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We have developed a novel detection system that couples clustered regularly interspaced short palindromic repeat-Cas recognition of target sequences, Cas-mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for simple detection of viral nucleic acid targets. After target recognition and Cas-mediated cleavage of biotinylated ssDNA probe molecules, the probe molecules are bound to magnetic beads. A complementary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to uncleaved probes on the magnetic beads. After separating hybridized quantum dots, the collected supernatant is illuminated by a portable ultraviolet flashlight, and it provides a simple “Yes-or-No” nucleic acid detection answer. By using a DNA target matching part of the African swine fever virus, detection limits of ∼0.5 and ∼1.25 nM are achieved in buffer and porcine plasma, respectively. The positive samples are readily confirmed by visual inspection, completely avoiding the need for complicated devices and instruments. This work establishes the feasibility of a simple assay for nucleic acid screening in both hospitals and point-of-care settings.

Clustered regularly interspaced short palindromic repeats, CRISPR, has recently emerged as a powerful molecular biosensing tool for nucleic acids and other biomarkers due to its unique properties such as collateral cleavage nature, room temperature reaction conditions, and high target-recognition specificity. Numerous platforms have been developed to leverage the CRISPR assay for ultrasensitive biosensing applications. However, to be considered as a new gold standard, several key challenges for CRISPR molecular biosensing must be addressed. In this paper, we briefly review the history of biosensors, followed by the current status of nucleic acid-based detection methods. We then discuss the current challenges pertaining to CRISPR-based nucleic acid detection, followed by the recent breakthroughs addressing these challenges. We focus upon future advancements required to enable rapid, simple, sensitive, specific, multiplexed, amplification-free, and shelf-stable CRISPR-based molecular biosensors.

A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR deTection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect-ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, the CRISPR-Cas12a/crRNA complex is injected into the fully enclosed microchannel. With the presence of a double-stranded DNA target, the CRISPR enzyme is activated and denatures the single-stranded DNA reporters from the micropillars. This collateral cleavage releases fluorescence reporters into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on the traditional dye-quencher-labeled probe, thus reducing the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed on-chip at isothermal conditions (37 °C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates sensitive and accurate DNA detection within 120 min and paves ways to the next-generation point-of-care diagnostics, responding to emerging and deadly pathogen outbreaks.

the critical need for rapid and sensitive molecular diagnostics to combat current and future pandemics. Sensitive polymerase chain reaction (PCR) based tests are the gold standard for molecular diagnostics but rely on bulky and expensive instruments. Thus, they are not suitable for self-diagnosis or point-of-care (POC) settings. [2] High-throughput sequencing can decipher the entire genomic landscape of the pathogens but is time consuming and requires bioinformatics for data interpretation. [3] Immunoassays, such as rapid antigen tests, are simple and rapid diagnostic methods but normally lack the sensitivity to reporting the low concentration biomarkers. [4] Assays with high limits of detection and low accuracy have been acceptable out of necessity, but there are many scenarios where a rapid, sensitive, POC device would be beneficial. Therefore, developing a simple to use, portable, and sensitive diagnostic platform is one important key to addressing the current challenges for molecular diagnostics.Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are bacterial systems evolved to combat bacteriophage infections by recognizing specific nucleic acid sequences to activate nucleolytic cleavage activities. The trans-cleavage of A gold nanoparticle (AuNP)-labeled CRISPR-Cas13a nucleic acid assay is developed for sensitive solid-state nanopore sensing. Instead of directly detecting the translocation of RNA through a nanopore, the system utilizes non-covalent conjugates of AuNPs and RNA targets. Upon CRISPR activation, the AuNPs are liberated from the RNA, isolated, and passed through a nanopore sensor. Detection of the AuNPs can be observed as increasing ionic current in the chip. Each AuNP that is detected is enumerated as an event, leading to quantitative of molecular targets. Leveraging the high signal-to-noise ratio enabled by the AuNPs, a detection limit of 50 fM before front-end target amplification is achieved using SARS-CoV-2 RNA segments as a Cas13 target. Furthermore, a dynamic range of six orders of magnitude is demonstrated for quantitative RNA sensing. This simplified AuNP-based CRISPR assay is performed at the physiological temperature without relying on thermal cyclers. In addition, the nanopore reader is similar in size to a smartphone, making the assay system suitable for rapid and portable nucleic acid biomarker detection in either low-resource settings or hospitals.

Using porous silicon (PSi) interferometer sensors, we show the first experimental implementation of the high contrast cleavage detection (HCCD) mechanism. HCCD makes use of dramatic optical signal amplification caused by cleavage of high-contrast nanoparticle labeled reporters instead of the capture of low-index biological molecules. An approximately 2 nm reflectance peak shift was detected after cleavage of DNA-quantum dot reporters from the PSi surface via exposure to a 12.5 nM DNase enzyme solution. This signal change is 20 times greater than the resolution of the spectrometer used for the interferometric measurements, and the interferometric measurements agree with the response predicted by simulations and fluorescence measurements. These proof of principle experiments show a clear path to achieving a real-time, highly sensitive readout for a broad range of biological diagnostic assays that generate a signal via nucleic acid cleavage triggered by specific molecular binding events.

A simple, portable, and low-cost microfluidic chip-Funnel Adapted Sensing Tube (FAST) is developed as an integrated, power-free, and pipette-free biosensor for viral nucleic acids. This FAST chip consists of four...

A planar, transparent, and adaptable nanosieve device is developed for efficient microalgae/bacteria separation. In the proposed method, a sacrificial layer is applied with dual photolithography patterning to achieve a 1D channel with a very low aspect ratio (1:10 000). A microalgae/bacteria mixture is then introduced into the deformable PDMS nanochannel. The hydrodynamic deformation of the nanochannel is regulated to allow the bacteria cells to pass through while leaving the microalgae cells trapped in the device. At a flow rate of 4 μL/min, the supernatant collected from the device is indistinguishable from a control solution, indicating that nearly all the microalgae cells are trapped in the device. Additionally, this device is capable of single cell auto-fluorescence tracking. These microalgae cells demonstrate minimal photobleaching over 250 s laser exposure and could be used to monitor hazardous compounds in the sample with a continuous flow. This method will be valuable to purify microalgae samples containing contaminations and study single-cell heterogeneity.