Enhanced CRISPR/Cas-Based Immunoassay through Magnetic Proximity Extension and Detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated systems have recently emerged as a focal point for developing next-generation molecular diagnosis, particularly for nucleic acid detection. However, the detection of proteins is equally critical across diverse applications in biology, medicine, and the food industry, especially for diagnosing and prognosing diseases like cancer, Alzheimer's and cardiovascular conditions. Despite recent efforts to adapt CRISPR/Cas systems for protein detection with immunoassays, these methods typically achieved sensitivity only in the femtomolar to picomolar range, underscoring the need for enhanced detection capabilities. To address this, we developed CRISPR-AMPED, an innovative CRISPR/Cas-based immunoassay enhanced by magnetic proximity extension and detection. This approach combines proximity extension assay (PEA) with magnetic beads that converts protein into DNA barcodes for quantification with effective washing steps to minimize non-specific binding and hybridization, therefore reducing background noise and increasing detection sensitivity. The resulting DNA barcodes are then detected through isothermal nucleic acid amplification testing (NAAT) using recombinase polymerase amplification (RPA) coupled with the CRISPR/Cas12a system, replacing the traditional PCR. This integration eliminates the need for thermocycling and bulky equipment, reduces amplification time, and provides simultaneous target and signal amplification, thereby significantly boosting detection sensitivity. CRISPR-AMPED achieves attomolar level sensitivity, surpassing ELISA by over three orders of magnitude and outperforming existing CRISPR/Cas-based detection systems. Additionally, our smartphone-based detection device demonstrates potential for point-of-care applications, and the digital format extends dynamic range and enhances quantitation precision. We believe CRISPR-AMPED represents a significant advancement in the field of protein detection.

Digitized Kinetic Analysis Enhances Genotyping Capacity of CRISPR-Based Biosensing.

CRISPR/Cas systems have been widely employed for nucleic acid biosensing and have been further advanced for mutation detection by virtue of the sequence specificity of crRNA. However, existing CRISPR-based genotyping methods are limited by the mismatch tolerance of Cas effectors, necessitating a comprehensive screening of crRNAs to effectively distinguish between wild-type and point-mutated sequences. To circumvent the limitation of conventional CRISPR-based genotyping, here, we introduce Single-Molecule kinetic Analysis via a Real-Time digital CRISPR/Cas12a-assisted assay (SMART-dCRISPR). SMART-dCRISPR leverages the differential kinetics of the signal increase in CRISPR/Cas systems, which is modulated by the complementarity between crRNA and the target sequence. It employs single-molecule digital measurements to discern mutations based on kinetic profiles that could otherwise be obscured by variations in the target concentrations. We applied SMART-dCRISPR to genotype notable mutations in SARS-CoV-2, point mutation (K417N) and deletion (69/70DEL), successfully distinguishing wild-type, Omicron BA.1, and Omicron BA.2 SARS-CoV-2 strains from clinical nasopharyngeal/nasal swab samples. Additionally, we introduced a portable digital real-time sensing device to streamline SMART-dCRISPR and enhance its practicality for point-of-care settings. The combination of a rapid and sensitive isothermal CRISPR-based assay with single-molecule kinetic analysis in a portable format significantly enhances the versatility of CRISPR-based nucleic acid biosensing and genotyping.

Single-Particle Spectroscopic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid Nanoparticles.

Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level. Here, we report an integrated spectroscopy-chromatography approach as a generalizable strategy to dissect the complexities of multicomponent LNP assembly. Our platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) to simultaneously profile population identity, hydrodynamic size, RNA loading levels, and distributions of helper lipid and PEGylated lipid of LNPs at the single-particle level and in a high-throughput manner. Using a benchmark siRNA LNP formulation, we demonstrate the capability of this platform by distinguishing seven distinct LNP populations, quantitatively characterizing size distribution and RNA loading level in wide ranges, and more importantly, resolving composition-size correlations. This SN-FSHS-CICS analysis provides critical insights into a substantial degree of heterogeneity in the packing density of RNA in LNPs and size-dependent loading-size correlations, explained by kinetics-driven assembly mechanisms of RNA LNPs.

Automated and miniaturized screening of antibiotic combinations via robotic-printed combinatorial droplet platform.

Antimicrobial resistance (AMR) has become a global health crisis in need of novel solutions. To this end, antibiotic combination therapies, which combine multiple antibiotics for treatment, have attracted significant attention as a potential approach for combating AMR. To facilitate advances in antibiotic combination therapies, most notably in investigating antibiotic interactions and identifying synergistic antibiotic combinations however, there remains a need for automated high-throughput platforms that can create and examine antibiotic combinations on-demand, at scale, and with minimal reagent consumption. To address these challenges, we have developed a Robotic-Printed Combinatorial Droplet (RoboDrop) platform by integrating a programmable droplet microfluidic device that generates antibiotic combinations in nanoliter droplets in automation, a robotic arm that arranges the droplets in an array, and a camera that images the array of thousands of droplets in parallel. We further implement a resazurin-based bacterial viability assay to accelerate our antibiotic combination testing. As a demonstration, we use RoboDrop to corroborate two pairs of antibiotics with known interactions and subsequently identify a new synergistic combination of cefsulodin, penicillin, and oxacillin against a model E. coli strain. We therefore envision RoboDrop becoming a useful tool to efficiently identify new synergistic antibiotic combinations toward combating AMR.

Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucleic Acid Amplification Testing Assays.

Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported. Moreover, lack of investigations into CRISPR/Cas in single-step assays results in incomplete understanding. To fill this knowledge gap, we conducted a systematic investigation by developing and comparing assays that share the identical recombinase polymerase amplification (RPA) but differ in CRISPR/Cas12a. We found that the addition of CRISPR/Cas12a indeed unlocks signal amplification but, at the same time, impedes RPA and that CRISPR/Cas12a concentration is a key parameter for attenuating RPA impediment and ensuring assay performance. Accordingly, we found that our protospacer adjacent motif (PAM)-free CRISPR/Cas12a-assisted RPA assay, which only moderately impeded RPA at its optimal CRISPR/Cas12a concentration, outperformed its counterparts in assay design, signal, sensitivity, and speed. We also discovered that a new commercial Cas12a effector could also drive our PAM-free CRISPR/Cas12a-assisted RPA assay and reduce its cost, though simultaneously lowering its signal. Our study and the new insights can be broadly applied to steer and facilitate further advances in CRISPR/Cas-based assays.

Emerging platforms for high-throughput enzymatic bioassays.

Enzymes have essential roles in catalyzing biological reactions and maintaining metabolic systems. Many in vitro enzymatic bioassays have been developed for use in industrial and research fields, such as cell biology, enzyme engineering, drug screening, and biofuel production. Of note, many of these require the use of high-throughput platforms. Although the microtiter plate remains the standard for high-throughput enzymatic bioassays, microfluidic arrays and droplet microfluidics represent emerging methods. Each has seen significant advances and offers distinct advantages; however, drawbacks in key performance metrics, including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity, remain. Herein, we compare recent high-throughput platforms using the aforementioned metrics as criteria and provide insights into remaining challenges and future research trends.

Facile and scalable tubing-free sample loading for droplet microfluidics.

Droplet microfluidics has in recent years found a wide range of analytical and bioanalytical applications. In droplet microfluidics, the samples that are discretized into droplets within the devices are predominantly loaded through tubings, but such tubing-based sample loading has drawbacks such as limited scalability for processing many samples, difficulty for automation, and sample wastage. While advances in autosamplers have alleviated some of these drawbacks, sample loading that can instead obviate tubings offers a potentially promising alternative but has been underexplored. To fill the gap, we introduce herein a droplet device that features a new Tubing Eliminated Sample Loading Interface (TESLI). TESLI integrates a network of programmable pneumatic microvalves that regulate vacuum and pressure sources so that successive sub-microliter samples can be directly spotted onto the open-to-atmosphere TESLI inlet, vacuumed into the device, and pressurized into nanoliter droplets within the device with minimal wastage. The same vacuum and pressure regulation also endows TESLI with cleaning and sample switching capabilities, thus enabling scalable processing of many samples in succession. Moreover, we implement a pair of TESLIs in our device to parallelize and alternate their operation as means to minimizing idle time. For demonstration, we use our device to successively process 44 samples into droplets-a number that can further scale. Our results demonstrate the feasibility of tubing-free sample loading and a promising approach for advancing droplet microfluidics.

Microfluidics-Implemented Biochemical Assays: From the Perspective of Readout.

Over the past decades, microfluidics has emerged as an increasingly important tool to perform biochemical assays for diagnosis and healthcare. The precise fluid control and molecule manipulation within microfluidics greatly contribute to developing assays with simplicity and convenience. The advantages of microfluidics, including decreased consumption of reagents and samples, lower operating and analysis time, much lower cost, and higher integration and automation over traditional systems, offer a great platform to meet the needs of point-of-care applications. In this Review, versatile strategies are outlined and recent advances in microfluidics-implemented assays are discussed from the perspective of readout, because a convenient and straightforward readout is what a biochemical assay requires and the end user desires. Functions and properties arising from each readout are reviewed and the advantages and limitations of each readout are discussed together with current challenges and future perspectives.

Inter-Limb Transfer of Grasp Force Perception With Closed-Loop Hand Prosthesis.

Sensory feedback of grasp forces provides important information about physical interactions between the hand and objects, enabling both reactive and anticipatory neural control mechanisms. The numerous studies have shown artificial sensory feedback of various forms improves force control during grasping tasks by prosthetic hand users through a closed-feedback loop. However, little is known about how perceptual information is transferred between an intact limb and a closed-loop prosthetic limb, and the extent to which training inter-limb transfer may improve myoelectric prosthetic control. We addressed these gaps by using a contralateral force-matching task in which able-bodied participants were asked to generate grasp forces with their native hand, and then match it using the contralateral hand or a soft-synergy prosthetic hand worn on the contralateral arm that was coupled with a mechanotactile feedback device. We found that absolute matching error and matching time were greater when using the prosthetic system than the native hand. However, with contralateral specific training, subjects were able to produce similar relative matching error with the prosthetic system and the native hand, especially at the untrained force level. These findings suggest that an association can be established between the perception produced by the prosthetic limb and the contralateral intact limb, and provide novel insights about potential applications to training and design of the closed-loop prosthesis.