Using Acoustic Differential Extraction to enhance analysis of sexual assualt evidence on a valveless glass microdevice
Författare
Redaktör
- Takehiko Kitamori
- Hiroyuki Fujita
- Shinji Hasabe
Summary, in English
The isolation of male and female DNA is an important step in the analysis of sexual assault
evidence. A vaginal swab with female epithelial cells and male sperm cells is obtained from the
female, and it is vital to separate the male and female fractions in order to obtain a single-source
DNA profile of the suspect. In the case of a low abundance of sperm cells, it is very important that
no cells are lost in the isolation step.
The conventional isolation method used in the forensic DNA laboratories, differential
extraction, is a time-consuming step, requiring up to 24 hours. It is neither highly amenable to
automation, nor can it be easily integrated with other steps of the analysis. Therefore, a novel
method of performing the isolation of male and female fractions of biological material from sexual
assault evidence has been developed, termed acoustic differential extraction (ADE).
After selectively lysing the female epithelial cells while keeping the sperm cells intact, the
sample, now containing sperm cells and female cell lysate, is infused in a 900 μm wide and 70 μm
deep microfabricated glass channel with miniature piezoelectric transducers mounted at the bottom
of the channel, as shown in Figure 1. Upon activation of the ultrasound, the sperm cells will be
trapped in a standing wave1 while free DNA will not be retained. The sperm cells, levitated in the
3D fluidic space above the transducer, can be washed with buffer and the unretained biological
material directed to an output reservoir. Using laminar flow valving2, the sperm cells can be
released and directed into a separate output reservoir in anticipation of DNA analysis, see Figure 2.
With the purpose of evaluating the ADE microdevice for the collection of the two output
fractions (male and female), preliminary work used a mock sexual assault sample created with
polystyrene microparticles as sperm cells and Evan's Blue dye as female cell lysate. The particles
were trapped over the transducer and the dye was directed to the female outlet reservoir as shown
in Figure 3. After washing the particles, the ultrasound was deactivated and the flow redirected in
order to collect the particles in the male outlet reservoir.
A biological sample consisting of sperm cells and lysed female epithelial cells was
subsequently tested by infusion into the device for five minutes while trapping the sperm cells over
the transducer (Figure 4). The trapped sperm cells were washed with PBS for five minutes, then
released and collected for analysis off-chip. DNA from the isolated cells was extracted with a
commercial DNA extraction kit and analyzed with a duplex quantitative PCR assay3 to show the
sample purity. An example of the qPCR data obtained is provided in Table 1.
The results show that a highly-enriched sperm cell fraction can be obtained with the ADE
technique. It is reasonable to expect that this technique can be integrated with on-chip downstream
sample processing, e.g. DNA extraction and amplification. This would greatly diminish the analysis
time from 24 hours to approximately 60 minutes. The time savings, in combination with the
possibility to create a fully automated system, gives the ADE technique the potential to significantly
alter the means by which sexual assault evidence is processed in crime laboratories today.
evidence. A vaginal swab with female epithelial cells and male sperm cells is obtained from the
female, and it is vital to separate the male and female fractions in order to obtain a single-source
DNA profile of the suspect. In the case of a low abundance of sperm cells, it is very important that
no cells are lost in the isolation step.
The conventional isolation method used in the forensic DNA laboratories, differential
extraction, is a time-consuming step, requiring up to 24 hours. It is neither highly amenable to
automation, nor can it be easily integrated with other steps of the analysis. Therefore, a novel
method of performing the isolation of male and female fractions of biological material from sexual
assault evidence has been developed, termed acoustic differential extraction (ADE).
After selectively lysing the female epithelial cells while keeping the sperm cells intact, the
sample, now containing sperm cells and female cell lysate, is infused in a 900 μm wide and 70 μm
deep microfabricated glass channel with miniature piezoelectric transducers mounted at the bottom
of the channel, as shown in Figure 1. Upon activation of the ultrasound, the sperm cells will be
trapped in a standing wave1 while free DNA will not be retained. The sperm cells, levitated in the
3D fluidic space above the transducer, can be washed with buffer and the unretained biological
material directed to an output reservoir. Using laminar flow valving2, the sperm cells can be
released and directed into a separate output reservoir in anticipation of DNA analysis, see Figure 2.
With the purpose of evaluating the ADE microdevice for the collection of the two output
fractions (male and female), preliminary work used a mock sexual assault sample created with
polystyrene microparticles as sperm cells and Evan's Blue dye as female cell lysate. The particles
were trapped over the transducer and the dye was directed to the female outlet reservoir as shown
in Figure 3. After washing the particles, the ultrasound was deactivated and the flow redirected in
order to collect the particles in the male outlet reservoir.
A biological sample consisting of sperm cells and lysed female epithelial cells was
subsequently tested by infusion into the device for five minutes while trapping the sperm cells over
the transducer (Figure 4). The trapped sperm cells were washed with PBS for five minutes, then
released and collected for analysis off-chip. DNA from the isolated cells was extracted with a
commercial DNA extraction kit and analyzed with a duplex quantitative PCR assay3 to show the
sample purity. An example of the qPCR data obtained is provided in Table 1.
The results show that a highly-enriched sperm cell fraction can be obtained with the ADE
technique. It is reasonable to expect that this technique can be integrated with on-chip downstream
sample processing, e.g. DNA extraction and amplification. This would greatly diminish the analysis
time from 24 hours to approximately 60 minutes. The time savings, in combination with the
possibility to create a fully automated system, gives the ADE technique the potential to significantly
alter the means by which sexual assault evidence is processed in crime laboratories today.
Avdelning/ar
Publiceringsår
2006
Språk
Engelska
Sidor
1055-1057
Publikation/Tidskrift/Serie
Proceedings of µTAS 2006 Conference
Volym
2
Dokumenttyp
Konferensbidrag
Förlag
Society for Chemistry and Micro-Nano Systems
Ämne
- Medical Engineering
Nyckelord
- differential extraction
- forensic science
- Acoustic trapping
Conference name
Micro Total Analysis Systems 2006
Conference date
2006-11-05 - 2006-11-09
Status
Published
ISBN/ISSN/Övrigt
- ISBN: 4-9903269-0-3-C3043