A microfluidic impedance cytometry device for robust identification of H. pluvialis.

H. pluvialis contains rich oleic acid and astaxanthin, which have important applications in the fields of biodiesel and biomedicine. Detection of live H. pluvialis is the prerequisite to obtaining oleic acid and astaxanthin. For this purpose, we successfully developed a reliable microfluidic impedance cytometry for the identification of live H. pluvialis. Firstly, we established a simulation model for detecting H. pluvialis based on their morphology and studied the effect of medium conductivity on the impedance of H. pluvialis at different frequencies. From the simulations, we determined that the optimal solution conductivity for the detection of H. pluvialis was 1500 µS cm-1 and studied the frequency responses of the impedance of H. pluvialis. Secondly, we fabricated the microchannels and stainless-steel detection electrodes and assembled them into microfluidic impedance cytometry. The frequency dependence of live and dead H. pluvialis was explored under different frequencies, and live and dead H. pluvialis were distinguished at a frequency of 1 MHz. The impedance of live H. pluvialis at the frequency of 1 MHz ranges from 33.73 to 52.23 Ω, while that of dead ones ranges from 13.05 to 19.59 Ω. Based on these findings, we accomplished the identification and counting of live H. pluvialis in the live and dead sample solutions. Furthermore, we accomplished the identification and counting of live H. pluvialis in the mixed samples containing Euglena and H. pluvialis. This approach possesses the promising capacity to serve as a robust tool in the identification of target microalgae, addressing a challenge in the fields of biodiesel and biomedicine.

Dielectrophoretic characterization and selection of non-spherical flagellate algae in parallel channels with right-angle bipolar electrodes.

Non-spherical flagellate algae play an increasingly significant role in handling problematic issues as versatile biological micro/nanorobots and resources of valuable bioproducts. However, the commensalism of flagellate algae with distinct structures and constituents causes considerable difficulties in their further biological utilization. Therefore, it is imperative to develop a novel method to realize high-efficiency selection of non-spherical flagellate algae in a non-invasive manner. Enthused by these, we proposed a novel method to accomplish the selection of flagellate algae based on the numerical and experimental investigation of dielectrophoretic characterizations of flagellate algae. Firstly, an arbitrary Lagrangian-Eulerian method was utilized to study the electro-orientation and dielectrophoretic assembly process of spindle-shaped and ellipsoid-shaped cells in a uniform electric field. Secondly, we studied the equilibrium state of spherical, ellipsoid-shaped, and spindle-shaped cells under positive DEP forces actuated by right-angle bipolar electrodes. Thirdly, we investigated the dielectrophoretic assembly and escape processes of the non-spherical flagellate algae in continuous flow to explore their influences on the selection. Fourthly, freshwater flagellate algae (Euglena, H. pluvialis, and C. reinhardtii) and marine ones (Euglena, Dunaliella salina, and Platymonas) were separated to validate the feasibility and adaptability of this method. Finally, this approach was engineered in the selection of Euglena cells with high viability and motility. This method presents immense prospects in the selection of pure non-spherical flagellate algae with high motility for chronic wound healing, bio-micromotor construction, and decontamination with advantages of no sheath, strong reliability, and shape-insensitivity.

Microfluidic impedance cytometry with flat-end cylindrical electrodes for accurate and fast analysis of marine microalgae.

Marine microalgae play an increasingly significant role in addressing the issues of environmental monitoring and disease treatment, making the analysis of marine microalgae at the single-cell level an essential technique. For this, we put forward accurate and fast microfluidic impedance cytometry to analyze microalgal cells by assembling two cylindrical electrodes and microchannels to form a three-dimensional detection zone. Firstly, we established a mathematical model of microalgal cell detection based on Maxwell's mixture theory and numerically investigated the effects of the electrode gap, microalgal positions, and ion concentrations of the solution on detection to optimize detection conditions. Secondly, 80 µm stainless steel wires were used to construct flat-ended cylindrical electrodes and were then inserted into two collinear channels fabricated using standard photolithography techniques to form a spatially uniform electric field to promote the detection throughput and sensitivity. Thirdly, based on the validation of this method, we measured the impedance of living Euglena and Haematococcus pluvialis to study parametric influences, including ion concentration, cell density and electrode gap. The throughput of this method was also investigated, which reached 1800 cells per s in the detection of Haematococcus pluvialis. Fourthly, we analyzed live and dead Euglena to prove the ability of this method to detect the physiological status of cells and obtained impedances of 124.3 Ω and 31.0 Ω with proportions of 15.9% and 84.1%, respectively. Finally, this method was engineered for the analysis of marine microalgae, measuring living Euglena with an impedance of 159.61 Ω accounting for 3.9%, dead Euglena with an impedance of 36.43 Ω accounting for 10.1% and Oocystis sp. with an impedance of 55.00 Ω accounting for about 81.0%. This method could provide a reliable tool to analyze marine microalgae for monitoring the marine environment and treatment of diseases owing to its outstanding advantages of low cost, high throughput and high corrosion resistance.

[Study on molecular epidemiology of Mycobacterium tuberculosis in Chinese army with PCR amplified fingerprinting methods].

OBJECTIVE: Typing of Mycobacterium tuberculosis strains and epidemiological studies in the army of southern China to provide scientific basis for prevention of pulmonary tuberculosis. METHODS: A rapid fingerprinting of M. tuberculosis strains method by polymerase chain reaction (PCR) with outward-directed primers that designed to the ends of the insertion sequence IS6110 was developed, and to analyze the relationship between the polymorphism of DNA fingerprinting and epidemiology of M. tuberculosis. RESULTS: One hundred and fifty-four M. tuberculosis detected were classified into eight types according to their characters of PCR amplified fingerprints. The main types were type I (36.4%), type II (31.8%), and type III (21.4%), while other types were less than 4 percentage. In those main type groups, patients aged 20 to 29 and 30 to 39 took up 31.8% and 27.9% respectively. For those main types, the distribution of those types in the first treated patients showed significant difference compared with that in the retreated patients, and the rate of drug-resistance was also statistically different. However, the distribution was not statistically significant to history of BCG vaccination and patients living in urban or rural area. The main drug-resistant strains were only Isoniazid-resistant or Rifampin-resistant strains, while the drug-resistant strains were 44.4%, 29.6% and 14.8% respectively in type I, type II and type III. CONCLUSION: PCR fingerprinting was a rapid, precise, sensitive, specific method to type M. tuberculosis, and could be used to study the epidemiology of tuberculosis; The prevalence of tuberculosis was primarily due to the transmission of type I, type II and type III in the army being studied from Southern China, to suggest that surveillance needs to be strengthened.