One out of every six American women has been the victim of a sexual assault in their lifetime. The failure to test and analyze evidence connected to sexual assault in a timely manner constitutes a growing problem for victims, public safety, and the criminal justice system. Expanded awareness the power of forensic technology to help in solving crimes creates new needs for scientific advances in the field.
In practice, processing of evidence from sexual assault kits generally requires separation Raghu man dating dna collection programs the victim's cells from the perpetrator's cells. As a result, they are not widely in use in community. The channels are then washed and sperm cells are specifically captured, while epithelial cells are removed due to their larger size and lack of an adhesion molecule on the channel surface.
A recent study identified an oligosaccharide i. Given that SLeX targets the sperm head, binding and capture of sperm was independent of sperm morphology. Specifically, sperm without a tail were also captured with SLeX agent primarily interacting with the sperm head.
Evaluation of SLeX binding kinetics and binding locations on sperm head. These experimental findings confirmed our results observed in silico, indicating that SLeX targets sperm head and its binding is independent of distinct sperm morphologies. To efficiently capture sperm in microchannels, we integrated SLeX with a microfluidic technology. Briefly, we designed microchannels that consist of three layers: Capture efficiency was assessed by varying three main parameters: We observed higher capture efficiencies at 0.
Further, this may point to a potential steric hindrance in higher mediator concentrations for SLeX immobilization.
As reported in the literature, more densely packed layers revealed lower surface activity. Therefore, we determined to use 0. In addition, we utilized BSA as a blocking agent, which has ideally dual To minimize nonspecific binding, we also utilized BSA in the specificity experiments to capture sperm from complex heterogeneous cell population including epithelial cells.
This experimental set achieved
Raghu man dating dna collection programs Next, we evaluated the effect of SLeX concentrations varying from 0. We observed that the increase in SLeX concentration enhanced sperm capture efficiency, and the highest SLeX concentration 0.
Overall, the highest sperm capture efficiency was achieved using i 0. We applied these parameters to the following experimental designs to capture sperm. In the experiments, we also observed that sperm cells were tightly captured in microchannels. Although sperm were trying to Raghu man dating dna collection programs, they were also stuck to the channel surface due to high capture capacity of SLeX material Video S2, Supporting Information.
Evaluation of surface chemistry and microfluidic chip parameters for sperm capture. Capture efficiency was evaluated by varying
Raghu man dating dna collection programs parameters: Further, this might be due to potential steric hindrance for SLeX immobilization to the surface. Further, the capture efficiency in 0. By performing contact angle measurements, we evaluated hydrophilicity properties after surface modification inset figure. The contact angle value of the bare glass surface altered from Sperm counts before and after the washing step were plotted through horizontal and vertical directions.
Before the washing step, a homogenous cell distribution was observed in a horizontal direction, whereas sperm cell count increased in the middle of the channels on the axis.
The cell count was altered in the horizontal direction Raghu man dating dna collection programs the washing step and most of the sperm close to the inlet washed away from the channel surface. On the other hand, the distribution trend at the vertical axis did not change after the washing step. These absorbance spectra values appear to be shifted from unbound SLeX molecule, 35 which might be caused by the generation of chemical bonding during immobilization process. The contact angle value of the bare glass surface reduced from These two different characterization methods confirmed that SLeX molecule was successfully immobilized to the microchannel surface.
In this experiment, we applied high and low sperm counts into the channels. During the imaging Raghu man dating dna collection programs, the entire channel was divided into 30 columns horizontal direction by 10 rows vertical direction. After the washing step, the cell count decreased in the first 5—10 lanes close to the inlet in the horizontal direction.
On the other hand, the vertical distribution did not change after the washing step. Before the washing step, we Raghu man dating dna collection programs nearly homogenous cell distribution in a horizontal direction. Through the vertical axis, we observed the same trend as with higher sperm count experiments, and the sperm cell count was higher in the middle of channel. After the washing step, the sperm count close to the inlet was altered Raghu man dating dna collection programs a horizontal direction, which was similarly observed in higher sperm count experiments.
After washing, the vertical axis also had a similar distribution trend, as observed before the washing step. In control experiments, we did not decorate the channels with surface chemistry, and the glass surface was only cleaned with EtOH before being assembled Figure 4.
High cell counts were defined as being between and sperm per channel, whereas the low cell count was around — sperm per channel. Evaluation of sperm cell binding control and limit of detection.
Nonspecific sperm cell binding was assessed with high — sperm per channel and low — sperm per channel cell numbers. The curve had a linearity of 0. COD and adjusted R 2respectively. Horizontal brackets and asterics demonstrate statistically significant differences between groups. Therefore, the microchannels were able to handle a Raghu man dating dna collection programs range of cell numbers and the capture capability of microfluidic chips was independent of high cell counts into the channels.
Since surface chemistry and channel parameters were all same, there might be two reasons: This could potentially contribute to changes in capture efficiency parameter due to different effective ratio of sperm removed from the surface after washing step.
Further, in the experiments, we observed a nonlinear trend with 0. The curve was also examined in two regions: Samples lower than sperm per channel range provided a capture efficiency between Vaginal samples in sexual assault kits typically contain vaginal epithelial cells from the victim and sperm cells from the perpetrator. To evaluate specificity performance of microfluidic chips, we designed two experimental sets: In both experimental sets, we worked with a heterogeneous cell population including Raghu man dating dna collection programs and buccal epithelial cells.
Thus, we evaluated whether SLeX is crucial in specific capture of sperm from mixed cell populations Figure 5. Overall, in these experiments, we obtained two critical outcomes: Specificity experiments and validation of Raghu man dating dna collection programs chips with forensic mock samples. Two sets of microfluidic chips were prepared: Black arrows represent epithelial cells ECs in the microchannels. Five different mock samples were introduced into the microchannels modified with SLeX, and the numbers of sperm were then counted before and after the wash steps.
The details were presented in the table. brackets demonstrate statistically significant differences between groups. Sperm cells were counted before and after washing steps. In the forensic mock samples, we observed a various number of sperm and epithelial cells.
Additionally, as reported in the literature, cotton content interferes with capture performance of assays, 21 and we observed similar hindrance when a large Raghu man dating dna collection programs swab was used. As demonstrated in the spiking experiments, we also confirmed that our microchannels were able to specifically capture sperm from a heterogeneous cell population, and device performance was not significantly changed while capturing sperm from aged forensic Raghu man dating dna collection programs samples.
The collected lysate solution was then
Raghu man dating dna collection programs through Proteinase K and Raghu man dating dna collection programs column protocols, as described in the Materials and Methods section.
After these protocols, the DNA concentration of each sample was measured and demonstrated in Table 1. We counted Raghu man dating dna collection programs number of sperm ranging from to in the microchannels. The differential extraction of sexual assault samples from sexual assault kits requires up to 8 Raghu man dating dna collection programs of skilled personnel to complete. Our technology solves a significant problem that has failed to find a solution in the past for efficient differential extraction of sperm.
Here, we integrated microfluidics with a unique oligosaccharide unit i. This Raghu man dating dna collection programs is still in development, and we expect the following improvements in the design and workflow. Fourth, in this study, sexual assault kit samples were visualized using a standard laboratory microscope. The next generation of this platform can potentially be integrated with a portable imaging system, 40 enabling easy access to the technology in remote locations for sexual assault evidence screening.
Fifth, we decorated channels with SLeX monomer units in the current study. Higher concentrations of SLeX more than 0. Ideally, a bioprinting strategy will further accelerate to increase the coverage rate of SLeX in the channels. Sixth, the present platform utilizes affordable components such as plastic layers, polymers, and glass slides, the cost of goods used for the fabrication and surface chemistry can potentially be reduced further with mass production.
The microfluidic chips consisted of three main components: Inlets and outlets of the chips 0. The microfluidic chips were
Raghu man dating dna collection programs constructed by assembling these three components.
Glass cover slides were used as a substrate material, where we performed surface chemistry for sperm capture. After the silanization step, the surfaces were rinsed with EtOH to remove unbound chemical residues and dried using either N 2 gas or filtered dry air. Before sampling, sperm were incubated at room temperature for Raghu man dating dna collection programs d.
Sperm samples were incubated for an hour while the imaging was being performed within the entire microchannel using a tiling function of the light
Raghu man dating dna collection programs with a motorized x — y stage Zeiss, Germany before the washing step. The captured cells within the microchannels were counted after the washing step. Sperm counts before and Raghu man dating dna collection programs the washing steps were manually calculated using these microscope images.
In specificity experiments, buccal epithelial cells were collected from female individuals and were mixed with sperm samples.
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