The threat of biological and chemical terror acts remains a growing worldwide concern. There is therefore a need to develop appropriate technology for the detection of chemical and biological warfare agents, with early identification intended for use by first responders. Here, we disclose the developed autonomous air sampling and detection system for evaluation of the presence of chemical and biological warfare agents that can be harmful to the population. The current device utilizes the designed mist generator-assisted air collection system (338 l min − 1 ) and biosensing chip technologies, such as electrochemical measurement, Au nanoparticle-based localized surface plasmon resonance, and rapid microfluidic chip PCR for detection of minute concentrations lower than the mean lethal dose (LD 50 ) of nerve gases (sarin and VX), toxic proteins (BTX/A/Hc and ricin), and pathogens (anthrax simulant). An operation time of only 5-15 min is needed for the collection and detection; sample preparation is already integrated into the system without the need for direct human intervention. In addition to the system's sensitivity and ease of use, its portability makes it highly beneficial for first responders, which could aid in immediate risk assessment and mitigation of on-site events.
Here, we report the developed cyclo olefin polymer (COP) microfluidic chip on a fabricated rotating heater stage that utilizes centrifugation-assisted thermal cycle in a ring-structured microchannel for polymerase chain reaction (PCR). The PCR solution could be driven by thermal convection and continuously exchanged high/low temperatures in a ring structured microchannel without the use of typical syringe pump. More importantly, the flow rate was controlled by the relative gravitational acceleration only. The platform enables amplification within 10 min at 5G and has a detection limit of 70.5 pg/channel DNA concentration (β-actin, 295 bp). The current rotating system is capable of testing four different samples in parallel. The microfluidic chip can be preloaded with the PCR premix solution for on-site utility, and, with all of the features integrated to the system, the test can be conducted without the need for specialized laboratory and trained laboratory staff. In addition, this innovative chemical reaction technique has the potential to be utilized in other micromixing applications.
Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment.Methods: A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept.Results: GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade.Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.
Drug screening and profiling is an important phase in drug discovery, development, and marketing. However, some profiling tests are not routinely done because of the needed additional technical skills and costly maintenance, which leads to cases of unexpected side effects or adverse drug reactions (ADRs). This study presents the design and operation of a microfluidic chip for single-cell level drug screening and profiling as an alternative platform for this purpose. Centrifugation was utilized to trap isolated single and groups of primary cultured neonatal rat cardiomyocytes in the same chip. In the off-spin operation of the chip, the cells can be observed under a microscope and movies of the beat motion can be recorded. The beat profiles of the cells were generated by image correlation analysis of the recorded video to study the contractile characteristics (beating rate, beating strength, and inter-beat duration). By utilizing this non-invasive tool, long term continuous monitoring, right after trapping, was made possible and cell growth and dynamics were successfully observed in the chip. Media and liquid replacement does not require further centrifugation but instead utilizes capillary flow only. The effect of carbachol (100 μM) and isoproterenol (4 μg mL(-1)) on single cells and groups of cells was demonstrated and the feature for immunostaining (β-actin) applicability of the chip was revealed. Furthermore, these findings can be helpful for the headway of non-invasive profiling of cardiomyocytes and for future chip design and operation of high-throughput lab-on-a-chip devices.
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