Ultrasensitive protein and exosome analysis based on a rolling circle amplification assisted-CRISPR/Cas12a strategy.

CRISPR/Cas12a system has attracted extensive concern in biosensing due to its high specificity and programmability. Nevertheless, existing Cas12a-based assays mainly focus on nucleic acid detection and have limitations in non-nucleic acid biomarker analysis. To broaden the application prospect of the CRISPR/Cas technology, a cascade Cas12a biosensing platform is reported by combining dual-functionalized gold nanoparticles (FGNPs)-assisted rolling circle amplification (RCA) and Cas12a trans-cleavage activity (GAR-Cas) for ultrasensitive protein and exosome analysis. FGNPs serve as a critical component in the transduction of protein or exosome recognition information into nucleic acid amplification events to produce Cas12a activators. In the GAR-Cas assay, by integrating the triple cascade amplification of FGNPs-assisted transduction, RCA, and Cas12a signal amplification, ultralow abundance of target molecules can arouse numerous concatemers to activate Cas12a trans-cleavage activity to release intense fluorescence, allowing the ultrasensitive detection of as low as 1 fg/mL (∼41 aM) cTnI and 5 exosomes per µL. Furthermore, the presented strategy can be applied to detect exosome levels from clinical samples, showing excellent performance in distinguishing cancer patients from healthy individuals. The GAR-Cas sensing platform exhibits great potential in clinical diagnosis and enlarges biosensing toolboxes based on CRISPR/Cas technology for non-nucleic acid target analysis.

An immunoassay-like recognition mechanism-based lateral flow strategy for rapid microRNA analysis.

A rapid lateral flow assay (LFA) is developed for the colorimetric and surface-enhanced Raman scattering (SERS) dual-mode detection of microRNA (miRNA) based on the robust immunoassay-like (immuno-like) recognition mechanism of S9.6 antibody to DNA/miRNA duplexes. Different from the traditional target-mediated sandwich-type hybridization-based LFA methods, the formation of S9.6 antibody/miRNA/DNA complexes is more rapid and stable, achieving 40 times higher sensitivity with only 10 min assaying time. Furthermore, taking benefit of the versatility of the immuno-like recognition mode, the multiplexed detection of miRNAs can be realized with the SERS signal readout, providing a versatile LFA design towards sensitive, specific, and multiplexed miRNA analysis.

Digital-like Enzyme Inhibition Mechanism-Based Strategy for the Digital Sensing of Heparin-Specific Biomarkers.

A new microbead (MB)-based digital flow cytometric sensing system is proposed for the sensitive detection of heparin-specific biomarkers, including heparin-binding protein (HBP) and heparinase. This strategy takes advantage of the inherent space-confined enzymatic behavior of T4 polynucleotide kinase phosphatase (T4 PNKP) around a single MB and the heparin's digital-like inhibitory effect on T4 PNKP. By integrating with an on-bead terminal deoxynucleotidyl transferase (TdT)-catalyzed fluorescence signal amplification technology, the concentration of HBP and heparinase can be digitally determined by the number of fluorescence-positive/-negative MBs which can be easily counted by flow cytometry. This is not only the first test to expand the application scenario of T4 PNKP to the digital detection of different biomarkers but also pioneers a new direction for fabricating digital biosensing platforms based on the enzyme inhibition mechanism.

Effect of fat replacement by flaxseed flour on the quality parameters of pork meatballs.

To improve the edible qualities of meatballs, various percentages of pork fat in meatballs were replaced by brown flaxseed flour (BFF) to decrease the fat contents and further optimize the fatty acid compositions. Five different meatball formulations that used 0%, 5%, 10%, 15%, and 20% of flaxseed flour additions were used in which the corresponding amounts pork fat were replaced. The proximate compositions, water activity, pH levels, colors, textures, cooking losses, fatty acid compositions, sensory properties, flavors, and oxidation stabilities of these meatballs were analyzed. Increasing the BFF addition amounts improved the protein and dietary fiber contents, pH levels, fatty acid profiles and oxidation stabilities, but decreased the fat contents, moisture levels, cooking losses, n6/n3 ratios, hardness, and lightness. The volatile flavors of meatballs with different BFF replacement levels were significantly different. According to the sensory evaluation, the use of 5% BFF increased the odor of meatballs without significantly affecting the other sensory scores. This work demonstrated that BFF may be a healthier alternative as pork fat replacer for preparing meatballs.

Advances in optical counting and imaging of micro/nano single-entity reactors for biomolecular analysis.

Ultrasensitive detection of biomarkers is of paramount importance in various fields. Superior to the conventional ensemble measurement-based assays, single-entity assays, especially single-entity detection-based digital assays, not only can reach ultrahigh sensitivity, but also possess the potential to examine the heterogeneities among the individual target molecules within a population. In this review, we summarized the current biomolecular analysis methods that based on optical counting and imaging of the micro/nano-sized single entities that act as the individual reactors (e.g., micro-/nanoparticles, microemulsions, and microwells). We categorize the corresponding techniques as analog and digital single-entity assays and provide detailed information such as the design principles, the analytical performance, and their implementation in biomarker analysis in this work. We have also set critical comments on each technique from these aspects. At last, we reflect on the advantages and limitations of the optical single-entity counting and imaging methods for biomolecular assay and highlight future opportunities in this field.

Nucleic Acid Substrate-Independent DNA Polymerization on the Exosome Membrane: A Mechanism Study and Application in Exosome Analysis.

As generally acknowledged, terminal deoxynucleotidyl transferase (TdT) can only elongate DNA substrates from their 3'-OH ends. Herein, for the first time, we report that TdT-catalyzed DNA polymerization can directly proceed on the exosome membrane without the mediation of any nucleic acids. We prove that both the glycosyl and phenolic hydroxyl groups on the membrane proteins can initiate the DNA polymerization. Accordingly, we have developed powerful strategies for high-sensitive exosome profiling based on a conventional flow cytometer and an emerging CRISPR/Cas system. By using our strategy, the featured membrane protein distributions of different cancer cell-derived exosomes can be figured out, which can clearly distinguish plasma samples of breast cancer patients from those of healthy people. This work paves new ways for exosome profiling and liquid biopsy and expands the understanding of TdT, holding great significance in developing TdT-based sensing systems as well as establishing protein/nucleic acid hybrid biomaterials.

Microchamber-Free Digital Flow Cytometric Analysis of T4 Polynucleotide Kinase Phosphatase Based on Single-Enzyme-to-Single-Bead Space-Confined Reaction.

Digital bioassays have attracted extensive attention in biomedical applications due to their ultrahigh sensitivity. However, traditional digital bioassays require numerous microchambers such as droplets or microwells, which restricts their application scope. Herein, we propose a microchamber-free flow cytometric method for the digital quantification of T4 polynucleotide kinase phosphatase (T4 PNKP) based on an unprecedented phenomenon that each T4 PNKP molecule-catalyzed reaction can be spatially self-confined on a single microbead, which ultimately enables the one-target-to-one-fluorescence-positive microbead digital signal transduction. The digital signal-readout mode can clearly detect T4 PNKP concentrations as low as 1.28 × 10-10 U/µL, making it most sensitive method to date. Significantly, T4 PNKP can be specifically distinguished from other phosphatases and nucleases in complex samples by digitally counting the fluorescence-positive microbeads, which cannot be realized by traditional bulk measurement-based methods. Taking advantage of the novel space-confined enzymatic feature of T4 PNKP, this digital mechanism can use T4 PNKP as the enzyme label to fabricate digital sensing systems toward various biomolecules such as digital enzyme-linked immunosorbent assay (ELISA). Therefore, this work not only enlarges the toolbox for high-sensitivity biomolecule detection but also opens new gates to fabricate next-generation digital assays.

Amplification-Free and Mix-and-Read Analysis of Multiplexed MicroRNAs on a Single Plasmonic Microbead.

In this work, a single microbead covered with a plasmonic layer is employed as the microreactor for the multiplexed miRNA analysis without nucleic acid amplification. On the plasmonic layer, the S9.6 antibody is adopted as the universal module for binding DNA/miRNA duplexes regardless of the sequence. Meanwhile, there is also a SERS reporter gold nanoparticle (GNP) pool, in which each group of GNPs is labeled with both a Raman coding molecule and a DNA probe for recognizing a given miRNA of interest. The target miRNAs will lead to the specific capture of the corresponding SERS reporter GNPs onto the plasmonic layer, which will enormously enhance the target miRNA-induced SERS signals. Finally, the enhanced SERS signals concentrated on the microbead will be mapped out by a confocal Raman microscope. The proposed method achieves the high-precision sensing of sub-pM target miRNA in a simple mix-and-read format and possesses multiplexed assay capability.

Click Chemistry-Actuated Digital DNA Walker Confined on a Single Particle toward Absolute MicroRNA Quantification.

A versatile single magnetic nanoparticle (MNP)-confined, click chemistry-actuated digital DNA walker (ddWalker) machine is devised for the absolute quantification of microRNA (miRNA). This delicate ddWalker allows one target molecule to fix one DNA walking leg on a single MNP, following the Poisson statistics through a specific click chemical DNA ligation, which will initiate single molecule DNA walking to stepwise cleave the molecular beacon tracks strictly constrained on the leg-hold MNP without cross-particle reaction, fluorescently "lighting up" the exact MNP. Accordingly, the initial miRNA input can be digitally and faithfully reflected by the number of fluorescent-positive MNPs counted by a total internal reflection fluorescent microscope, enabling the absolute and precise miRNA quantification down to the femtomolar level without external calibration. This flexible ddWalker design provides a new digital signaling concept and elegantly expands the toolbox for digital biosensing.

An emulsion-free digital flow cytometric platform for the precise quantification of microRNA based on single molecule extension-illuminated microbeads (dFlowSeim).

A robust digital flow cytometric platform is developed towards the precise quantification of microRNAs based on single molecule extension-illuminated microbeads (dFlowSeim).

High-sensitive sensing of plant microRNA by integrating click chemistry with an unusual on-bead poly(T)-promoted transcription amplification.

MicroRNAs (miRNAs) act as pivotal regulators in plants. Therefore, sensing strategies with high specificity and high sensitivity are desired for plant miRNA analysis in order to unveil the exact biofunctions of miRNAs. Toward this goal, a fluorescent assay is developed based on a two-step signal amplification strategy. In the first step, target miRNA-templated cycling click nucleic acid ligation is employed for target recognition and amplification, the product of which can bind to magnetic microbeads (MBs) and introduce the T7 promoter sequence to the surface. In the second step, the poly(T) containing transcription template partially hybridizes with the T7 promoter sequence on the ligated strand and then regulates the on-bead transcription in a cycling manner with the participation of T7 RNA polymerase. Surprisingly, different from other reported templates, the poly(T) template improves the transcription efficiency to an unexpectedly high level by releasing ultra-long RNA chains in the reaction system. Finally, the RNA intercalating dye, RiboGreen, is utilized to specifically light up the as-produced RNA chains for low-background signal readout. Benefiting from the elaborate design, the detection limit of plant miRNA is down to ∼0.1 amol. This strategy provides a highly specific and highly sensitive platform for plant miRNA detection, which promises potential in the practical applications of miRNA-related biofunctions.

On-bead enzyme-catalyzed signal amplification for the high-sensitive detection of disease biomarkers.

The high-sensitive and rapid detection of critical biomarkers, e.g., disease-related nucleic acids and proteins, is always desired. Compared with the routine homogenous detection strategies, the on-bead flow cytometry (FCM)-based assays have drawn a lot of interests owing to their unique advantages. On one hand, microbeads (MBs) are employed for the enrichment of fluorescent signals, allowing the size encoding for multiplexed detection of biomarkers. On the other hand, FCM enables the fast read-out of the total fluorescent signals enriched on the MBs and the decoding of MBs' size information. For an improved sensitivity and versatile application scenarios, the signal amplification on MBs is required. However, the enzyme-catalyzed on-bead reactions remain challenging owing to the critical reaction conditions on the MBs/solution interface. Toward the high-sensitive detection of target biomolecules in real-samples, a series of on-bead enzyme-catalyzed signal amplification strategies have been developed. After careful optimization of the reaction conditions, the proposed sensors are proven to have ultra-high sensitivities to fulfill the requirement of real-sample detection.

Trends of Bead Counting-Based Technologies Toward the Detection of Disease-Related Biomarkers.

Nowadays, the biomolecular assay platforms built-up based on bead counting technologies have emerged to be powerful tools for the sensitive and high-throughput detection of disease biomarkers. In this mini-review, we classified the bead counting technologies into statistical counting platforms and digital counting platforms. The design principles, the readout strategies, as well as the pros and cons of these platforms are introduced in detail. Finally, we point out that the digital bead counting technologies will lead the future trend for the absolute quantification of critical biomarkers, and the integration of new signal amplification approaches and routine optical/clinical instruments may provide new opportunities in building-up easily accessible digital assay platforms.

A Versatile Dynamic Light Scattering Strategy for the Sensitive Detection of Plant MicroRNAs Based on Click-Chemistry-Amplified Aggregation of Gold Nanoparticles.

Plant microRNAs (miRNAs) are naturally 2'-O-methylated at the 3'-terminal; as a consequence, they cannot be efficiently detected by traditional target-triggered polymerization reactions. Here, a simple but robust enzyme-free sensing strategy was developed for plant miRNA analysis by using dynamic light scattering (DLS) to monitor the crosslinking of gold nanoparticles (AuNPs) amplified by click chemical ligation. Combining the enzyme-free cycling chemical-ligation-mediated signal amplification, and the intrinsic outstanding ability of DLS for discriminating the extremely low level of particle aggregation in a large pool of monodisperse AuNPs, high sensitivity was achieved and as low as 78.6 fm plant miRNA could be easily detected.

An Enzyme-Free MicroRNA Assay Based On Fluorescence Counting of Click Chemical Ligation-Illuminated Magnetic Nanoparticles with Total Internal Reflection Fluorescence Microscopy.

MicroRNAs (miRNAs) have been considered as promising cancer biomarkers. However, the simple but sensitive detection of low levels of miRNAs in biological samples still remains challenging. Herein, we wish to report an entirely enzyme-free, simple, and highly sensitive miRNA assay based on the counting of cycling click chemical ligation (3CL)-illuminated fluorescent magnetic nanoparticles (MNPs) with a total internal reflection fluorescence microscopy (TIRFM). In this strategy, each miRNA molecule can trigger many cycles of click chemical ligation reactions to produce plentiful ligated oligonucleotides (ODNs) with both 5'-biotin and 3'-fluorophore, resulting in efficient signal amplification. It is worth noting that only the ligated ODNs can bring fluorophores onto streptavidin-functionalized MNPs (STV-MNPs). Notably, merely 10 fluorescent molecules on each 50 nm MNP can make it bright enough to be clearly visualized by the TIRFM, which can significantly improve the detection sensitivity for miRNA. Through fluorescence counting of individual MNPs and integrating their fluorescence intensities, the amount of target miRNA can be quantitatively determined. This miRNA assay can be accomplished in a mix-and-read manner just by simply mixing the enzyme-free 3CL reaction system with the MNPs before TIRFM imaging, which avoids tedious immobilization, washing, and purification steps. Despite the extremely simple operation, this strategy exhibits high sensitivity with a quite low detection limit of 50 fM target miRNA as well as high specificity to well discriminate miRNA sequences with a single-base variation. Furthermore, the applicability of this method in real biological samples is also verified through the accurate detection of the miRNA target in cancer cells.

Click Chemical Ligation-Initiated On-Bead DNA Polymerization for the Sensitive Flow Cytometric Detection of 3'-Terminal 2'-O-Methylated Plant MicroRNA.

A versatile flow cytometric strategy is developed for the sensitive detection of plant microRNA (miRNA) by coupling the target-templated click nucleic acid ligation (CNAL) with on-bead terminal enzymatic DNA polymerization (TEP). Unlike ligase-catalyzed ligation reaction, the plant miRNA-templated enzyme-free CNAL between two single-stranded DNA (ssDNA) probes, respectively modified with Aza-dibenzocyclooctyne (Aza-DBCO) and N3, can not only simplify the operation, but also achieve a much higher ligation efficiency. More importantly, the undesirable nonspecific ligation between the Aza-DBCO- and N3-modified ssDNA, can be effectively eliminated by adding Tween-20, which allows the use of cycling CNAL (CCNAL) in a background-free manner. So each plant miRNA can template many rounds of CNAL reaction to produce numerous ligation products, forming efficient signal amplification. The ligated ssDNA can be anchored on the magnetic beads (MBs) with the 3'-OH termini exposed outside. Then terminal deoxynucleotidyl transferase (TdT), a sequence-independent and template-free polymerase, would specifically catalyze the DNA polymerization along these 3'-OH termini on the MBs, forming poly(T) tails up to thousands of nucleotides long. Each poly(T) tail allows specific binding of numerous 6-carboxyfluorescein (FAM)-labeled poly(A)25 oligonucleotides to accumulate a lot of fluorophores on the MBs, leading to the second step of signal amplification. By integrating the advantages of CCNAL-TEP for highly efficient signal amplification and robust MBs signal readout with powerful flow cytometer, high sensitivity is achieved and the detection limit of plant miRNA has been pushed down to a low level of 5 fM with high specificity to well discriminate even single-base difference between miRNA targets.

An enzyme-free flow cytometric bead assay for the sensitive detection of microRNAs based on click nucleic acid ligation-mediated signal amplification.

A versatile flow cytometric bead assay (FCBA) coupled with a completely enzyme-free signal amplification mechanism is developed for the sensitive detection of microRNAs (miRNAs). This new strategy integrates click chemistry-mediated ligation chain reaction (CLCR) with hybridization chain reaction (HCR) for enzyme-free signal amplification on magnetic beads (MBs), and a flow cytometer for the robust fluorescence readout of the MBs. Firstly, target miRNA can initiate CLCR on the surface of MBs based on the click chemical ligation between dibenzocyclooctyne (DBCO)- and azide-modified single-stranded DNA (ssDNA) probes, and the amount of ligated ssDNA sequences on the MBs will be proportional to the dosage of target miRNA. Afterward, each of the ligated ssDNA products can trigger a cascade chain reaction of hybridization events between two alternating fluorophore-tagged hairpin probes, resulting in another signal amplification pathway with an amplified accumulation of fluorophores on the MBs. Finally, the fluorophore-anchored MBs are directly and rapidly analyzed by using a flow cytometer without any separation or elution processes. Herein, the click nucleic acid ligation only occurs on the surface of MBs, so the nonspecific ligations are greatly inhibited compared with that of ligation reaction performed in homogeneous solution. Furthermore, the signal amplification by CLCR-HCR is highly efficient but totally enzyme-free, which may overcome the potential drawbacks of conventional enzyme-catalyzed signal amplification protocols and lead to a high sensitivity. The CLCR-HCR-based FCBA has pushed the detection limit of let-7a miRNA down to the femtomolar (fM) level, showing great potential in miRNA-related biological studies and disease diagnosis.