The COVID-19 pandemic has been the most critical public health issue in modern history due to its highly infectious and deathly potential, and the limited access to massive, low-cost, and reliable ...testing has significantly worsened the crisis. The recovery and the vaccination of millions of people against COVID-19 have made serological tests highly relevant to identify the presence and levels of SARS-CoV-2 antibodies. Due to its advantages, microfluidic-based technologies represent an attractive alternative to the conventional testing methodologies used for these purposes. In this work, we described the development of an automated ELISA on-chip capable of detecting anti-SARS-CoV-2 antibodies in serum samples from COVID-19 patients and vaccinated individuals. The colorimetric reactions were analyzed with a microplate reader. No statistically significant differences were observed when comparing the results of our automated ELISA on-chip against the ones obtained from a traditional ELISA on a microplate. Moreover, we demonstrated that it is possible to carry out the analysis of the colorimetric reaction by performing basic image analysis of photos taken with a smartphone, which constitutes a useful alternative when lacking specialized equipment or a laboratory setting. Our automated ELISA on-chip has the potential to be used in a clinical setting and mitigates some of the burden caused by testing deficiencies.
Circulating tumor cells (CTCs) have the potential of becoming the gold standard marker for cancer diagnosis, prognosis and monitoring. However, current methods for its isolation and characterization ...suffer from equipment variability and human operator error that hinder its widespread use. Here we report the design and construction of a fully automated high-throughput fluorescence microscope that enables the imaging and classification of cancer cells that were labeled by immunostaining procedures. An excellent agreement between our machine vision-based approach and a state-of-the-art microscopy equipment was achieved. Our integral approach provides a path for operator-free and robust analysis of cancer cells as a standard clinical practice.
The detection and analysis of circulating tumor cells (CTCs) may enable a broad range of cancer-related applications, including the identification of acquired drug resistance during treatments. ...However, the non-scalable fabrication, prolonged sample processing times, and the lack of automation, associated with most of the technologies developed to isolate these rare cells, have impeded their transition into the clinical practice. This work describes a novel membrane-based microfiltration device comprised of a fully automated sample processing unit and a machine-vision-enabled imaging system that allows the efficient isolation and rapid analysis of CTCs from blood. The device performance was characterized using four prostate cancer cell lines, including PC-3, VCaP, DU-145, and LNCaP, obtaining high assay reproducibility and capture efficiencies greater than 93% after processing 7.5 mL blood samples spiked with 100 cancer cells. Cancer cells remained viable after filtration due to the minimal shear stress exerted over cells during the procedure, while the identification of cancer cells by immunostaining was not affected by the number of non-specific events captured on the membrane. We were also able to identify the androgen receptor (AR) point mutation T878A from 7.5 mL blood samples spiked with 50 LNCaP cells using RT-PCR and Sanger sequencing. Finally, CTCs were detected in 8 out of 8 samples from patients diagnosed with metastatic prostate cancer (mean ± SEM = 21 ± 2.957 CTCs/mL, median = 21 CTCs/mL), demonstrating the potential clinical utility of this device.
Microfluidics is a multidisciplinary field that is changing the way how biological research is being performed, enabling new applications and accelerating the existing ones. However, one of its most ...important drawbacks is the lack of technological tools to facilitate its widespread use. In this paper we present a new pressure and flow control device for the high-performance control of microfluidic applications. The main advantage of our device is that all pneumatic and electronic components, including the communications and control circuitry, are fully embedded inside the apparatus. Therefore, this device can be used as a standalone laboratory equipment and it can also be remotely operated from a personal computer. The microfluidic application used to demonstrate the performance of the proposed tool is the generation of alginate (1% sodium alginate solution) droplets in mineral oil. The generation of a stable stream of uniform droplets validates the good performance of our control device.
The detection and analysis of circulating tumor cells (CTCs) may enable a broad range of cancer-related applications, including the identification of acquired drug resistance during treatments. ...However, the non-scalable fabrication, prolonged sample processing times, and the lack of automation, associated with most of the technologies developed to isolate these rare cells, have impeded their transition into the clinical practice. This work describes a novel membrane-based microfiltration device comprised of a fully automated sample processing unit and a machine-vision-enabled imaging system that allows the efficient isolation and rapid analysis of CTCs from blood. The device performance was characterized using four prostate cancer cell lines, including PC-3, VCaP, DU-145, and LNCaP, obtaining high assay reproducibility and capture efficiencies greater than 93% after processing 7.5 mL blood samples from healthy donors, spiked with 100 cancer cells. Cancer cells remained viable after filtration due to the minimal shear stress exerted over cells during the procedure, while the identification of cancer cells by immunostaining was not affected by the number of non-specific events captured on the membrane. We were also able to identify the androgen receptor (AR) point mutation T878A from 7.5 mL blood samples spiked with 50 LNCaP cells using RT-PCR and Sanger sequencing. Finally, CTCs were detected in 8 of 8 samples from patients diagnosed with metastatic prostate cancer (mean \(\pm\) SEM = 21 \(\pm\) 2.957 CTCs/mL, median = 21 CTC/mL), thereby validating the potential clinical utility of the device.
Celery (Apium graveolens var. dulce) is affected by several plant diseases, such as Fusarium oxysporum f. sp. apii (Foa). Four Foa races have been found in the US. The goals of this study were to ...determine which races are present in Costa Rica and to quantify the tolerance of the imported commercial cultivars of celery produced in the country. Isolates from 125 symptomatic celery plants from three different geographical locations were analyzed, 65 of which were selected for phylogenetic analysis. All isolates presented a short sequence of five nucleotides that differentiates Foa race 3 in the IGS rDNA region. Three different haplotypes closely related to race 3 were found, which were highly virulent, produced great losses, and affected all cultivars (resistant to races 2 and 4) of imported commercial celery. Additionally, five different cultivars of celery were evaluated against seven pathogen isolates identified as race 3 in greenhouse conditions. Two of the cultivars showed significantly less chlorosis, wilting, mortality, and higher fresh weight. Most of the Foa isolates significantly increased chlorosis, wilting, and mortality compared to non-inoculated control. Celery producers in Costa Rica lack access to seeds resistant to the Foa race 3 present in the country.
Celery (Apium graveolens var. dulce) is affected by several plant diseases, such as Fusarium oxysporum f. sp. apii (Foa). Four Foa races have been found in the US. The goals of this study were to ...determine which races are present in Costa Rica and to quantify the tolerance of the imported commercial cultivars of celery produced in the country. Isolates from 125 symptomatic celery plants from three different geographical locations were analyzed, 65 of which were selected for phylogenetic analysis. All isolates presented a short sequence of five nucleotides that differentiates Foa race 3 in the IGS rDNA region. Three different haplotypes closely related to race 3 were found, which were highly virulent, produced great losses, and affected all cultivars (resistant to races 2 and 4) of imported commercial celery. Additionally, five different cultivars of celery were evaluated against seven pathogen isolates identified as race 3 in greenhouse conditions. Two of the cultivars showed significantly less chlorosis, wilting, mortality, and higher fresh weight. Most of the Foa isolates significantly increased chlorosis, wilting, and mortality compared to non-inoculated control. Celery producers in Costa Rica lack access to seeds resistant to the Foa race 3 present in the country.
The existence of circulating endothelial progenitor cells (EPC) in adult humans is under intensive investigation due to its potential clinical application. In the present study we have attempted to ...identify the EPC with colony forming capacity and compare their characteristics with those of the monocytic-macrophage lineage.
42 healthy donors were analysed (7 Bone Marrow, 20 steady-state Peripheral Blood (PB), 4 apheresis products from mobilised PB and 9 buffy-coat products). Median age was 38 years and M/F ratio was 19/23. EPC were obtained by culturing MNC in IMDM with VEGF and beta-FGF. At day 7 the colonies were counted and flow cytometry and immunohistochemistry studies were carried out. Sequential studies were performed on days +21, +28 and +35 of culture with vascular growth factors. Monocytic cells were obtained by adhesion to plastic surface or CD14+ cell selection using magnetic microbeads (Miltenyi, Biotech). Then, monocytic cells were cultured using the same conditions as EPC until day +21 or alternatively with VEGF, beta-FGF and IGF. Von Willebrand gene expression was analysed by RT-PCR and the formation of vascular structures was analyzed by Matrigel assays on both cell sources, EPC and monocytic cells. The mean number of EPC colonies at day 7 was significantly higher in BM (813±695) than in steady-state PB (21.2±2.5), while mobilized PB displayed intermediate values (272±274). By contrast, using the same medium as EPC, monocytic cells did not form colonies at day 7, but cord-like structures could be seen in 7 out 9 cases. However, when the cells were cultured by adding IGF to the medium, a greater number of colonies could be observed. By immunohistochemistry colonies were positive for CD45, CD31 and lysozyme but negative for vWf. Flow cytometry analysis showed that colonies were positive for CD4, CD13, CD14, CD31, CD33, CD45, lysozyme and VE-cadherin, weak positive for CD15 and CD105, and did not express CD16, CD34, CD133 or KDR. This phenotypic profile remained unchanged at all time-points analysed. The immunophenotype of cultured monocytes at day +21 was identical to that of pre-cultured monocytes and both were similar to those obtained in EPC, even for the “specific” markers lysozyme and VE-cadherin. PCR analysis showed small amounts of vWF transcripts in EPC as well as in monocytes but the expression increased after culture with VEGF. Finally, when matrigel assays were carried out, we observed that monocytes formed cord- and tubular-like structures to a higher extent than EPC. Our results in both cell subtypes suggest that the so “called” EPC belongs to the monocytic cell lineage and both populations express some “specific” endothelial antigens such as CD31 or VE-cadherin, as well as monocytic markers such as lysozyme.
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