Development of Point-of-Care Lateral Flow Assay Devices for Salivary Endotoxin Detection
Authors: Han Daewoo , Xie Sancai , Steckl Andrew 
Published: July 2021

Endotoxins, also known as lipopolysaccharides (LPS), are important macromolecules in outer membrane of Gram-negative bacteria, which are generated when the bacteria are in growing and stationary phases, and disintegrate after death. While endotoxins induce a strong immune response in hosts, it also can serve as an important biomarker to diagnose various bacterial diseases. Among various pathogenic bacteria, Porphyromonas gingivalis is attracting much attention in recent years. P. gingivalis is a major Gram-negative bacterium that is responsible for chronic periodontitis, among > 500 bacteria species found in subgingival plaque. Periodontal disease presents a chronic inflammation caused by bacterial infection, leading to gum disease and even loss of teeth. LPS from P. gingivalis (PG LPS) induces significant host responses in gingival tissue by increasing the production of inflammatory biomarkers, such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor alpha (TNF- a) in gingival fibroblasts.[1] Furthermore, because of the close relationship between P. gingivalis and other important systemic diseases such as cardiovascular diseases (e.g. atherosclerosis[2]) and neurodegenerative Alzheimer’s disease[3] that has emerged in recent years, quantitative evaluation of PG LPS has become a key measure in oral health as well as in the whole body health system.
Considering the increasing significance of P. gingivalis LPS, high demand for a novel point-of-care device has emerged for both early diagnosis and frequent monitoring PG LPS levels. Traditional qualitative detection of bacterial biomarkers is carried out mostly utilizing the well-known PCR method, which requires trained personnel and relatively longer time span in a laboratory setting. Lateral flow assay is one of the most promising POC platforms with many benefits, such as low cost, simple fabrication process, user-friendly colorimetric interface, rapid turnaround time, and no need for external instruments.[4] We have developed two different approaches for LFA-based POC devices to detect and quantify PG LPS concentrations. First, traditional immunoassay based LFA was developed to detect the PG LPS. Because PG LPS is a large macromolecule, multiple binding sites can be present on PG LPS. The sandwich assay mechanism was adopted using two different antibodies against PG LPS (Fig. 1a). Monoclonal antibodies are conjugated on the surface of gold nanoparticles (AuNP) to capture PG LPS molecules in the sample solution, and polyclonal antibodies are printed on the nitrocellulose membrane to form a test line by capturing PG LPS bound AuNP conjugate from the sample solution. After dispensing the sample solution on LFA, the test line is formed based on the PG LPS presence in the sample solution (Fig. 1b). Higher LPS concentration leads to a stronger test line. The second approach is to replace antibodies with aptamers as a bio-recognition molecule for target analytes. Aptamer-based assay uses a short synthetic DNA or RNA molecules that replace the traditional antibody/antigen immunoassay. Aptamer can be designed and synthesized in vitro with excellent sensitivity and selectivity to various target molecules. In addition, excellent long-term and thermal stability can also be provided because aptamers are chemically synthetized molecules. As shown in Fig. 2a, duplex aptamer was covalently conjugated to AuNPs. When LPS is introduced, the aptamer binds to the target LPS and is released from the duplex aptamer. In the competitive binding approach (Fig. 2b), a stronger test line is formed when less LPS is present. As shown in Fig. 2c, an increasing difference in test line intensity is observed as the LPS concentration is varied between 0 and 1 ng/mL. Interestingly, although usually the competitive assay is less sensitive than sandwich assay, in this case so far the competitive aptamer-based LFA presented higher sensitivity than that of sandwich immunoassay-based LFA test results. To further increase the assay sensitivity, future work will focus on enhancing the sandwich aptamer based LFA device using two different aptamers binding to the different sites on PG LPS.

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Dalirirad, S., D. Han, and A.J. Steckl, Aptamer-Based Lateral Flow Biosensor for Rapid Detection of Salivary Cortisol. ACS Omega, 2020.doi: 10.1021/acsomega.0c03223

Figure 1