The Design And Implementation Of Microfluidic Chip Electrophoresis Fluorescence Detection System Based On Grating

An optical grating based and low-cost microfluidic chip electrophoresis fluorescence detection device has been designed in our work.Laser induced fluorescence(LIF)method was used in the device.We used 488nm all solid-state laser as excitation light source and 6001/mm holographic diffractive diffraction grating as dispersive element.The final spectral image was detected by CCD.In our experiments,DNA fragments was successfully separated and detected by the device.The device consisted of optical system and DNA analysis software,to provide microfluidic chip electrophoresis hardware platform and DNA map analysis algorithm respectively.The specific contents of this paper were as follows:(1)Using Zemax to simulate the grating.Explored the imaging quality under different light incident angle,grating parameters,focusing lens type,and the focal length of lens.The simulation results showed that the spectral resolution was positively correlated with the incident angle of the light,the engraved lines of the grating and the focal length of the focusing lens.The quality of image used by double glued achromatic focusing lens was higher than that uesd by normal focusing lens.According to the resluts of simulation,we finally selected 10.4° as fluorescence incidence angle and 60mm double glued achromatic focusing lens as convergence lens.(2)Introduced the design principle of optical system,characteristics of optical elements and optical path combination model.Then elaborated the optical construction process and the debugging method.Optical debugging contained unit degugging and system coordination.Optical power meter has been used as debugging tool to debug the excitation optical path and mercury lamp was used as light source to debug the dispersion optical path.Then the fluorescence spectral image of 10'6 mol/L fluorescein sodium has been obtained,verifmg the effection of the detection device.(3)Tested the sensitivity of the system.We used 10-7 mol/L,10-8 mol/L,10-9 mol/L fluorescein sodium as samples,analyzing the spectral images under different laser power,solution concentration and exposure time.The results indicated that fluorescence signal of 10-9 mol/L fluorescein sodium could still be detected at laser power of 1.2mW and 50ms exposure time.The calculated SNR was 168.44dB and SBR was 1.82×105.That implied the detection limit reached 10-9 mol/L.(4)Mercury lamp was selected to complete the wavelength calibration of the system.And the peak position of mercury lamp spectrum has been calculated by direct reading method,centroid method and fitting method respectively.According to the linear dispersion characteristics of grating,we confirmed the first-order linear relationship between spectral wavelength and the position of the CCD imaging pixel.A veriety of fitting methods were compared.Finally,we found the Gaussian function together with the first order polynomial fitting was best,with the smallest SSE of 0.00323 and used this result as the final calibration function.In addition,the spectral resolution of the system was studied.We referred the minimum half-peak width as the spectral resolution and the result of measurement was 0.9889nm.(5)This paper also studied a series of DNA map analysis and processing alogrithms to solve the problem that the resolution of the original map was not high and the signal-to-noise was small.Data segment selection method was applied to extract effective spectral data for reducing invalid signal interference and speeding up map processing.An impoved histogram method is proposed,which combined the traditional histogram method and the segmentation method.With both advantages,base fragments peaks were precisely located on the same baseline.Then we used median filtering,Gaussian filtering and wavelet filtering to reduce the impact of impulse noise,high and low frequency noise,white noise and Gaussian noise.In order to solve the problem of fluorescence spectrum crosstalk and multiple fluorescent substances passing through the detection window at the same time,a spectral correction model with nonnegative constrained least squares(NCLS)was adopted to realize linear resolution of polychromatic spectra.The covariate blind deconvolution method was used to reduce the broadening effect of the base fragments peak and improve the resolution of the base fragments peak.Finally,all the above algorithm has been applied to MATLAB implementation,and integrated into the GUIDE programming software interface.A complete DNA analysis software has been consituted.(6)Finally,the microfluidic chip electrophoresis experiments were carried out.Monochromatic electrophoresis experiments were carried out on the marker which contained 20 base fragments.The results showed 20 base fragments peaks and the base sorting was achieved.The system was able to distinguish a difference of 5 bp of the base fragment.In addition,it was found that the size of the base fragment had a high linear relationship with the detection time.Five samples of five DNA fragments were the analyzed by NCLS,and five base fragments peaks were obtained.The ladder samples of 4 bp and 5 bp fragment differences were subjected to electrophoresis experiments,and the resolved base peaks were achieved.This results showed that the system could distinguish at least 0.26 bp base fragments.