With the trend of moving molecular tests from clinical laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established diagnostics with the ease, cost effectiveness and speed of isothermal amplification and detection methods. A promising new nucleic acid detection method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.

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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...

Endotoxin is a deadly pyrogen, rendering it crucial to monitor with high accuracy and efficiency. However, current endotoxin detection relies on multistep processes that are labor-intensive, time-consuming, and unsustainable. Here, we report an aptamer-based biosensor for the real-time optical detection of endotoxin. The endotoxin sensor exploits the distance-dependent scattering of gold nanoparticles (AuNPs) coupled to a gold nanofilm. This is enabled by the conformational changes of an endotoxin-specific aptamer upon target binding. The sensor can be used in an ensemble mode and single-particle mode under dark-field illumination. In the ensemble mode, the sensor is coupled with a microspectrometer and exhibits high specificity, reliability (i.e., linear concentration to signal profile in logarithmic scale), and reusability for repeated endotoxin measurements. Individual endotoxins can be detected by monitoring the color of single AuNPs via a color camera, achieving single-molecule resolution. This platform can potentially advance endotoxin detection to safeguard medical, food, and pharmaceutical products.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) systems have repeatedly shown to have excellent performance in nucleotide sensing applications. High specificity and selectivity of Cas effector proteins is determined by the CRISPR RNAs (crRNAs) interchangeable spacer sequence, as well as position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide change, and are therefore unsuitable for CRISPR assisted sensing methods. Here we demonstrate a method to discriminate single CpG site methylation in DNA, which is an epigenetic alteration, by the use of methylation sensitive restriction enzymes (MSREs) followed by Cas12a assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single-stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene were for the first time determined. The modularity of both Cas12a and MSREs provide a high level of versatility to the Cas12a MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.