Micropillar enhanced FRET-CRISPR biosensor for nucleic acid detection.

CRISPR technology has gained widespread adoption for pathogen detection due to its exceptional sensitivity and specificity. Although recent studies have investigated the potential of high-aspect-ratio microstructures in enhancing biochemical applications, their application in CRISPR-based detection has been relatively rare. In this study, we developed a FRET-based biosensor in combination with high-aspect-ratio microstructures and Cas12a-mediated trans-cleavage for detecting HPV 16 DNA fragments. Remarkably, our results show that micropillars with higher density exhibit superior molecular binding capabilities, leading to a tenfold increase in detection sensitivity. Furthermore, we investigated the effectiveness of two surface chemical treatment methods for enhancing the developed FRET assay. A simple and effective approach was also developed to mitigate bubble generation in microfluidic devices, a crucial issue in biochemical reactions within such devices. Overall, this work introduces a novel approach using micropillars for CRISPR-based viral detection and provides valuable insights into optimizing biochemical reactions within microfluidic devices.

Micro- and nanosystems for the detection of hemorrhagic fever viruses.

Hemorrhagic fever viruses (HFVs) are virulent pathogens that can cause severe and often fatal illnesses in humans. Timely and accurate detection of HFVs is critical for effective disease management and prevention. In recent years, micro- and nano-technologies have emerged as promising approaches for the detection of HFVs. This paper provides an overview of the current state-of-the-art systems for micro- and nano-scale approaches to detect HFVs. It covers various aspects of these technologies, including the principles behind their sensing assays, as well as the different types of diagnostic strategies that have been developed. This paper also explores future possibilities of employing micro- and nano-systems for the development of HFV diagnostic tools that meet the practical demands of clinical settings.

Micropillar enhanced FRET-CRISPR biosensor for nucleic acid detection.

CRISPR technology has gained widespread adoption for pathogen detection due to its exceptional sensitivity and specificity. Although recent studies have investigated the potential of high-aspect-ratio microstructures in enhancing biochemical applications, their application in CRISPR-based detection has been relatively rare. In this study, we developed a FRET-based biosensor in combination with high-aspect-ratio microstructures and Cas12a-mediated trans-cleavage for detecting HPV 16 DNA fragments. Remarkably, our results show that micropillars with higher density exhibit superior molecular binding capabilities, leading to a tenfold increase in detection sensitivity. Furthermore, we investigated the effectiveness of two surface chemical treatment methods for enhancing the developed FRET assay. A simple and effective approach was also developed to mitigate bubble generation in microfluidic devices, a crucial issue in biochemical reactions within such devices. Overall, this work introduces a novel approach using micropillars for CRISPR-based viral detection and provides valuable insights into optimizing biochemical reactions within microfluidic devices.

Gold Nanoparticle-Labeled CRISPR-Cas13a Assay for the Sensitive Solid-State Nanopore Molecular Counting.

A gold nanoparticle (AuNP) labeled CRISPR-Cas13a nucleic acid assay has been developed for sensitive solid-state nanopore sensing. Instead of directly detecting the translocation of RNA through a nanopore, our 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.

Computer vision enabled funnel adapted sensing tube (FAST) for power-free and pipette-free nucleic acid detection.

A simple, portable, and low-cost microfluidic system-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 reaction chambers separated by carbon fiber rods, and the reagents in each chamber are transferred and mixed by manually removing the rods. Rather than using electrical heaters, only a hand warmer pouch is used for an isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a reaction. The signal produced by the RPA-CRISPR reaction is observed by the naked eye using an inexpensive flashlight as a light source. The FAST chip is fabricated using water-soluble polyvinyl alcohol (PVA) as a sacrificial core, which is simple and environmentally friendly. Using a SARS-CoV-2 fragment as a target, a ∼10 fM (6 × 103 copies per µL) detection limit is achieved. To generalize standard optical readout for individuals without training, a linear kernel algorithm is created, showing an accuracy of ∼100% for identifying both positive and negative samples in FAST. This power-free, pipette-free, disposable, and simple device will be a promising tool for nucleic acid diagnostics in either clinics or low-resource settings.

On-Demand Fully Enclosed Superhydrophobic-Optofluidic Devices Enabled by Microstereolithography.

Superhydrophobic surface-based optofluidics have been introduced to biosensors and unconventional optics with unique advantages, such as low light loss and power consumption. However, most of these platforms were made with planar-like microstructures and nanostructures, which may cause bonding issues and result in significant waveguide loss. Here, we introduce a fully enclosed superhydrophobic-based optofluidics system, enabled by a one-step microstereolithography procedure. Various microstructured cladding designs with a feature size down to 100 µm were studied and a "T-type" overhang design exhibits the lowest optical loss, regardless of the excitation wavelength. Surprisingly, the optical loss of superhydrophobic-based optofluidics is not solely decided by the solid area fraction at the solid/water/air interface, but also the cross-section shape and the effective cladding layer composition. We show that this fully enclosed optofluidic system can be used for CRISPR-labeled quantum dot quantification, intended for in vitro and in vivo CRISPR therapeutics.

Challenges and Opportunities for Clustered Regularly Interspaced Short Palindromic Repeats Based Molecular Biosensing.

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.

High contrast cleavage detection for enhancing porous silicon sensor sensitivity.

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.

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing.

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.

Magnetic Bead-Quantum Dot (MB-Qdot) Clustered Regularly Interspaced Short Palindromic Repeat Assay for Simple Viral DNA Detection.

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.

High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system.

Here we report the development of a high throughput, all-solution phase, and isothermal detection system for African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with a fluorescence-based point-of-care (POC) system for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 h. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 h of incubation, resulting in an improved detection limit of 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact and simple ASFV detection, intended for low resource settings.

Perspective of Molecular Diagnosis in Healthcare: From Barcode to Pattern Recognition.

Barcode technology has a broad spectrum of applications including healthcare, food security, and environmental monitoring, due to its ability to encode large amounts of information. With the rapid development of modern molecular research, barcodes are utilized as a reporter with different molecular combinations to label many biomolecular targets, including genomic and metabolic elements, even with multiplex targeting. Along with the advancements in barcoded bioassay, the improvements of various designs of barcode components, encoding and decoding strategies, and their portable adoption are indispensable in satisfying multiple purposes, such as medical confirmation and point-of-care (POC) testing. This perspective briefly discusses the current direction and progress of barcodes development and provides a hypothesis for barcoded bioassay in the near future.

A Review of Cerebral Shunts, Current Technologies, and Future Endeavors.

Objective. The use of cerebrospinal shunts is the standard of care for hydrocephalus. However, shunts are extremely vulnerable to failure and lack noninvasive methods to monitor their viability. We review current shunt technologies and attempts to improve their function. Methods. A PubMed search was performed to find literature on shunts and shunt function. Company brochures and websites were also used. Results. Fixed and variable pressure valves from four major companies are discussed. Also reviewed are siphon resistive devices, intracranial pressure sensors, and recent attempts on the development of cerebrospinal fluid sensors, including a micromechanical flow sensor we have recently developed. Conclusions. While variable pressure valves and siphon resistive devices have both had considerable success in dealing with variable intracranial pressure, a more sophisticated, continuous monitoring system is needed to ensure shunt viability and patient safety. An integrated flow sensor may provide the ability to track fluid flow and determine shunt functionality.