CRISPR-Cas enzymes: The toolkit revolutionizing diagnostics.

The programmable nature of sequence-specific targeting by CRISPR-Cas nucleases has revolutionized a wide range of genomic applications and is now emerging as a method for nucleic acid detection. We explore how the diversity of CRISPR systems and their fundamental mechanisms have given rise to a wave of new methods for target recognition and readout. These cross-disciplinary advances found at the intersection of CRISPR biology and engineering have led to the ability to rapidly generate solutions for emerging global challenges like the COVID-19 pandemic. We further discuss the advances and potential for CRISPR-based detection to have an impact across a continuum of diagnostic applications.

High-Resolution Patterning of Hydrogel Sensing Motifs within Fibrous Substrates for Sensitive and Multiplexed Detection of Biomarkers.

There has been an increasing and urgent demand to develop nucleic acid bioassays which not only offer high analytical performance but which are also amenable with point-of-care testing. Hydrogels present a versatile class of materials with biocompatible antifouling properties and the ability to be engineered for a range of advanced sensing applications. Fibrous substrates like nitrocellulose offer low-cost and durable platforms to run complex bioassays while enabling portability and ease of handling. We demonstrate herein the ability to synergistically combine these two materials into a portable biosensing platform by leveraging projection lithography. We demonstrate the direct polymerization of hydrogel sensing motifs within a range of fibrous substrates with precise control over their shape, size, location, and functionality. Spatial encoding of the hydrogel motifs enables the multiplex detection of multiple biomarkers on the same test. As a proof-of-concept, we apply the platform to the detection of microRNA, an emerging class of circulating biomarkers with promising potential for early diagnosis and monitoring of cancer. The assay offers a large dynamic range (over three orders of magnitude), high sensitivity (limit of detection of 2.5 amol), as well as versatility and ease of handling. Finally, the bioassay is validated using real biological samples, namely, total RNA extracted from the sera of late-stage breast cancer patients, demonstrating its utility and compatibility with clinical biosensing applications.

Design of Hydrogel Particle Morphology for Rapid Bioassays.

Hydrogel microparticles have been extensively used in the field of medical diagnostics for detecting targets ranging from proteins to nucleic acids. However, little is known about how the shape of hydrogel particles impacts the signal from a bioassay. In this article, we analyze the flux into porous hydrogel particles to develop scaling laws for the signal from a point-of-care bioassay. The signal can be increased by increasing the ratio of the surface area of the hydrogel particle to the two-dimensional projected imaging area used for analysis. We show that adding internal surface area to hydrogel particles increases the assay signal in a biotin-streptavidin bioassay. We also demonstrate the application of this technique to a protein-based assay for thyroid-stimulating hormone, reducing the limit of detection of the assay sixfold by changing particle shape. We anticipate that these strategies can be used broadly to optimize hydrogel-based systems for point-of-care diagnostics.

Constricted microfluidic devices to study the effects of transient high shear exposure on platelets.

Due to the critical roles that platelets play in thrombosis during many biological and pathological events, altered platelet function may be a key contributor to altered hemostasis, leading to both thrombotic and hemorrhagic complications. Platelet adhesion at arterial shear rates occurs through binding to von Willebrand Factor via the glycoprotein (GP) GPIb receptor. GPIb binding can induce platelet activation distinguishable by P-selectin (CD62P) surface expression and αIIbß3 activation, resulting in platelet aggregation and formation of the primary hemostatic plug to stop bleeding. Previous studies have used cone and plate viscometers to examine pathologic blood flow conditions, applied shear rates that are relatively low, and examined exposure times that are orders of magnitude longer compared to conditions present in ventricular assist devices, mechanical heart valves, or pathologic states such as stenotic arteries. Here, we evaluate the effect of short exposure to high shear on granule release and receptor shedding utilizing a constricted microfluidic device in conjunction with flow cytometry and enzyme-linked immunosorbent assay. In this study, platelets were first perfused through microfluidic channels capable of producing shear rates of 80 000-100 000 s-1 for exposure times of 0-73 ms. We investigated platelet activation by measuring the expression level of CD62P (soluble and surface expressed), platelet factor 4 (PF4), and beta-thromboglobulin (ßTG). In addition, we measured potential platelet receptor shedding of GPVI and GPIb using flow cytometry. The results showed that a single pass to high shear with short exposure times (milliseconds) had no effect on the levels of CD62P, GPVI and GPIb, or on the release of alpha granule content (PF4, ßTG, and sP-selectin).

Encoded Hydrogel Microparticles for Sensitive and Multiplex microRNA Detection Directly from Raw Cell Lysates.

In recent years, microRNAs (miRNAs) have emerged as promising diagnostic markers because of their unique dysregulation patterns under various disease conditions and high stability in biological fluids. However, current methods of analyzing miRNA levels typically require RNA isolation, which is cumbersome and time-consuming. To achieve high-throughput and accurate miRNA profiling, this study eliminates the need for purification steps by detecting miRNA directly from raw cellular lysate using nonfouling polyethylene glycol microparticles. In contrast to recent studies on direct miRNA measurements from cell lysate, our hydrogel-based system provides high-confidence quantification with robust performance. The lysis buffer for the assay was optimized to maximize reaction and labeling efficiency, and this assay has a low limit of detection (<1000 cells) without target amplification. Additionally, the capability for multiplexing was demonstrated through analyzing the levels of three endogenous miRNAs in 3T3 cell lysate. This versatile platform holds great potential for rapid and reliable direct miRNA quantification in complex media, and can be further extended to single-cell analysis by exploiting the flexibility and scalability of our system.