Frontiers in Serodiagnostics: A New Plasmon-Enhanced Fluorescence Biosensor Technology Suitable for Lab-on-a-Chip Applications

Abstract

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Host response to microbes may be detected in human serum using a variety of plasmonic-based technology platforms, including localized surface plasmon resonance, propagating surface plasma resonance, surface-enhanced Raman scattering, surface-enhanced infrared absorption spectroscopy, and various surface-enhanced fluorescence methods. One recently devised adaptation of Grating-Coupled Surface Plasmon Resonance (GC-SPR) is referred to as Grating-Coupled Fluorescent Plasmonics (GC-FP). The feasibility of using this new technology for detecting immune responses to microbes has only recently been demonstrated in the context of Lyme disease and Covid-19 infections. While still in a relatively early stage of technology development, this GC-SPR-based method holds promise in democratizing lab-on-a-chip serologic testing for widespread deployment in point-of-care (POC) diagnostic environments.

Introduction

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Standard microbial detection approaches typically demand relatively expensive assay kits, sophisticated instrumentation, and expert handling, as exemplified by gene sequencing, polymerase chain reaction (PCR), hemagglutination assay, Western blotting (WB), lateral flow immunoassay (LFIA), and enzyme-linked immunosorbent assay (ELISA). Additionally, the pre-developed protocols for particular pathogens are usually limited to specific strains or types of microbes. Conventional techniques such as ELISA and LFIA often provide sufficient sensitivity to detect individual analytes at physiologic concentrations. However, the cost per analyte scales linearly and becomes impractical when evaluating complex multi-analyte systems. WB allows simultaneous measurement of multiple antibodies but is limited by sensitivity, specificity, resolution of the electrophoretic separation medium, and assay complexity. Increasingly there is a need for higher content, more sensitive, more specific, less expensive, and easier-to-use alternatives to ELISA, LFIA, and WB for antigen detection. Plasmonic-based biosensing technologies may potentially provide such an alternative approach to microbial antigen detection, offering highly sensitive and rapid diagnosis, with minimal sample pretreatment, easy operation, and inexpensive instrumentation (Niu et al., 2015; Shrivastav et al., 2021).

GC-SPR and GC-FP Technology

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Surface plasmon resonance (SPR)-based immunosensors provide a nondestructive optical analysis approach which is useful for examining antigen-antibody interactions for biomolecules deposited in a thin layer on the surface of a gold-coated sensor chip (Rossi et al., 2018). Many optical biosensors utilize the principle of SPR, the resonant oscillation of conduction electrons at the interface between negative and positive permittivity material stimulated by incident light. The surface plasmon polariton (SPP) is a non-radiative electromagnetic surface wave that propagates in a direction parallel to the negative permittivity/dielectric material interface. Since the wave is localized at the boundary of the conductor and the external medium (e.g., air, buffer, or serum), these oscillations are particularly sensitive to any perturbation of this boundary, such as can occur through the adsorption of macromolecules to the conducting surface. Standard SPR-based sensors offer the advantage of being label-free, enzyme-free, real-time, and readily implemented in cost-effective integrated devices.

Surface plasmon resonance (SPR)-based immunosensors provide a nondestructive optical analysis approach which is useful for examining antigen-antibody interactions for biomolecules deposited in a thin layer on the surface of a gold-coated sensor chip (Rossi et al., 2018). Many optical biosensors utilize the principle of SPR, the resonant oscillation of conduction electrons at the interface between negative and positive permittivity material stimulated by incident light. The surface plasmon polariton (SPP) is a non-radiative electromagnetic surface wave that propagates in a direction parallel to the negative permittivity/dielectric material interface. Since the wave is localized at the boundary of the conductor and the external medium (e.g., air, buffer, or serum), these oscillations are particularly sensitive to any perturbation of this boundary, such as can occur through the adsorption of macromolecules to the conducting surface. Standard SPR-based sensors offer the advantage of being label-free, enzyme-free, real-time, and readily implemented in cost-effective integrated devices.



One widely used approach to deliver coupling between incident light and SPP for SPR sensing of biological macromolecules is through the use of a prism. Unfortunately, however, the awkward and complex optical system required for prism-based devices often limits its versatility and ease of integration into miniaturized arrays for lab-on-a-chip applications. In contrast to prism-based SPR, for grating-based coupling devices (GC-SPR), resonance conditions are provided by diffraction of the incident light, offering greater opportunity for miniaturization and integration into lab-on-a-chip platforms (Figure 1). However, one drawback to grating-based relative to prism-based systems is their overall poorer detection sensitivity and consequently poorer target limit of detection (LOD).

In GC-FP, the interaction of fluorophores with surface plasmons amplifies fluorescence signal (brightness) accompanying molecular binding events by several orders of magnitude. GC-FP technology enables multiplexed biomarker screening of blood serum using a GC-SPR microchip that can be imaged with a complementary metal-oxide-semiconductor (CMOS) or charge-coupled device (CCD) camera and subsequently algorithmically scored to aid in achieving a diagnosis. The SPR effect can enhance the reporter signal by 2-3 orders of magnitude and can aid in quantifying a variety of biological attributes, such as a heterogeneous population of antibodies within the sample, using an intuitive microarray format.

Applications: COVID-19 and Lyme Disease Serodiagnostics

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Several key biomedical indications demand sensitive antibody-based testing in the clinical setting, including characterization of individual immune response arising from vaccination and the assessment of herd immunity in population-based health studies, as exemplified by the recent COVID-19 pandemic, as well as the diagnosis of infections arising from low-titer organisms, as exemplified by Lyme disease. It is becoming increasingly apparent that most clinically relevant disease states can be better characterized by tracking panels of biomarkers, as a particular clinical phenotype may be definable by hundreds of changes at the molecular and cellular level. The feasibility of detecting panels of biomarkers in COVID-19 and Lyme disease using GC-FP has recently been demonstrated, as described below.

COVID-19

Recently, humanity has been attempting to deal with the public health emergency of the 2019 SARS CoV-2 (COVID-19) pandemic, which is believed to have originated from Wuhan, China, in December 2019, before rapidly spreading across the globe (Pascarella et al., 2020). The disease is characterized by flu-like symptoms, which can become especially life-threatening in high-risk individuals. Specifically, symptoms include conjunctivitis, cough, fatigue, fever, gastrointestinal disturbances, headache, labored breathing, loss of taste, loss of smell, and sore throat. As of August 2021, more than 200 million people have contracted the disease worldwide, with a global death toll of roughly 4.5 million. COVID-19 is transmitted from human to human through the air in respiratory droplets and upon physical contact with contaminated environmental surfaces. In order to minimize loss of life from this new pandemic and to increase preparedness for any future reemergence of COVID-19 variants, as well as other future pandemics, rapid and timely diagnostic assays are urgently required (Pascarella et al., 2020).

Plasmonic-based biosensor assays potentially offer advantages with respect to viral diagnostics, including low assay cost, low instrument cost, fast implementation, and ease of use. GC-FP has recently been employed to monitor antibody-antigen binding interactions for multiple targets in individual COVID-19 patient serum samples (Cady et al., 2021; Cognetti et al., 2021). Recombinant nucleocapsid protein, the S1 fragment of the spike protein, the extracellular domain of the spike protein, the receptor-binding domain of the spike protein, the S1 domain of the 2005 SARS coronavirus spike protein, and human Influenza B nucleoprotein IgG were spotted in replicates of three onto gold-coated silicon microchips containing a plasmonic diffraction grating, using a robotic spotter. Human serum albumin (HSA) and human IgG were included on the microarrays as negative and positive spotting controls, respectively. Microarrays were blocked to minimize nonspecific binding, then incubated with diluted serum sample, followed by far-red emitting fluorophore-labeled IgG/IgM antibodies, with washes after the serum and antibody incubations. Microarrays were analyzed using a GC-SPR instrument developed by Ciencia, Inc. (East Hartford, CT), equipped with an incident collimated 633 nm laser beam.

Initial results established 100% selectivity and sensitivity (n = 23) in detecting serum IgG levels raised against three common COVID-19 antigens, spike S1, spike S1S2, and the nucleocapsid protein. The assay was revealed to provide quantitative data across serum dilutions ranging from 1:25–1:1,600. This feasibility study also demonstrated a strong correlation of results with commercially available ELISA and Luminex-based immunoassays, though ELISA provided a superior LOD. The time required to perform antibody detection by GC-FP was determined to be roughly 30 minutes, which is significantly shorter than the standard 2-3 hours required to complete ELISA or Luminex-based assays. The investigators demonstrated the feasibility of detecting COVID-19 from dried blood spots, reducing the complexity of sample collection, handling, transport, and storage relative to standard venipuncture-based whole blood collection.

Lyme Disease

Lyme disease afflicts 300,000 - 450,000 people annually, primarily in the Great Lakes and Northeastern regions of the United States and portions of Canada. The disease is primarily caused by the bacterium Borrelia burgdorferi, though rarely it can also be spread by Borrelia mayonii (Moore et al., 2016). It is transmitted to humans through the bite of infected black-legged ticks (Figure 2), which also infect deer populations in the cited regions. Common symptoms arising from infection include headache, fever, fatigue, and a characteristic skin rash. If the disease is not treated, the infection can ultimately spread to joints (arthritis), the heart (carditis, heart arrhythmia), and the nervous system (meningitis, neuropathy). Lyme disease is generally diagnosed based upon patient symptoms, the presence of a characteristic rash (erythema migrans), and the likelihood of exposure to infected ticks in the environment. Laboratory testing using validated antigen assays, as briefly described below, is required to confirm the presence of the disease. In most instances, Lyme disease can be successfully treated using a regime of antibiotics over the course of several weeks.

The U.S. Center for Disease Control (CDC) recommends a standard two-tiered test (STTT) for diagnosing Lyme disease. The first tier employs either ELISA or an immunofluorescence assay, while the second tier is WB. However, the STTT used to diagnose Lyme disease displays relatively poor sensitivity, especially during the early stages of disease progression, often reporting false-negative results. Conversely, false-positive test results may occur from circulating antibodies remaining in the patient's system for years' post-infection and after resolution of the infection.

A protein microarray has recently been fabricated to measure potentially diagnostic host serum antibody targets to Lyme disease using GC-FP technology (Chou et al., 2020; 2021). Recombinant Borrelia burgdorferi proteins (BmpA, OspD, OspC, DbpA, DbpB, RevA, ErpG, ErpL, ErpY, VlsE, BBA65, BBA69, BBA70, BBA73, P41, and p48) were spotted in replicates of four onto gold-coated silicon microchips containing a plasmonic diffraction grating, using a robotic spotter. Bovine serum albumin (BSA) and human IgG were included on the microarrays as negative and positive spotting controls, respectively. Microarrays were blocked to minimize nonspecific binding, then incubated with diluted serum sample, followed by far-red emitting fluorophore-labeled IgG and IgM antibodies, with washes after the serum and each antibody incubation. Microarrays were analyzed using a GC-SPR instrument developed by Ciencia, Inc, equipped with an incident collimated 633 nm laser beam.

The cited analytical approach requires low microliter volumes of human serum to facilitate multiplexed biomarker screening of femtomole levels of host antibodies using a compact microarray surface. The approach produces semi-quantitative results (~6-fold linear dynamic range, Chou et al., 2020) that can be further processed to obtain a diagnostic score. The semi-quantitative, high-sensitivity nature of GC-FP enabled the screening of antibody targets for predictive value in determining Lyme disease status and the creation of a diagnostic algorithm that was more sensitive, specific, and informative than standard ELISA and WB assays. The diagnostic algorithm appeared to be more sensitive than the current CDC STTT standard for detecting early Lyme disease while maintaining 100% specificity. Furthermore, analysis of relative IgG and IgM serum antibody levels to predict disease status, such as acute-versus-convalescent stages of infection, appears feasible using GC-FP.

Fluorescent Conjugates

Biosensors are typically comprised of three main components: the target, recognition, and transducing portions. The target is the analyte of interest, which is detected when it is captured by the recognition element through some specific interaction. Upon binding to the target molecule, the recognition element of the sensor undergoes a change in one of its physical or chemical properties, such as conductivity, refractive index (RI), absorbance maxima, or pH value. This property change is translated to a readable signal with the aid of a transducer.

The selection of suitable fluorophore conjugates depends upon a particular instrument's design and set-up. The cited platforms for microbial host response detection described in this review rely upon a Helium-Neon or laser diode light source. In order to separate incident light at the excitation wavelength from the SPP-stimulated fluorescence emission signal, a set of bandpass filters is typically employed. For He-Ne and laser diode laser sources, the selected bandpass filters have typically been centered at about 670 nm, matching the emission maximum of the Alexa Fluor^® 647 fluorophore (Note: AAT Bioquest's XFD647 fluorophore has an identical structure as Alexa Fluor^® 647 dye). However, conceivably longer wavelength bandpass filters could be employed for fluorophores emitting further in the red region of the spectrum. AAT Bioquest offers more than 100 different fluorescent, biotinylated, and enzyme-labeled goat anti-human secondary antibodies that are potentially suitable for GC-FP biosensor technology. The compendium of fluorophore conjugates that could potentially be utilized for GC-FP using commercially available instrumentation is summarized in Table 1.

Conclusion

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A wide range of conventional viral sensors have been devised over the years, often for biomedical and agricultural applications, which have been based upon chromatography, mass-sensitive, or electrochemical (amperometric, potentiometric, impedimetric, and calorimetric) signal transduction approaches (Bauch et al., 2014; Shrivastav et al., 2021). Plasmonics-based sensors offer several advantages compared to these conventional approaches, including real-time monitoring to probe the binding dynamics involved in various biomolecular interactions, short response time, device reusability, and simple sample preparation, as well as the use of relatively few electrical components in the detection platform, which is conducive to assay miniaturization. Limitations encountered with the class of biosensors include the nonspecific nature of the binding surface, which can be somewhat abrogated by immobilizing an analyte selective layer over the plasmonic film, mass transportation limitations, and steric hindrance during the target binding event. GC-FP is a relatively new SPR-based detection technology currently being benchmarked in R&D laboratories. Only time will tell whether this technology successfully runs the gauntlet of regulatory tests necessary for its full authorization as a POC lab–on–a–chip diagnostic platform.

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