Nano and Micro Technologies
Balazs Kaszala
Undergraduate Researcher
Georgia Institute of Technology
Glen Allen, Virginia, United States
Kanz A. Elkhiyari (she/her/hers)
Undergraduate Researcher
Georgia Institute of Technology
Peachtree Corners, Georgia, United States
Neda Rafat
Senior Scientist
Merck, United States
Asma Hashim
Graduate Researcher
Georgia Institute of Technology, United States
Aniruddh Sarkar
Assistant Professor
Georgia Institute of Technology, United States
Sub-Track: Sensors/Nanodevices for Diagnostics/Biomedical Imaging
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), affects nearly 2 billion people. The vast majority of new cases occur in resource-poor settings in low and middle income countries1. Although TB is treatable, early and accurate diagnosis remains a bottleneck. There are methods in place to detect active cases, but 90-95% of worldwide cases are latent - meaning that the patients are asymptomatic1.
Currently, the two most widely used methods for latent TB (LTB) diagnosis are the tuberculin skin test (TST) and the interferon-gamma release assay (IGRA). Although the TST is low-cost and can be performed at the point-of-care (POC), it is prone to both false positives and false negatives due to interpretation errors and requires 48-72hrs to yield results. Alternatively, the IGRA is the gold standard detection method for LTB diagnosis and is performed by measuring the release of interferon-gamma (IFN-γ) by Mtb-specific T-cells in a blood sample. This method is sensitive but is hindered by its high equipment cost and need for specialized laboratories, making it impractical in resource-limited settings3.
Thus, globally scalable LTB diagnosis necessitates the development of a test combining the affordability and ease-of-use of the TST with the sensitivity of the IGRA. We have earlier developed an inexpensive POC-compatible multiplexed immunoassay for antibodies using enzymatic metallization on glass and flexible polymeric substrates and quantified via cellphone-based computer vision4. Here, we adapt this assay to develop an inexpensive, high-throughput POC method of detection for IFN-γ from a single drop of sample.
The experiment involved the immobilization of an anti-human interferon-gamma (IFN-γ) antibody onto microwells each capable of holding up to 3 µL of solution, which were defined by laser-cut polydimethylsiloxane (PDMS) on a gamma sterilized Petri dish made of polystyrene (Fig. 1a). To minimize non-specific binding, the wells were initially treated with a blocking solution of 1% bovine serum albumin (BSA) in phosphate-buffered saline with 0.05% Tween-20 (PBST). Serial dilutions of the human IFN-γ antigen were subsequently added to the wells. Following that, biotinylated anti-human IFN-γ antibody was introduced, followed by Streptavidin Poly-HRP (Fig. 1b). The resulting signal was then developed using enzymatic metallization and captured using a cellphone camera. Finally, the acquired images were subjected to analysis to determine the darkness of the metallization in each well by calculating the average grayscale value.
Results and Discussion:
The device exhibited dark metallization at the highest IFN-γ concentration, with diminishing signals in serial dilutions (Fig. 1c). The limit of detection (LOD) was found to be 700 pg/mL (Fig. 1d). This can be compared to the gold standard conventional enzyme-linked immunosorbent assay (ELISA) LOD of 22 pg/mL (Fig. 1e). The device's miniaturization drastically reduces reagent and sample volumes, making it potentially 4X less costly than ELISA in consumable cost, and utilizing up to 33X less patient sample volume. Moreover, this POC method eliminates the need for expensive specialized equipment such as plate readers, relying solely on a cellphone for signal readout and quantification, thus reducing equipment cost by 50X. This indicates promising potential for effective and affordable LTB diagnosis in resource-limited settings. Optimization efforts will focus on further enhancing sensitivity through alternative antibodies and signal amplification with engineered nanoparticles5,6. Clinical evaluation using patient samples will also be performed.
Conclusion:
In this study, we have introduced a novel POC approach for the detection of IFN-γ in LTB, offering a cost-effective and high-throughput method tailored for resource-limited settings. This approach successfully demonstrated a LOD as low as 700 pg/mL, which is within the reference range of the gold standard conventional ELISA. Notably, it utilizes significantly lower sample volumes and less expensive materials compared to conventional techniques while eliminating the need for specialized laboratory equipment.
Although the initial results are promising, further optimization of the platform is underway to include multiplexed cytokine detection and enhance sensitivity via exploring alternative antibodies, buffers, and potential signal-enhancing methods employing engineered nanoparticles. Additionally, analysis of patient samples will ascertain the device’s clinical efficacy.
With its potential to revolutionize TB diagnosis in resource-limited settings, this novel method for IFN-γ detection holds significant promise in contributing to improved TB management and reducing the burden of this global health challenge.