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Abstract Most CRISPR-based biosensors rely on labeled reporter molecules and expensive equipment for signal readout. A recent approach quantifies analyte concentration by sizing λ DNA reporters via gel electrophoresis, providing a simple solution for label-free detection. Here, we report an alternative strategy for label-free CRISPR-Cas12a, which relies on Cas12atrans-nicking induced supercoil relaxation of dsDNA plasmid reporters to generate a robust and ratiometric readout. The ratiometric CRISPR (rCRISPR) measures the relative percentage of supercoiled plasmid DNA to the relaxed circular DNA by gel electrophoresis for more accurate target concentration quantification. This simple method is two orders of magnitude more sensitive than the typical fluorescent reporter. This self-referenced strategy solves the potential application limitations of previously demonstrated DNA sizing-based CRISPR-Dx without compromising the sensitivity. Finally, we demonstrated the applicability of rCRISPR for detecting various model DNA targets such as HPV 16 and real AAV samples, highlighting its feasibility for point-of-care CRISPR-Dx applications. 

Abstract CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn’t cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3′ overhang dsDNA substrates at least 3 times faster than 5′ overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule. 

Abstract Biosensors are analytical tools for monitoring various parameters related to living organisms, such as humans and plants. Liquid metals (LMs) have emerged as a promising new material for biosensing applications in recent years. LMs have attractive physical and chemical properties such as deformability, high thermal and electrical conductivity, low volatility, and low viscosity. LM‐based biosensors represent a new strategy in biosensing particularly for wearable and real‐time sensing. While early demonstrations of LM biosensors focus on monitoring physical parameters such as strain, motion, and temperature, recent examples show LM can be an excellent sensing material for biochemical and biomolecular detection as well. In this review, the recent progress of LM‐based biosensors for personalized healthcare and disease monitoring via both physical and biochemical signaling is survey. It is started with a brief introduction of the fundamentals of biosensors and LMs, followed by a discussion of different mechanisms by which LM can transduce biological or physiological signals. Next, it is reviewed example LM‐based biosensors that have been used in real biological systems, ranging from real‐time on‐skin physiological monitoring to target‐specific biochemical detection. Finally, the challenges and future directions of LM‐integrated biosensor platforms is discussed. 

Abstract CRISPR‐based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR‐Cas12a‐based sensing platform to detect ssDNA targets by sizing double‐stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massivetrans‐nuclease activity of Cas12a toward λ DNA is due to the presence of single‐stranded looped structures along the λ DNA sequence. In addition, we observed a strong binding affinity between Cas12a and λ DNA, which further promotes thetrans‐cleavage activity and helps achieve sub‐picomolar detection sensitivity, ≈100 times more sensitive than the fluorescent counterpart. The concept of utilizing the physical size change of λ DNA unlocks the possibility of using a variety of dsDNA as CRISPR reporters. 

Abstract Management of breast cancer in limited-resource settings is hindered by a lack of low-cost, logistically sustainable approaches toward molecular and cellular diagnostic pathology services that are needed to guide therapy. To address these limitations, we have developed a multimodal cellphone-based platform—the EpiView-D4—that can evaluate both cellular morphology and molecular expression of clinically relevant biomarkers directly from fine-needle aspiration (FNA) of breast tissue specimens within 1 h. The EpiView-D4 is comprised of two components: (1) an immunodiagnostic chip built upon a “non-fouling” polymer brush-coating (the “D4”) which quantifies expression of protein biomarkers directly from crude cell lysates, and (2) a custom cellphone-based optical microscope (“EpiView”) designed for imaging cytology preparations and D4 assay readout. As a proof-of-concept, we used the EpiView-D4 for assessment of human epidermal growth factor receptor-2 (HER2) expression and validated the performance using cancer cell lines, animal models, and human tissue specimens. We found that FNA cytology specimens (prepared in less than 5 min with rapid staining kits) imaged by the EpiView-D4 were adequate for assessment of lesional cellularity and tumor content. We also found our device could reliably distinguish between HER2 expression levels across multiple different cell lines and animal xenografts. In a pilot study with human tissue (n = 19), we were able to accurately categorize HER2-negative and HER2-positve tumors from FNA specimens. Taken together, the EpiView-D4 offers a promising alternative to invasive—and often unavailable—pathology services and may enable the democratization of effective breast cancer management in limited-resource settings. 

Abstract Smartphone is emerging as a portable analytical biosensing platform in many point-of-care (POC) applications such as disease diagnostics, environmental monitoring, and food toxin screening. With the recent advancement of imaging technologies on the smartphone, the manual control of acquisition settings (e.g., exposure time, frame rate, focusing distance, etc.) has already been expanded from the photo to the video capturing mode. In modern smartphone models, high frame rate (above 100 fps) can be achieved to bring in a new temporal dimension to the smartphone-supported POC tests by recording high-definition videos. This opens up a new analytical method defined as smartphone videoscopy. In this review, the recent development of smartphone videoscopy is summarized based on different POC applications. Representative examples of smartphone videoscopy systems and how these time-dependent measurements could open up new opportunities for POC diagnostics are discussed in detail. The advances demonstrated so far illustrate the promising future of smartphone videoscopy in biosensing, POC diagnostics, and time-resolved analysis in general.