% ameliorating signal intermittency. We developed an assay for cancer-associated miRNA biomarkers with single-molecule resolution, single-base mutation selectivity, and 10-attomolar detection limit. Additionally, we observed differential surface motion trajectories of QDs when their surface attachment stringency is altered by changing a single base in a cancer-specific miRNA sequence.

Microscopies Enabled by Photonic Metamaterials.

In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between light and biological matter. A common objective is to concentrate the electromagnetic energy associated with light into nanometer-scale volumes that, in many cases, can extend below the conventional Abbé diffraction limit. Dating back to the first application of surface plasmon resonance (SPR) for label-free detection of biomolecular interactions, resonant optical structures, including waveguides, ring resonators, and photonic crystals, have proven to be effective conduits for a wide range of optical enhancement effects that include enhanced excitation of photon emitters (such as quantum dots, organic dyes, and fluorescent proteins), enhanced extraction from photon emitters, enhanced optical absorption, and enhanced optical scattering (such as from Raman-scatterers and nanoparticles). The application of photonic metamaterials as a means for enhancing contrast in microscopy is a recent technological development. Through their ability to generate surface-localized and resonantly enhanced electromagnetic fields, photonic metamaterials are an effective surface for magnifying absorption, photon emission, and scattering associated with biological materials while an imaging system records spatial and temporal patterns. By replacing the conventional glass microscope slide with a photonic metamaterial, new forms of contrast and enhanced signal-to-noise are obtained for applications that include cancer diagnostics, infectious disease diagnostics, cell membrane imaging, biomolecular interaction analysis, and drug discovery. This paper will review the current state of the art in which photonic metamaterial surfaces are utilized in the context of microscopy.

Accelerated Digital Biodetection Using Magneto-plasmonic Nanoparticle-Coupled Photonic Resonator Absorption Microscopy.

Rapid, ultrasensitive, and selective quantification of circulating microRNA (miRNA) biomarkers in body fluids is increasingly deployed in early cancer diagnosis, prognosis, and therapy monitoring. While nanoparticle tags enable detection of nucleic acid or protein biomarkers with digital resolution and subfemtomolar detection limits without enzymatic amplification, the response time of these assays is typically dominated by diffusion-limited transport of the analytes or nanotags to the biosensor surface. Here, we present a magnetic activate capture and digital counting (mAC+DC) approach that utilizes magneto-plasmonic nanoparticles (MPNPs) to accelerate single-molecule sensing, demonstrated by miRNA detection via toehold-mediated strand displacement. Spiky Fe3O4@Au MPNPs with immobilized target-specific probes are "activated" by binding with miRNA targets, followed by magnetically driven transport through the bulk fluid toward nanoparticle capture probes on a photonic crystal (PC). By spectrally matching the localized surface plasmon resonance of the MPNPs to the PC-guided resonance, each captured MPNP locally quenches the PC reflection efficiency, thus enabling captured MPNPs to be individually visualized with high contrast for counting. We demonstrate quantification of the miR-375 cancer biomarker directly from unprocessed human serum with a 1 min response time, a detection limit of 61.9 aM, a broad dynamic range (100 aM to 10 pM), and a single-base mismatch selectivity. The approach is well-suited for minimally invasive biomarker quantification, enabling potential applications in point-of-care testing with short sample-to-answer time.

Digital-resolution and highly sensitive detection of multiple exosomal small RNAs by DNA toehold probe-based photonic resonator absorption microscopy.

Small noncoding RNAs (snRNA) have been emerging as promising diagnostic biomarkers for detecting early stage cancer. Currently existing methods for snRNA detection, including northern blot, reverse transcription-polymerase chain reaction, microarrays and RNA-Seq, are limited to time-consuming, low sensitivity, expensive instrumentation or complex analysis of data. Herein, we present a rapid quantitative analysis of multiple liver cancer-associated exosomal snRNA by a nucleic acid toehold probe-based photonic resonator absorption microscopy (PRAM) assay, with digital resolution and high sensitivity. The assay relies on the use of three toehold probe-encoded gold nanoparticles (AuNPs) and addressable photonic crystal (PC) sensing chips. The presence of target snRNA will initiate toehold-mediated strand displacement reactions that trigger the capture of gold particles onto the PC surface, which is subsequently imaged by PRAM for digital counting of detected snRNA molecules. We achieved highly sensitive and selective detection of three snRNA targets in buffer with a 30 min assay protocol, with detection limits of 4.56 fM, 4.68 fM and 0.69 pM. Having confirmed our assay's performance for detection of snRNA targets spiked into exosomal RNA extracts, we demonstrated its capability for quantitative detection of the same targets from patient blood plasma samples. The approach offers a rapid, simple workflow that operates at room temperature with a single step without enzymatic amplification, while the detection instrument can be implemented as a low-cost portable system for point of care environments.

Label-Free Digital Detection of Intact Virions by Enhanced Scattering Microscopy.

Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 103 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.

Single-step, wash-free digital immunoassay for rapid quantitative analysis of serological antibody against SARS-CoV-2 by photonic resonator absorption microscopy.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of Coronavirus Disease 2019 (COVID-19), poses extraordinary threats and complex challenges to global public health. Quantitative measurement of SARS-CoV-2 antibody titer plays an important role in understanding the patient-to-patient variability of immune response, assessing the efficacy of vaccines, and identifying donors for blood transfusion therapy. There is an urgent and ever-increasing demand for serological COVID-19 antibody tests that are highly sensitive, quantitative, rapid, simple, minimally invasive, and inexpensive. In this work, we developed a single-step, wash-free immunoassay for rapid and highly sensitive quantitative analysis of serological human IgG against SARS-CoV-2 which requires only a single droplet of serum. By simply incubating 4 µL human serum samples with antibody-functionalized gold nanoparticles, a photonic crystal optical biosensor coated with the recombinant spike protein serves as a sensing platform for the formation of sandwich immunocomplex through specific antigen-antibody interactions, upon which the detected IgG molecules can be counted with digital precision. We demonstrated a single-step 15-min assay capable of detecting as low as 100 pg mL-1 human COVID-19 IgG in serum samples. The calculated limit of detecting (LOD) and limit of quantification (LOQ) is 26.7 ± 7.7 and 32.0 ± 8.9 pg mL-1, respectively. This work represents the first utilization of the Activate Capture + Digital Counting (AC + DC)-based immunoassay for rapid and quantitative analysis of serological COVID-19 antibody, demonstrating a route toward point-of-care testing, using a portable detection instrument. On the basis of the sandwich immunoassay principle, the biosensing platform can be extended for the multiplexed detection of antigens, additional IgGs, cytokines, and other protein biomarkers.

Critical Review: digital resolution biomolecular sensing for diagnostics and life science research.

One of the frontiers in the field of biosensors is the ability to quantify specific target molecules with enough precision to count individual units in a test sample, and to observe the characteristics of individual biomolecular interactions. Technologies that enable observation of molecules with "digital precision" have applications for in vitro diagnostics with ultra-sensitive limits of detection, characterization of biomolecular binding kinetics with a greater degree of precision, and gaining deeper insights into biological processes through quantification of molecules in complex specimens that would otherwise be unobservable. In this review, we seek to capture the current state-of-the-art in the field of digital resolution biosensing. We describe the capabilities of commercially available technology platforms, as well as capabilities that have been described in published literature. We highlight approaches that utilize enzymatic amplification, nanoparticle tags, chemical tags, as well as label-free biosensing methods.

Activate capture and digital counting (AC + DC) assay for protein biomarker detection integrated with a self-powered microfluidic cartridge.

We demonstrate a rapid, 2-step, and ultrasensitive assay approach for quantification of target protein molecules from a single droplet test sample. The assay is comprised of antibody-conjugated gold nanoparticles (AuNPs) that are "activated" when they are mixed with the test sample and bind their targets. The resulting liquid is passed through a microfluidic channel with a photonic crystal (PC) biosensor that is functionalized with secondary antibodies to the target biomarker, so that only activated AuNPs are captured. Utilizing recently demonstrated hybrid optical coupling between the plasmon resonance of the AuNP and the resonance of the PC, each captured AuNP efficiently quenches the resonant reflection of the PC, thus enabling the captured AuNPs to be digitally counted with high signal-to-noise. To achieve a 2-step assay process that is performed on a single droplet test sample without washing steps or active pump elements, controlled single-pass flow rate is obtained with an absorbing paper pad waste reservoir embedded in a microfluidic cartridge. We use the activate capture and digital counting (AC + DC) approach to demonstrate HIV-1 capsid antigen p24 detection from a 40 µL spiked-in human serum sample at a one thousand-fold dynamic range (1-103 pg mL-1) with only a 35-minute process that is compatible with point-of-care (POC) analysis. The AC + DC approach allows for ultrasensitive and ultrafast biomolecule detection, with potential applications in infectious disease diagnostics and early stage disease monitoring.