Development of On-Site Diagnostic Technology Using a Saliva-collected Swab

Abstract

Many technologies have been developed for point-of-care (POC) testing for rapid detection, not at the hospital or laboratory level. Various lateral flow immunoassay (LFIA) methods that can be detected with the naked eye using paper-based chips are being developed for POC testing. Also, various molecular diagnostic-based diagnostic methods such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) have been developed in place of conventional PCR that requires temperature control. The recent POC methods show as high sensitivity and accuracy as laboratory levels through many previous studies. To collect samples required for on-site diagnosis, this study facilitated the use and application of saliva, a biological fluid that can be easily and non-invasive collected. Saliva is more advantageous at the collection level than other bio-fluids and contains as many biomarkers as blood or serum. However, since the diagnostic sensitivity is lowered due to the high background effect of saliva, many studies proceed with the pretreatment process before diagnosis to remove impurities, which is disadvantageous for on-site diagnosis. Previous saliva collectors take longer to saturate saliva and use filtration, centrifugation, or resuspension for extraction. For easy saliva collection, the use of swabs is familiar to users and could quickly absorb relatively uniform small-volume samples. The viscous mucin in the saliva sample adheres to the fibrous structure of the swab and is removed a lot before the following detection steps. First, for the saliva-based immunoassay, the "gap-LFIA" structure modified from the conventional paper chip structure allows the whole saliva collected using a cotton swab to be applied. The absorption pad of the gap-LFIA was divided so that the swab could be inserted into the gap, and when an additional solution was added, the analyte collected by the swab could flow into the paper chip membrane. In this study, cotinine, a biomarker for smoking as a major metabolite of nicotine, was used as a model molecule for the gap-LFIA. In the conventional paper chip, applying the whole saliva directly without a pretreatment process not only blocks the membrane of the paper chip but also has a problem of increasing the error of the result. Using the gap-LFIA method, 10 ng/mL of cotinine in the saliva could be measured with the naked eye, and smokers and non-smokers were more clearly distinguished and deviation reduced. The detection of salivary cotinine can be useful in differentiating smokers from non-smokers. The existing methods of cotinine detection, a biomarker for smoking, require complex pretreatment procedures. Hence, in this study, gap-LFIA used the strip-based lateral flow immunoassay method for rapid, easy, and reliable on-site detection of cotinine as a test for smoking. The method used a simple cotton swab for saliva sample collection and detected cotinine in saliva within 15 minutes. For molecular diagnosis, a silica-based tip column connecting with a disposal syringe was developed and nucleic acid was purified directly from the saliva so that it could be directly used as a template in the amplification process such as PCR. The nucleic acid extraction conditions with high chaotropic salts were optimized to increase the convenience and recovery as much as possible instead of the nucleic acids extraction kit that can only be used on the lab scale. Influenza and COVID-19 in saliva were detected using RT-qPCR and experiments targeting Staphylococcus aureus were also detected at low concentrations and a tip column for extracting nucleic acids for field diagnosis that can be commonly used for both RNA and DNA was developed. The study has developed the preparation process of saliva optimized to be possible in the field without additional machines. The sensitivity and accuracy of immune-sensor and molecular diagnosis in the POC test using saliva samples collected by swab were improved. Both the Gap-LFIA structure and tip column can be applied to various targets, and it is highly utilized because it can increase the sensitivity of the desired detection sensor.