Review 1: "A Label-free Optical Biosensor-Based Point-of-Care Test for the Rapid Detection of Monkeypox Virus"

Ugur Aygun

doi:doi:10.1162/2e3983f5.d4c6b965

A Label-free Optical Biosensor-Based Point-of-Care Test for the Rapid Detection of Monkeypox Virus

by Mete Aslan, Elif Seymour, Howard Brickner, Alex E. Clark, Iris Celebi, Michael B. Townsend, Panayampalli S. Satheshkumar, Megan Riley, Aaron F. Carlin, M. Selim Ünlü, and Partha Ray

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Published on Jul 05, 2024
www.medrxiv.org

Description

ABSTRACT Diagnostic approaches that combine the high sensitivity and specificity of laboratory-based digital detection with the ease of use and affordability of point-of-care (POC) technologies could revolutionize disease diagnostics. This is especially true in infectious disease diagnostics, where rapid and accurate pathogen detection is critical to curbing the spread of disease. We have pioneered an innovative label-free digital detection platform that utilizes Interferometric Reflectance Imaging Sensor (IRIS) technology. IRIS leverages light interference from an optically transparent thin film, eliminating the need for complex optical resonances to enhance the signal by harnessing light interference and the power of signal averaging in shot-noise-limited operation to achieve virtually unlimited sensitivity. In our latest work, we have further improved our previous ‘Single-Particle’ IRIS (SP-IRIS) technology by allowing the construction of the optical signature of target nanoparticles (whole virus) from a single image. This new platform, ‘Pixel-Diversity’ IRIS (PD-IRIS), eliminated the need for z-scan acquisition, required in SP-IRIS, a time-consuming and expensive process, and made our technology more applicable to POC settings. Using PD-IRIS, we quantitatively detected the Monkeypox virus (MPXV), the etiological agent for Monkeypox (Mpox) infection. MPXV was captured by anti-A29 monoclonal antibody (mAb 69-126-3) on Protein G spots on the sensor chips and were detected at a limit-of-detection (LOD) - of 200 PFU/ml (∼3.3 attomolar). PD-IRIS was superior to the laboratory-based ELISA (LOD - 1800 PFU/mL) used as a comparator. The specificity of PD-IRIS in MPXV detection was demonstrated using Herpes simplex virus, type 1 (HSV-1), and Cowpox virus (CPXV). This work establishes the effectiveness of PD-IRIS and opens possibilities for its advancement in clinical diagnostics of Mpox at POC. Moreover, PD-IRIS is a modular technology that can be adapted for the multiplex detection of pathogens for which high-affinity ligands are available that can bind their surface antigens to capture them on the sensor surface.

RR\ID Evidence Scale rating by reviewer:

Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: This manuscript presents the development and validation of the Pixel-Diversity Interferometric Reflectance Imaging Sensor (PD-IRIS), a novel diagnostic platform designed to enhance detection capabilities for infectious diseases, specifically targeting the Monkeypox virus (MPXV). The authors have made an important effort in advancing point-of-care (POC) diagnostic technology by introducing a label-free digital detection system that shows superior sensitivity and specificity compared to the traditional ELISE method. The manuscript well-organized, and the technology's potential impact on public health, particularly in low-resource settings, is compellingly argued.

The evidence provided convincingly supports the main claims regarding PD-IRIS's efficacy and applicability. Detailed methodological descriptions, comparative results with ELISA, specificity tests against other viruses, and the use of real patient samples collectively reinforce the platform's enhanced diagnostic capabilities.

Key strengths include:

The innovative approach of PD-IRIS as a label-free, high-sensitive platform eliminates the need for z-scan acquisition, a major bottleneck in translating sensitive label-free interferometric imaging systems to point of care settings. Traditionally, z-stack acquisition requires costly and complex three-dimensional positioning systems, which are not practical for resource-limited environments. PD-IRIS overcomes this by using a pixel-diversity technique that captures the necessary optical information in a single image capture, eliminating the need for mechanical z-scanning. This simplifies the hardware, reduces costs, and speeds up diagnostics, making it more suitable for point-of-care use.
The methodology presented in the study is comprehensively detailed, supporting the reproducibility of the results. The use of the anti-A29 monoclonal antibody for specific detection of MPXV, along with a comparative analysis against ELISA, solidifies the foundational claims of the study.
The demonstrated sensitivity of PD-IRIS, with a limit of detection better than that of traditional ELISA, is particularly noteworthy. This high level of sensitivity, combined with the specificity demonstrated against other viruses like HSV-1 and Cowpox virus, underscores the potential of PD-IRIS to improve infectious disease diagnostics, particularly in resource-limited settings where rapid and accurate testing is crucial.
The relevance and timeliness of this research are profound, given the urgent global need for rapid and accurate diagnostics highlighted by recent Monkeypox outbreaks. This context enriches the significance of PD-IRIS as a critical tool in enhancing disease management and response strategies.

Limitations and Suggestions: Though not the primary focus at this stage, evaluating the long-term stability and usability of the PD-IRIS sensor chips under various storage and operational conditions is vital, particularly for deployment in low-resource settings where these factors significantly impact practical utility. While the comparison to ELISA provides a basic benchmark, expanding this to include other contemporary diagnostics like PCR and various point-of-care technologies could enrich the evaluation of PD-IRIS’s effectiveness. Additionally, the platform’s reliance on specific viral antigens, prone to mutations, suggests a need for ongoing assessment of antibody binding efficiency to ensure sustained test sensitivity and specificity amidst viral evolution. Addressing these aspects would enhance the platform's robustness and clinical relevance.

In conclusion, PD-IRIS represents a significant contribution to diagnostic technologies, with considerable potential impact global health security. Its development could lead to critical improvements in the rapid and onsite management of disease outbreaks, particularly in regions prone to infectious diseases. As this technology undergoes further validation and refinement, it holds promise for playing an important role in global infectious disease management and containment strategies.