Digital single cancer biomarker detection by CRISPR/Cas sensing: Towards dynamic profiling for CpG methylation quantification

Introduction Cancer, as a disease, starts with a genetic change in the genome that results in the downregulation of genes involved in “normal” cell behavior. This genetic change can either be induced by a change in the DNA code (mutation) or epigenetic alterations, such as cytosine methylation. Hypermethylation of cytosines has shown to be a critical hallmark in many cancer cells. More specifically, the loss of expression of genes occurs about 10 times more often by hypermethylation of promotor CpG islands than by (point) mutations. For early cancer diagnostics, accurate and rapid detection of these epigenetic CpG methylation mutations involved in tumor development is crucial. Previously, we have presented an amplification-free in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. While this method showed a lot of potential in terms of specificity, selectivity and ease of use, the limit of detection presented (100 pM) was still far away from the concentrations of DNA found in liquid biopsies such as urine. Since all commonly available pre-amplification techniques used for CRISPR sensing will result in a loss of epigenetic modifications we here present a methods to lower the limit of detection of this Cas12a assay, while keeping methylation selectivity

Flow-Free Microfluidic Device for Quantifying Chemotaxis in Spermatozoa

Current male fertility diagnosis tests focus on assessing the quality of semen samples by studying the concentration, total volume, and motility of spermatozoa. However, other characteristics such as the chemotactic ability of a spermatozoon might influence the chance of fertilization. Here we describe a simple, easy to fabricate and handle, flow-free microfluidic chip to test the chemotactic response of spermatozoa made out of a hybrid hydrogel (8% gelatin/1% agarose). A chemotaxis experiment with 1 mu M progesterone was performed that significantly demonstrated that boar spermatozoa are attracted by a progesterone gradient

Effect of microfluidic processing on the viability of boar and bull spermatozoa

The use of microfluidics in artificial reproductive technologies for manipulation or assessment of spermatozoa is unique in the sense that it is not always an end point measurement and the sample may be used afterward. During microfluidic processing, spermatozoa are exposed to shear stress, which may harm viability and functioning of spermatozoa. The shear stresses during general microfluidic processing steps were calculated and compared to estimated shear stresses during ejaculation. The viability of boar and bull spermatozoa after microfluidic processing was studied and compared to the typical handling method (centrifugation) and to a control (the sample in a tube at the same temperature). The boar spermatozoa showed a small but significant decrease in viability of 6% after microfluidic handling. Bull spermatozoa proved to be less susceptible to shear stress and were not significantly affected by microfluidic processing. These data indicate that the impact of microfluidic processing on the viability of boar and bull spermatozoa is less than the literature values reported for flow cytometry and comparable to the impact of centrifugatio

Virus removal from semen with a pinched-flow fractionation microfluidic chip

In the veterinary industry, due to the use of artificial insemination, a single infected boar can transfer a disease to a multitude of sows which can lead to a disease outbreak [1]. An example is the epidemic of classical swine fever (CSF) in the Netherlands in 1997-1998, where the semen of infected boars led to CSF outbreaks in farms all over the country and beyond [2]. To reduce the chance of such an event happening in the future, we present a pinched-flow fractionation device to remove viruses from semen while retaining a high percentage of spermatozoa

Virus removal from semen with a pinched flow fractionation microfluidic chip

Nowadays pigs are bred with artificial insemination to reduce costs and transportation. To prevent the spread of diseases, it is important to test semen samples for viruses. Screening techniques applied are enzyme-linked immunosorbent assays and/or polymerase chain reaction, which are labor-intensive and expensive methods. In contrast to the current used screening techniques, it is possible to remove viruses physically from semen. However, existing methods for virus removal techniques have a low yield of spermatozoa. Therefore, we have developed a microfluidic chip that performs size-based separation of viruses and spermatozoa in boar semen samples, thereby having the potential to reduce the risk of disease spreading in the context of artificial insemination in the veterinary industry. As the head of a spermatozoon is at least twenty times larger than a virus particle, the particle size can be used to achieve separation, resulting in a semen sample with lower viral load and of higher quality. To achieve the size separation, our microfluidic device is based on pinched-flow fractionation. A model virus, cowpea chlorotic mottle virus, was used and spiked to porcine semen samples. With the proposed microfluidic chip and the optimized flow parameters, at least 84 ± 4% of the model viruses were removed from the semen. The remaining virus contamination is caused by the model virus adhering to spermatozoa instead of the separation technique. The spermatozoa recovery was 86 ± 6%, which is an enormous improvement in yield compared to existing virus removal techniques