Microscale immiscible phase magnetic processing for bioanalytical applications

Rodriguez-Mateos, Pablo; Ngamsom, Bongkot; Iles, Alexander; Pamme, Nicole

doi:10.1016/j.trac.2022.116867

Cited by 4 publications

(2 citation statements)

References 67 publications

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“…The use of the T. pallidum-IMRS primers when modi ed with uorescent tags can be used to achieve a visual read-out of pathogen DNA this can then be utilized in the development of a Lateral Flow Assay (LFA) as has been described [28]. Also a simple and integrated platform based on microscale immiscible ltration and isothermal ampli cation for colorimetric detection of T. pallidum DNA can be developed as it has been described [29].…”

Section: Discussionmentioning

confidence: 99%

Highly Sensitive Molecular Assay for the Detection of Treponema pallidum infection

Shiluli,

Kamath,

Kanoi

et al. 2024

Preprint

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Introduction: According to the World Health Organization (WHO) more than 7 million new Treponema pallidum (T. pallidum) infections were reported among people aged 15–49 years in 2020 globally, the majority of them in developing countries. Syphilis, which is caused by T. pallidum is transmitted through contact with active lesions of a sexual partner or from an infected pregnant woman to her foetus. Gold standard T. pallidum laboratory diagnosis methods include dark-field microscopy, silver staining, direct fluorescence immunoassays and the rabbit infectivity test. However, these tests are associated with false positive or negative results. The gold standard 16S ribosomal (rRNA) gene polymerase chain reaction (PCR) is used for routine amplification of T. pallidum conserved genes. Here we report on an ultrasensitive syphilis diagnostic method, based on de novo genome mining of the T. pallidum DNA to identify identical multi repeat sequences (IMRS) as amplification primers. Methods: We used genome-mining approaches to find IMRS distributed throughout the T. pallidum genome to design a primer pair that target four repeat sequences. Genomic T. pallidum DNA was diluted from 8.14×104 to 8.14×10− 2 genome copies/𝜇l and used as template in the IMRS-based amplification assay. For performance comparison, 16S rRNA PCR assay was employed. Probit analysis was used to calculate the lower limit of detection of the T. pallidum-IMRS PCR and the conventional 16S rRNA PCR assays. Results: Probit analysis confirmed that the T. pallidum-IMRS primers offered higher test sensitivity of 0.03 fg/𝜇L compared to the 16S rRNA PCR (0.714 pg/𝜇L). Using the T. pallidum-IMRS primers, we were able to observe considerable isothermal amplification of genomic DNA at a starting concentration of 0.01 pg/µL. Conclusion: De novo genome mining of T. pallidum IMRS as amplification primers can serve as a platform for developing ultrasensitive diagnostics for Syphilis and potentially a wide range of infectious pathogens.

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Section: Discussionmentioning

confidence: 99%

Highly Sensitive Molecular Assay for the Detection of Treponema pallidum infection

Shiluli,

Kamath,

Kanoi

et al. 2024

Preprint

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“…In recent years, droplet magnetofluidics has emerged as a promising avenue for developing user-friendly, efficient, and mobile diagnostic technologies. − Utilizing magnetic beads that bind to nucleic acids, this technique facilitates transport of nucleic acids through distinct stationary reagent droplets, enabling intricate bioassays to be conducted using affordable cartridges and streamlined instruments. − Owing to its affordability and simplicity, droplet magnetofluidics presents a valuable solution, especially in resource-constrained environments . While current research primarily centers around pathogen detection, its use in methylation biomarker detection is gaining traction, , particularly using the methylation-on-beads (MOB) assay, a process streamlined for DNA sample handling and methylation-specific qPCR (qMSP) using magnetic beads. , Nevertheless, initial studies into droplet magnetofluidic methylation biomarker detection prioritized proving the concept.…”

Section: Introductionmentioning

confidence: 99%

Cancer Methylation Biomarker Detection in an Automated, Portable, Multichannel Magnetofluidic Platform

Hasnain,

Stark,

Trick

et al. 2024

ACS Nano

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Early detection of cancer is critical to improving clinical outcomes, especially in territories with limited healthcare resources. DNA methylation biomarkers have shown promise in early cancer detection, but typical workflows require highly trained personnel and specialized equipment for manual and lengthy processing, limiting use in resource-constrained areas. As a potential solution, we introduce the Automated Cartridge-based Cancer Early Screening System (ACCESS), a compact, portable, multiplexed, automated platform that performs droplet magnetofluidic-and methylation-specific qPCR-based assays for the detection of DNA methylation cancer biomarkers. Development of ACCESS focuses on esophageal cancer, which is among the most prevalent cancers in low-and middle-income countries with extremely low survival rates. Upon implementing detection assays for two esophageal cancer methylation biomarkers within ACCESS, we demonstrated successful detection of both biomarkers from esophageal tumor tissue samples from eight esophageal cancer patients while showing specificity in paired normal esophageal tissue samples. These results illustrate ACCESS's potential as an amenable epigenetic diagnostic tool for resource-constrained areas toward early detection of esophageal cancer and potentially other malignancies.

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Integrated microscale immiscible phase extraction and isothermal amplification for colorimetric detection of Neisseria gonorrhoeae

Rodriguez-Mateos,

Ngamsom,

Ameyo

et al. 2023

Anal Bioanal Chem

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Gonorrhea is the second most common sexually transmitted infection (STI) with around 87 million cases worldwide estimated in 2016 by the World Health Organization. With over half of the cases being asymptomatic, potential life-threatening complications and increasing numbers of drug-resistant strains, routine monitoring of prevalence and incidence of infections are key preventive measures. Whilst gold standard qPCR tests have excellent accuracy, they are neither affordable nor accessible in low-resource settings. In this study, we developed a lab-on-a-chip platform based on microscale immiscible filtration to extract, concentrate and purify Neisseria gonorrhoeae DNA with an integrated detection assay based on colorimetric isothermal amplification. The platform was capable of detecting as low as 500 copies/mL from spiked synthetic urine and showed no cross-reactivity when challenged with DNAs from other common STIs. The credit card–size device allows DNA extraction and purification without power or centrifuges, and the detection reaction only needs a low-tech block heater, providing a straightforward and visual positive/negative result within 1 h. These advantages offer great potential for accurate, affordable and accessible monitoring of gonorrhea infection in resource-poor settings.

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Microscale immiscible phase magnetic processing for bioanalytical applications

Cited by 4 publications

References 67 publications

Highly Sensitive Molecular Assay for the Detection of Treponema pallidum infection

Highly Sensitive Molecular Assay for the Detection of Treponema pallidum infection

Cancer Methylation Biomarker Detection in an Automated, Portable, Multichannel Magnetofluidic Platform

Integrated microscale immiscible phase extraction and isothermal amplification for colorimetric detection of Neisseria gonorrhoeae

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