Study On Plankton Cell Counting Technology Based On Microfluidic Microfluorescence

Planktonic algae are the main primary producers in water bodies,water health indicators,the planktonic algae cell count detection,especially the live algae cell count detection,can be the most direct and effective response to their growth status.Fast and accurate measurement of planktonic algae cell count has important scientific and application value for artificial culture,water quality condition monitoring,algal bloom disaster warning,etc.Flow cytofluorimetry has the advantage of fast and accurate,but the injection structure and focusing mode are too cumbersome to be applied to miniaturized field rapid measurements.The flow-controlled chip technology realizes the functions of feeding,focusing and sorting by constructing a microchannel pipeline on a chip of several square centimeters,and this technology simplifies the complex feeding structure of the flow fluorescence method.In view of this,this paper proposes a method for planktonic algal cell density detection based on microfluidics-microfluorescence technology.Using chlorophyll fluorescence in the characteristic wavelength of algal cell stimulated emission,the microfluidic chip and microfluorescence detection technology are combined to achieve rapid and accurate detection of the number of planktonic algal cells and living cells by detecting the fluorescence of single algal cells at a specific volume,and the main research contents and results of the study are shown below.（1）A microfluidic microscopic algal fluorescence measurement system was constructed.Based on the analysis of the difficulties in measuring the fluorescence of planktonic algal cells,the microfluidic system was designed to achieve rapid and accurate sampling of planktonic algal cells;the microscopic fluorescence optical measurement technology was studied,and the confocal microscopic fluorescence optical path was designed to achieve efficient excitation and collection of single-cell fluorescence;the efficient acquisition technology of weak fluorescence was studied,and the The integration amplifier circuit was designed to achieve highly sensitive detection of weak fluorescence signals.Finally,high signal-to-noise ratio acquisition of fluorescence signal of algal cells with particle size above 3μm was achieved.（2）Based on the acquisition of algal cell fluorescence peak information for planktonic algal cell counting,a microfluidic microscopic fluorescence algal cell count measurement method was established,and the measurement range and error sources of the method were analyzed.At low density,the relative errors of the counting results with both microscopic inspection method and Coulter counting method did not exceed3.49%,and the results of testing algal cells of different species and particle sizes showed that the relative errors of the method were less than 3.96%in the measurement within the density range of 1.3×10⁶Cells·L-1,and the accuracy was not affected by the suspended matter,algal cell species and size,within the 10%allowable error The method can be used to increase the upper limit of detection density to5×10⁶Cell·L^-1.（3）The chlorophyll fluorescence method was applied to the live discrimination of single algal cells,and a method based on microfluidic microscopic variable fluorescence live algal cell measurement was studied and established.For the key problems of single algal cell measurement,we focused on variable light pulse technology and weak variable fluorescence oversampling detection technology to achieve high sensitivity measurement of variable fluorescence of single algal cells;using round sieve algae solution as the experimental object,the method of microscopic inspection of live staining was compared,and the results showed that the relative error of the method relative to microscopic inspection of live algal counts were not more than4.98%,and the relative standard deviation was The results showed that the relative error of live algae cell density detection results under the mixture of different inactivation ratios was less than 7.37%,and the detection results showed that the method has good accuracy in different ratios of live algae.（4）The microfluidic microfluorescence algal cell count measurement method was applied to the outfield Chaohu Lake water sample test and the study of flocculation and algae removal materials,and the microscopic examination method was used for comparison to verify the accuracy of the practical application of the method,and to study the algae removal performance and optimal dosage of flocculation and algae removal materials.The results showed that the relative errors in the field water samples were within 9.33%,and the relative errors in the application of the research flocculation and algae removal materials were less than 7.31%,which indicated that the method has good practicality and accuracy,and the algae removal effect of chitosan was found to be better than that of seafoam in the research flocculation and algae removal materials,and the removal rate of chitosan could reach 98.5%at the dosage of 0.8mg·L^-1,and it did not lead to The removal rate of chitosan can reach 98.5%at the dosage of 0.8 mg·L^-1,and it does not lead to turbidity in the water,and the removal rate is more than 90%under the effect of both compound modifications,which has no significant enhancement effect compared with chitosan alone.The microfluidic microfluorescence technique proposed in this paper achieves the counting of planktonic algal cells and living cells by using the unique chlorophyll fluorescence information of algae,which has the advantages of simple feeding module and optical structure,and provides a new way for rapid and accurate detection of algal cell density.