飞秒激光加工结合Ga2O3纳米线修饰硅基微孔阵列用于混合斑精子快速分离与法医学DNA分型检测OA

Objective Mixed stains are the most critical biological evidence in sexual assault cases.However,due to the marked size difference and complex coexistence between spermatozoa(4-6 µm)and vaginal epithelial cells(40-60 µm),conventional differential lysis suffers from time-consuming operation,residual epithelial cell DNA,or sperm loss,making it difficult to meet the demands of efficient forensic examination.This study aimed to prepare a double-layer filter using an inverted conical silicon-based micropore array to achieve rapid and efficient separation of spermatozoa from epithelial cells in mixed stains based on cell size differences and femtosecond laser processing technology,thereby obtaining complete and uncontaminated DNA typing.Methods Inverted-taper micropore arrays(pore spacing 100 µm,pore density 104/cm2)were fabricated on 300-µm single-crystal silicon wafers using femtosecond laser(wavelength 1 030 nm,frequency 10 kHz,magnification×10,current intensity 6 A,drilling time 0.5 s),with 9 micropores randomly selected for pore size measurement,and further modified by Ga2O3 nanowires via chemical vapor deposition(960℃,2 min).Thus,a double-layer filter was assembled:Layer Ⅰ intercepted epithelial cells(approximately 20 µm),and Layer Ⅱ retained spermatozoa(approximately 8 µm).Five groups of simulated mixed stain samples(n=5)were prepared by mixing healthy male semen(1×10⁴/mL)and female oral epithelial cells(1×10⁴/mL)at ratios of 1∶1,1∶5,1∶2,3∶10,and 3∶50),and then filtered under negative pressure.Sperm DNA was extracted with M48 magnetic beads,short tandem repeats(STRs)were amplified using the VeriFiler Plus PCR kit(27 cycles),and typed by capillary electrophoresis on a 3500XL genetic analyzer.The main outcome measures included micropore diameter and its uniformity[coefficient of variation(CV)],inter-batch stability(relative deviation),separation time,and completeness of DNA typing.Results Under optimal conditions of femtosecond laser processing,the upper pore diameter was approximately 20 µm,and the lower pore diameter was(4.72±0.38)µm(CV=8.05%,n=9);the mean lower pore diameters of 3 independent batches were 4.68,4.75,and 4.73 µm,respectively(inter-batch RSD<1.5%).After Ga2O3 nanowire growth(2 min),the pore diameter decreased from 20 µm to approximately 8 µm(8.02±0.58 µm,CV=7.23%,n=9).For all 5 groups of mixed stain samples at different ratios,separation was completed within 5 to 15 min using the constructed double-layer filter(far shorter than the 12 to 24 h required by conventional differential lysis).DNA typing showed that all 23 autosomal STR loci were effectively detected,with complete typing of high polymorphism loci such as FGA and D21S11 loci,heterozygous peak height ratios>0.7,and symmetric and sharp peaks.The sample map was completely consistent with those of the male volunteer controls,with no interference from female epithelial cell typing,extremely low background noise,and accurate and reliable typing results.Conclusion A silicon-based double-layer filter is successfully constructed with femtosecond laser-processing technology and Ga2O3 nanowire modification.The device can efficiently complete the separation of spermatozoa from epithelial cells in mixed stains within 15 min,obtaining sperm DNA with purity and STR profiling completeness that meet forensic identification requirements.This provides a rapid,reliable,and widely applicable new technology for addressing the pain points of conventional differential lysis,such as long-time consumption,easy contamination,and insufficient accuracy.