| 1. |
- Changtor, Phanupong, 1994-, et al.
(author)
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Integration of IFAST-based nucleic acid extraction and LAMP for on-chip rapid detection of Agroathelia rolfsii in soil
- 2024
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In: Biosensors & bioelectronics. - 0956-5663 .- 1873-4235. ; 250
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Journal article (peer-reviewed)abstract
- Agroathelia rolfsii (A. rolfsii) is a fungal infection and poses a significant threat to over 500 plant species worldwide. It can reduce crop yields drastically resulting in substantial economic losses. While conventional detection methods like PCR offer high sensitivity and specificity, they require specialized and expensive equipment, limiting their applicability in resource -limited settings and in the field. Herein, we present an integrated workflow with nucleic acid extraction and isothermal amplification in a lab -on -a -chip cartridge based on immiscible filtration assisted by surface tension (IFAST) to detect A. rolfsii fungi in soil for point -of -need application. Our approach enabled both DNA extraction of A. rolfsii from soil and subsequent colorimetric loop -mediated isothermal amplification (LAMP) to be completed on a single chip, termed IFAST-LAMP. LAMP primers targeting ITS region of A. rolfsii were newly designed and tested. Two DNA extraction methods based on silica paramagnetic particles (PMPs) and three LAMP assays were compared. The best -performing assay was selected for on -chip extraction and detection of A. rolfsii from soil samples inoculated with concentrations of 3.75, 0.375 and 0.0375 mg fresh weight per 100-g soil (%FW). The full on -chip workflow was achieved within a 1-h turnaround time. The platform was capable of detecting as low as 3.75 %FW at 2 days after inoculation and down to 0.0375 %FW at 3 days after inoculation. The IFAST-LAMP could be suitable for field -applicability for A. rolfsii detection in low -resource settings.
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| 2. |
- Ngamsom, Bongkot, et al.
(author)
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A sample-to-answer COVID-19 diagnostic device based on immiscible filtration and CRISPR-Cas12a-assisted detection
- 2022
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In: Talanta Open. - : Elsevier BV. - 2666-8319. ; 6
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Journal article (peer-reviewed)abstract
- In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip device, namely ‘IFAST-LAMP-CRISPR’, as an affordable, rapid and high-precision molecular diagnostic means for detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The herein proposed ‘sample-to-answer’ platform integrates RNA extraction, amplification and molecular detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and guanidine hydrochloride, streamline sample preparation (including RNA extraction, concentration and purification) in 15 min with minimal hands-on steps. The pre-amplification in combination with CRISPR-Cas12a detection assays targeting the nucleoprotein (N) gene achieved visual identification of ≥ 470 copies mL−1 genomic SARS-CoV-2 samples in 45 min. On-chip assays showed the ability to isolate and detect SARS-CoV-2 RNA from 100 genome copies mL−1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity- and sensitivity-comparable alternative to the costly gold-standard reverse transcription-polymerase chain reaction (RT-PCR) assay, requiring only a simple heating source. Initial testing illustrates the platform viability both on nasopharyngeal swab and saliva samples collected using the easily accessible Swan-brand cigarette filter, providing a complete workflow for COVID-19 diagnostics in low-resource settings.
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| 3. |
- Rodriguez Mateos, Pablo, 1995-, et al.
(author)
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Integrated microscale immiscible phase extraction and isothermal amplification for colorimetric detection of Neisseria gonorrhoeae
- 2023
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In: Analytical and Bioanalytical Chemistry. - 1618-2642 .- 1618-2650. ; 415:21, s. 5129-5137
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Journal article (peer-reviewed)abstract
- 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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| 4. |
- Rodriguez Mateos, Pablo, 1995-, et al.
(author)
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Microscale immiscible phase magnetic processing for bioanalytical applications
- 2023
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In: TrAC. Trends in analytical chemistry. - : Elsevier BV. - 0165-9936 .- 1879-3142. ; 158
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Research review (peer-reviewed)abstract
- Bioanalytical processes such as ligand-binding assays, nucleic acid amplification tests and cancer cell identification methods require multiple steps including sampling, transport, sample preparation, chemical reaction/s, detection and interpretation. These necessitate trained personnel and laboratory infrastructure, and are often time-consuming and labor-intensive. Integrated platforms capable of combining the aforementioned steps are generally challenging to realize. Approaches employing immiscible phases constrained in the microscale and paramagnetic particles have emerged as pathways to extract proteins, nucleic acids and whole cells from complex matrices via affinity purification and magnetic actuation. In recent years, further steps for detection and readout of these bioanalytes have been incorporated, providing versatile lab-on-a-chip platforms for multiple integrated bioanalytical workflows and with great potential as point-of-need devices. In this manuscript, we review and summarize the different strategies employed and lay out further avenues for development and implementation.
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