Femtosecond laser-fabricated silicon micropore arrays modified with Ga<sub>2</sub>O<sub>3</sub> nanowires for rapid sperm separation from mixed stains and forensic DNA typing

Ning Shuhua; Yao Yiren; Pu Xiaohe; Wang Qingshan; Liu Shaoxiang; Feng Shuanglong

doi:10.16016/j.2097-0927.202605067

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Femtosecond laser-fabricated silicon micropore arrays modified with Ga₂O₃ nanowires for rapid sperm separation from mixed stains and forensic DNA typing

Basic Medicine | 更新时间：2026-10-19

- Femtosecond laser-fabricated silicon micropore arrays modified with Ga₂O₃ nanowires for rapid sperm separation from mixed stains and forensic DNA typing
- Journal of Army Medical University Vol. 48, Issue 19, Pages: 2745-2754(2026)
- 作者机构：
  
  1.重庆警察学院侦查系，重庆
  2.公安部鉴定中心，北京
  3.重庆市公安局物证鉴定中心，重庆
  4.中国科学院重庆绿色智能技术研究院，重庆
- 作者简介：
  
  Wang Qingshan，E-mail： wqshan105@163.com
  Liu Shaoxiang，E-mail： liushaoxiang@cigit.ac.cn
- 基金信息：
  
  the Key Special Project of Technological Innovation and Application Development of Chongqing(CSTB2024TIAD-KPX0109);the Open Project of Institute of Forensic Science of China(2019CSEEKFKT01);the Innovation and Development Joint Fund of Natural Science Foundation of Chongqing(CSTB2023NSCQ-LZX0173)
- DOI：10.16016/j.2097-0927.202605067
  CLC： D919.2;R321.1;R446.9
- Received：25 May 2026，
  
  Revised：2026-08-02，
  
  Accepted：04 August 2026，
  
  Published：15 October 2026
- 稿件说明：
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宁淑华，姚伊人，蒲小禾，等.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［J］.陆军军医大学学报，2026，48（19）：2745-2754.DOI：10.16016/j.2097-0927.202605067.

Ning S H，Yao Y R，Pu X H，et al.Femtosecond laser-fabricated silicon micropore arrays modified with Ga₂O₃ nanowires for rapid sperm separation from mixed stains and forensic DNA typing［J］.J Army Med Univ，2026，48（19）：2745-2754.DOI：10.16016/j.2097-0927.202605067.
宁淑华，姚伊人，蒲小禾，等.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［J］.陆军军医大学学报，2026，48（19）：2745-2754.DOI：10.16016/j.2097-0927.202605067. DOI：

Ning S H，Yao Y R，Pu X H，et al.Femtosecond laser-fabricated silicon micropore arrays modified with Ga₂O₃ nanowires for rapid sperm separation from mixed stains and forensic DNA typing［J］.J Army Med Univ，2026，48（19）：2745-2754.DOI：10.16016/j.2097-0927.202605067. DOI：

摘要

目的

混合斑是性侵案件中最关键的生物学证据，但因精子（4~6 μm）与阴道上皮细胞（40~60 μm）尺寸差异明显且共存复杂，传统差异裂解法存在操作耗时、上皮细胞DNA残留或精子损耗等问题，难以满足法医高效检验需求。本研究基于细胞尺寸差异与飞秒激光加工技术，利用倒锥形硅基微孔阵列构建双层过滤装置，以实现混合斑中精子与上皮细胞的快速高效分离，并获取完整、无污染的DNA分型。

方法

300 μm单晶硅片作为基底，采用飞秒激光（波长1 030 nm，频率10 kHz，场镜倍数×10，电流6 A，打孔时间0.5 s）加工倒锥形微孔阵列（孔间距100 μm，孔密度10

个/cm

），随机选取9个微孔测量孔径。通过化学气相沉积（960 ℃，2 min）技术生长Ga

纳米线修饰孔道，并构建双层过滤装置：第一层拦截上皮细胞（约20 μm），第二层截留精子（约8 μm）。制备以健康男性精液（1×10

/mL）及女性口腔上皮细胞（1×10

/mL）配制成5组不同比例（1∶1、1∶5、1∶2、3∶10、3∶50）的模拟混合斑样本（

=5），经负压过滤分离后，使用M48磁珠提取精子DNA，采用VeriFiler Plus PCR试剂盒（27个循环）进行短串联重复序列（short tandem repeats，STRs）复合扩增，并以3 500XL基因分析仪进行毛细管电泳分型检测。主要测量指标包括：微孔孔径及其均一性［变异系数（coefficient of variation，CV）］、批次间稳定性（相对偏差）、分离时间及DNA分型图谱完整性。

结果

飞秒激光最佳加工条件下，上孔径约20 μm，下孔径（4.72±0.38）μm（CV=8.05%，

=9），3个独立批次平均下孔径分别为4.68、4.75、4.73 μm（批次间相对偏差

1.5%）。Ga

纳米线生长2 min后，孔径由20 μm缩小至约8 μm［（8.02±0.58）μm，CV=7.23%，

=9］。全部5组不同比例混合样本经双层装置过滤后，均于5~15 min内完成分离（远短于传统差异裂解法的12~24 h）；DNA分型检验显示，23个常染色体STRs位点全部有效检出，

FGA

基因座、

D21S11

基因座等高多态性位点分型完整，杂合子等位基因峰高比值

0.7，峰形对称尖锐，样本图谱与男性志愿者对照完全一致，未见女性上皮细胞分型干扰，背景噪声极低，分型结果准确可靠。

结论

本研究所建立的飞秒激光结合Ga

纳米线修饰的硅基双层微孔分离技术，能够在15 min内高效完成混合斑中精子与上皮细胞的分离，获得纯度及STR分型完整度满足法医鉴定要求的精子DNA，为解决传统差异裂解法耗时长、易污染、精度不足的痛点提供了一种快速、可靠且基层可推广的新技术手段。

Abstract

Objective

Mixed stains are the most critical biological evidence in sexual assault cases. However

due to the marked size difference and c

omplex 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 10

/cm

) 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 Ga

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 (

=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 femto

second laser processing

the upper pore diameter was approximately 20 μm

and the lower pore diameter was (4.72±0.38) μm (CV=8.05%

=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 Ga

nanowire growth (2 min)

the pore diameter decreased from 20 μm to approximately 8 μm (8.02±0.58 μm

CV=7.23%

=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 Ga

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.

关键词

Keywords

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Femtosecond laser-fabricated silicon micropore arrays modified with Ga2O3 nanowires for rapid sperm separation from mixed stains and forensic DNA typing

DOI：10.16016/j.2097-0927.202605067

摘要

Abstract

关键词

Keywords

references

Femtosecond laser-fabricated silicon micropore arrays modified with Ga₂O₃ nanowires for rapid sperm separation from mixed stains and forensic DNA typing