Flow Cytometry-Assisted Mix-and-Read Assay for Ultrasensitive Detection of Protein Kinase Activity by use of Zr⁴⁺-Functionalized Mesoporous SiO₂ Microspheres

A flow cytometry-assisted mix-and-read assay is developed for ultrasensitive detection of protein kinase activity by use of Zr⁴⁺-functionalized mesoporous SiO₂ microspheres (ZrMMs). This strategy integrates the distinct advantages of ZrMMs for highly specific recognition as well as high capacity binding of kinase-induced fluorescent phosphopeptides and flow cytometry for powerful and separation-free bead analysis, leading to an ultrahigh sensitivity for kinase analysis in a extremely simple mix-and-read manner. Furthermore, this ultrasensitive design is well suitable for detection of cell kinase activities in complex biological samples and for screening of potential protein kinase inhibitors, which is of great significance for the development of targeted therapy, clinical diagnosis, and studies of cellular signal transduction pathways

Graphene Surface-Anchored Fluorescence Sensor for Sensitive Detection of MicroRNA Coupled with Enzyme-Free Signal Amplification of Hybridization Chain Reaction

A new enzyme-free signal amplification-based assay for microRNA (miRNA) detection is developed by using hybridization chain reaction (HCR) coupled with a graphene oxide (GO) surface-anchored fluorescence signal readout pathway. MiRNAs can efficiently initiate HCR between two species of fluorescent hairpin probes. After HCR, both of the excess hairpin probes and the HCR products will be anchored on the GO surface. The fluorescence of the hairpin probes can be completely quenched by GO, whereas the HCR products maintain strong fluorescence. Therefore, integrating HCR strategy for signal amplification with selective fluorescence quenching effects of GO provides a versatile miRNA assay

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

Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs’ actions in a single cell, which is of great importance for the study of miRNAs’ biofunction as well as for the related biomedical studies

Highly Sensitive and Specific Multiplexed MicroRNA Quantification Using Size-Coded Ligation Chain Reaction

As important regulators of gene expression, microRNAs (miRNAs) are emerging as novel biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. There is a great demand for flexible multiplexed miRNA quantification methods that can quantify very low levels of miRNA targets with high specificity. For further analysis of miRNA signatures in biological samples, we describe here a highly sensitive and specific method to detect multiple miRNAs simultaneously in total RNA. First, we rationally design one of the DNA probes modified with two ribonucleotides, which can greatly improve the ligation efficiency of DNA probes templated by miRNAs. With the modified DNA probes, the ligation chain reaction (LCR) can be well applied to miRNA detection and as low as 0.2 fM miRNA can be accurately determined. High specificity to clearly discriminate a single nucleotide difference among miRNA sequences can also be achieved. By simply coding the DNA probes with different length of oligo (dA) for different miRNA targets, multiple miRNAs can be simultaneously detected in one LCR reaction. In our proof of principle work, we detect three miRNAs: let-7a, mir-92a, and mir-143, which can also be simultaneously detected in as small as 2 ng of total RNA sample

Upconversion Nanophosphor: An Efficient Phosphopeptides-Recognizing Matrix and Luminescence Resonance Energy Transfer Donor for Robust Detection of Protein Kinase Activity

Protein kinases play important regulatory roles in intracellular signal transduction pathways. The aberrant activities of protein kinases are closely associated with the development of various diseases, which necessitates the development of practical and sensitive assays for monitoring protein kinase activities as well as for screening of potential kinase-targeted drugs. We demonstrate here a robust luminescence resonance energy transfer (LRET)-based protein kinase assay by using NaYF₄:Yb,Er, one of the most efficient upconversion nanophosphors (UCNPs), as an autofluorescence-free LRET donor and a tetramethylrhodamine (TAMRA)-labeled substrate peptide as the acceptor. Fascinatingly, besides acting as the LRET donor, NaYF₄:Yb,Er UCNPs also serve as the phosphopeptide-recognizing matrix because the intrinsic rare earth ions of UCNPs can specifically capture the fluorescent phosphopeptides catalyzed by protein kinases over the unphosphorylated ones. Therefore, a sensitive and generic protein kinase assay is developed in an extremely simple mix-and-read format without any requirement of surface modification, substrate immobilization, separation, or washing steps, showing great potential in protein kinases-related clinical diagnosis and drug discovery. To the best of our knowledge, this is the first report by use of rare earth-doped UCNPs as both the phospho-recognizing and signal reporting elements for protein kinase analysis

Dual-Readout Fluorescent Assay of Protein Kinase Activity by Use of TiO₂‑Coated Magnetic Microspheres

A simple, highly sensitive, and dual-readout fluorescent assay is developed for the detection of protein kinase activity based on the specific recognition utility of TiO₂-coated Fe₃O₄/SiO₂ magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When the fluorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they can bind specifically to the TiO₂ layer of TMSPs by means of phosphate groups, resulting in fluorophore enrichment on the TMSP surfaces. The accumulated fluorophores on the TMSPs are proportional to the kinase activity, and the fluorescence signal readout could be run through either direct fluorescent imaging of the TMSPs or measurement of the fluorescence intensity by simply detaching the fluorescent phosphopeptides into the solution. The TMSPs exhibit extremely high selectivity for capturing phosphorylated peptides over the nonphosphorylated ones, resulting in an ultrahigh fluorescence signal-to-background ratio of 42, which is the highest fluorescence change thus far in fluorescent assays for detection of protein kinase activities. Therefore, the proposed fluorescent assay presents high sensitivity, low detection limit of 0.1 milliunit/μL, and wide dynamic range from 0.5 milliunit/μL to 0.5 unit/μL with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fluorescent assay can simultaneously quantify multiple kinase activities with their specific peptides labeled with different dyes. This new strategy is also successfully applied to monitoring drug-triggered PKA activation in cell lysates. Therefore, the TMSP-based fluorescent assay is very promising in high-throughput screening of kinase inhibitors and in highly sensitive detection of kinase activity, and thus it is a valuable tool for development of targeted therapy, clinical diagnosis, and studies of fundamental life science

Light-Triggered Disruption of PAG-Based Amphiphilic Random Copolymer Micelles

The amphiphilic random copolymer of P­(NVP-<i>co</i>-NHPSS) with photocleavable N–O sulfonate side groups has been prepared to investigate the light-triggered disruption of copolymer micelles. Methods of absorption and emission spectra, solution transmittance, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were applied. It was found that P­(NVP-<i>co</i>-NHPSS) could form polymeric nanoaggregates in aqueous solution. And the photocleavage of the N–O bond within copolymer micelles upon 365 nm UV light could be conveniently controlled by changing the irradiation intensity, leading to the disruption of copolymer micelles and the photocontrolled release of Nile red encapsulation. And by encapsulating NaLuF₄:Gd/Yb/Tm UCNPs inside copolymer micelles, the response of the photocleavable N–O bond to the 980 nm laser was much weaker than the response to 365 nm light; however, the photocontrolled release of Nile red could still be effectively triggered by the NIR light of the 980 nm laser