Aggregation-Augmented Magnetism of Lanthanide-Doped Nanoparticles and Enabling Magnetic Levitation-Based Exosome Sensing.

Due to the presence of unpaired electron orbitals in most lanthanide ions, lanthanide-doped nanoparticles (LnNPs) exhibit paramagnetism. However, as to biosensing applications, the magnetism of LnNPs is so weak that can hardly be employed in target separation. Herein, it is discovered that the magnetism of the LnNPs is highly associated with their concentration in a confined space, enabling aggregation-augmented magnetism to make them susceptive to a conventional magnet. Accordingly, a magnetic levitation (Maglev) sensing system is designed, in which the target exosomes can specifically introduce paramagnetic LnNPs to the microbeads' surface, allowing aggregation-augmented magnetism and further leverage the microbeads' levitation height in the Maglev device to indicate the target exosomes' content. It is demonstrated that this Maglev system can precisely distinguish healthy people's blood samples from those of breast cancer patients. This is the first work to report that LnNPs hold great promise in magnetic separation-based biological sample sorting, and the LnNP-permitted Maglev sensing system is proven to be promising for establishing a new generation of biosensing devices.

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.

Tyramine-Invertase Bioconjugate-Amplified Personal Glucose Meter Signaling for Ultrasensitive Immunoassay.

Highly sensitive and facile detection of low levels of protein markers is of great significance for the early diagnosis and efficacy monitoring of diseases. Herein, aided by an efficient tyramine-signal amplification (TSA) mechanism, we wish to report a simple but ultrasensitive immunoassay with signal readout on a portable personal glucose meter (PGM). In this study, the bioconjugates of tyramine and invertase (Tyr-inv), which act as the critical bridge to convert and amplify the protein concentration information into glucose, are prepared following a click chemistry reaction. Then, in the presence of a target protein, the sandwich immunoreaction between the immobilized capture antibody, the target protein, and the horseradish peroxidase (HRP)-conjugated detection antibody is specifically performed in a 96-well microplate. Subsequently, the specifically loaded HRP-conjugated detection antibodies will catalyze the amplified deposition of a large number of Tyr-inv molecules onto adjacent proteins through highly efficient TSA. Then, the deposited invertase, whose dosage can faithfully reflect the original concentration of the target protein, can efficiently convert sucrose to glucose. The amount of finally produced glucose is simply quantified by the PGM, realizing the highly sensitive detection of trace protein markers such as the carcinoembryonic antigen and alpha fetoprotein antigen at the fg/mL level. This method is simple, cost-effective, and ultrasensitive without the requirement of sophisticated instruments or specialized laboratory equipment, which may provide a universal and promising technology for highly sensitive immunoassay for in vitro diagnosis of diseases.

Composite Nanomaterials of Conjugated Polymers and Upconversion Nanoparticles for NIR-Triggered Photodynamic/Photothermal Synergistic Cancer Therapy.

Phototherapies such as photodynamic therapy (PDT) and photothermal therapy (PTT) have attracted great attention in the field of cancer treatment. However, the individual PDT or PTT makes it difficult to achieve optimal antitumor effects compared to the PDT/PTT combined therapy. Also, the effect of PDT is usually limited by the penetration depth of the UV-vis light source. Herein, we designed and synthesized novel composite nanoparticles UCNPs-CPs, which are constructed from two conjugated polymers and upconversion nanoparticles ß-NaYF4:Yb,Tm (UCNPs) via a coordination reaction. By virtue of the excellent spectral overlap between absorption of conjugated polymers and emission of UCNPs, the UCNPs can absorb NIR light and effectively excite conjugated polymers by energy transfer to produce massive reactive oxygen species under 980 nm excitation and heat energy under 808 nm laser irradiation, achieving photodynamic/photothermal synergistic therapy. The in vitro cellular investigation proves that the dual modal phototherapy exhibits enhanced antitumor ability compared to single PDT or PTT. Furthermore, UCNPs-CPs inhibit tumor growth 100% in a 4T1 breast tumor mice model with both NIR laser irradiation, indicating that UCNPs-CPs is an excellent platform for synergistic PDT/PTT treatment. Thus, this study provides a promising strategy for NIR-triggered dual modal phototherapy.

A Handheld Visible Resonance Raman Analyzer Used in Intraoperative Detection of Human Glioma.

There is still a lack of reliable intraoperative tools for glioma diagnosis and to guide the maximal safe resection of glioma. We report continuing work on the optical biopsy method to detect glioma grades and assess glioma boundaries intraoperatively using the VRR-LRRTM Raman analyzer, which is based on the visible resonance Raman spectroscopy (VRR) technique. A total of 2220 VRR spectra were collected during surgeries from 63 unprocessed fresh glioma tissues using the VRR-LRRTM Raman analyzer. After the VRR spectral analysis, we found differences in the native molecules in the fingerprint region and in the high-wavenumber region, and differences between normal (control) and different grades of glioma tissues. A principal component analysis-support vector machine (PCA-SVM) machine learning method was used to distinguish glioma tissues from normal tissues and different glioma grades. The accuracy in identifying glioma from normal tissue was over 80%, compared with the gold standard of histopathology reports of glioma. The VRR-LRRTM Raman analyzer may be a new label-free, real-time optical molecular pathology tool aiding in the intraoperative detection of glioma and identification of tumor boundaries, thus helping to guide maximal safe glioma removal and adjacent healthy tissue preservation.

A novel full solar light spectrum responsive antimicrobial agent of WS2 quantum dots for photocatalytic wound healing therapy.

Photocatalytic antimicrobial therapy (PCAT) is considered to be a potential therapeutic treatment for bacterial-infection diseases. However, the antibacterial efficiency is unsatisfactory due to the limited application scope of photocatalysis. In this work, full-spectrum responsive tungsten disulfide quantum dots (WS2 QDs) are prepared for killing bacteria and enabling wound healing through photocatalytic reactive oxygen species (ROS) generation and glutathione (GSH) depletion. On the one hand, these ultrasmall WS2 QDs exhibit an excellent full spectrum (UV-Vis-NIR)-responsive photocatalytic effect by hindering the recombination of electron-hole pairs, thereby achieving the full use of the energy spectrum. Furthermore, the full-spectrum photocatalytic property of the as-prepared WS2 QDs can be effectively strengthened by redox reaction to deplete GSH for accelerated wound healing. In a word, the as-prepared nanoplatform exhibits the ability to act as an admirable antibacterial reagent with full-spectrum catalytic performance for photocatalytic wound healing therapy. Therefore, this work will not only provide an effective full-spectrum photocatalytic reagent for anti-bacteria therapy and wound healing, but also provide a rational idea for the development of other novel antibacterial agents for applications in the biomedical field.

Tumor microenvironment-activated single-atom platinum nanozyme with H2O2 self-supplement and O2-evolving for tumor-specific cascade catalysis chemodynamic and chemoradiotherapy.

Nanozyme-based tumor collaborative catalytic therapy has attracted a great deal of attention in recent years. However, their cooperative outcome remains a great challenge due to the unique characteristics of tumor microenvironment (TME), such as insufficient endogenous hydrogen peroxide (H2O2) level, hypoxia, and overexpressed intracellular glutathione (GSH). Methods: Herein, a TME-activated atomic-level engineered PtN4C single-atom nanozyme (PtN4C-SAzyme) is fabricated to induce the "butterfly effect" of reactive oxygen species (ROS) through facilitating intracellular H2O2 cycle accumulation and GSH deprivation as well as X-ray deposition for ROS-involving CDT and O2-dependent chemoradiotherapy. Results: In the paradigm, the SAzyme could boost substantial ∙OH generation by their admirable peroxidase-like activity as well as X-ray deposition capacity. Simultaneously, O2 self-sufficiency, GSH elimination and elevated Pt2+ release can be achieved through the self-cyclic valence alteration of Pt (IV) and Pt (II) for alleviating tumor hypoxia, overwhelming the anti-oxidation defense effect and overcoming drug-resistance. More importantly, the PtN4C-SAzyme could also convert O2·- into H2O2 by their superior superoxide dismutase-like activity and achieve the sustainable replenishment of endogenous H2O2, and H2O2 can further react with the PtN4C-SAzyme for realizing the cyclic accumulation of ∙OH and O2 at tumor site, thereby generating a "key" to unlock the multi enzymes-like properties of SAzymes for tumor-specific self-reinforcing CDT and chemoradiotherapy. Conclusions: This work not only provides a promising TME-activated SAzyme-based paradigm with H2O2 self-supplement and O2-evolving capacity for intensive CDT and chemoradiotherapy but also opens new horizons for the construction and tumor catalytic therapy of other SAzymes.

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.

Intraoperative detection of human meningioma using a handheld visible resonance Raman analyzer.

To report for the first time the preliminary results for the evaluation of a VRR-LRR™ analyzer based on visible resonance Raman technique to identify human meningioma grades and margins intraoperatively. Unprocessed primary and recurrent solid human meningeal tissues were collected from 33 patients and underwent Raman analysis during surgeries. A total of 1180 VRR spectra were acquired from fresh solid tissues using a VRR-LRR™ analyzer. A confocal HR Evolution (HORIBA, France SAS) Raman system with 532-nm excitation wavelength was also used to collect data for part of the ex vivo samples after they were thawed from - 80 °C for comparison. The preliminary analysis led to the following observations. (1) The intensity ratio of VRR peaks of protein to fatty acid (I2934/I2888) decreased with the increase of meningioma grade. (2) The ratio of VRR peaks of phosphorylated protein to amid I (I1588/I1639) decreased for the higher grade of meningioma. (3) Three RR vibration modes at 1378, 3174, and 3224 cm-1 which were related to the molecular vibrational bands of oxy-hemeprotein, amide B, and amide A protein significantly changed in peak intensities in the two types of meningioma tissues compared to normal tissue. (4) The changes in the intensities of VRR modes of carotenoids at 1156 and 1524 cm-1 were also found in the meningioma boundary. The VRR-LRR™ analyzer demonstrates a new approach for label-free, rapid, and objective identification of primary human meningioma in quasi-clinical settings. The accuracy for detecting meningioma tissues using support vector machines (SVMs) was over 70% based on Raman peaks of key biomolecules and up to 100% using principal component analysis (PCA).

Colorimetric and fluorometric dual-readout protein kinase assay by tuning the active surface of nanoceria.

Herein, we demonstrate that the active surface of nanoceria can be fine-tuned by phosphorylated peptides. Accordingly, a colorimetric and fluorometric dual-readout strategy is rationally developed for assaying protein kinase activity. This feature not only enables the versatile monitoring of peptide phosphorylation but also broadens the application scope of nanoceria.

Effect of atmospheric cold plasma treatment on antioxidant activities and reactive oxygen species production in postharvest blueberries during storage.

BACKGROUND: Blueberry is universally acknowledged as a kind of berry rich in antioxidants. Cold plasma, an emerging non-thermal treatment technology, has been proved to be able to maintain or improve the antioxidant level while inactivating the microorganisms on the surface of fruits and vegetables. Postharvest blueberries were treated with atmospheric cold plasma (ACP; 12 kV, 5 kHz) for 0 s (Control), 30 s (ACP-30), 60 s (ACP-60), and 90 s (ACP-90) in this study, and the effects of ACP on the antimicrobial properties, antioxidant activities, and reactive oxygen species (ROS) production were investigated during storage at 4 ± 1 °C for 40 days. RESULTS: Total aerobic bacteria and mold populations on ACP-treated blueberries decreased significantly in a time-dependent manner (P < 0.05), and decreased by 0.34-1.24 and 0.57-0.87 log10 CFU g-1 respectively on ACP-60-treated blueberries during storage. The decay rate of blueberries was decreased by 5.8-11.7% and the decrease of blueberry firmness was slowed down by ACP-60. But the total phenol, anthocyanin, and ascorbic acid contents increased, and superoxide dismutase, catalase, and peroxidase activities were enhanced in ACP-treated blueberries. The free radical scavenging activity and total antioxidant capacity (T-AOC) were enhanced. Hydrogen peroxide (H2 O2 ) and superoxide anion (O2 - ) production rates declined by 27.3% and 41.3% at day 40 of storage, respectively. CONCLUSION: It is suggested that ACP may be a promising non-thermal treatment technology for postharvest sterilization and preservation of blueberry under suitable conditions. © 2020 Society of Chemical Industry.

Integrated Single Microbead-Arrayed µ-Fluidic Platform for the Automated Detection of Multiplexed Biomarkers.

An automated, single microbead-arrayed µ-fluidic immunoassay (AMIA) device is innovatively devised in this study, which enables the highly sensitive and simultaneous detection of multiplex biomarkers with fully automatic operations. The AMIA platform not only achieves automated assay processing and multiplexed target detection by integrating single microbead manipulation, sample loading, multistep washing, and immunoreaction on a microfluidic chip but also confers high sensitivity due to the highly efficient signal enriching effect on a single microbead by the use of only a routine sandwich immunoreaction. As such, as low as the pg/mL level of multiplexed protein biomarkers can be simultaneously determined in a quite small volume of serum (∼20 µL is enough), which can well meet the clinical demand for disease screening and prognosis. What is more, the detection results of several clinically important biomarkers in clinical samples with the AMIA platform exhibit excellent consistency with those obtained by using a standard clinical test. Thus, in virtue of the excellent features in terms of high sensitivity, multiplexing capability, generality, and high degree of automation, the AMIA provides a practical and user-friendly platform for assaying different biomarkers in clinical diagnostics and point-of-care testing.

A terminal extension-actuated isothermal exponential amplification strategy toward the ultrasensitive and versatile detection of enzyme activity in a single cell.

Terminal deoxynucleotidyl transferase (TdT) plays an important role in regulating a wide range of genomic processes. The sensitive and accurate detection of cellular TdT activity, particularly at the single-cell level, is highly significant for leukemia-associated biomedical and biological studies. Nevertheless, owing to the limited sensitivity of the existing TdT assays, the quantification of TdT activity at the single-cell level remains a big challenge. Herein, a simple but ultrasensitive method for assaying TdT activity is proposed based on terminal extension actuated loop-mediated isothermal amplification (TEA-LAMP). By using the TdT-induced extension product as an actuator, TdT activity is amplified twice by terminal extension and LAMP in an exponential manner and finally converted to a remarkably amplified fluorescent signal. In this study, as low as 2 × 10-8 U/µL TdT can be clearly detectable with the elegant TEA-LAMP strategy. Such an ultrahigh sensitivity enables the direct determination of TdT activity in individual single cells. In the meantime, by employing TdT as a co-factor, this strategy can also be applied to detecting other enzymes that can catalyze the DNA terminal hydroxylation. This work not only reports the up-to-now most sensitive TdT detection strategy at a single-cell level but also opens the new gate for versatile enzyme activity detection.

Optical biopsy identification and grading of gliomas using label-free visible resonance Raman spectroscopy.

Glioma is one of the most refractory types of brain tumor. Accurate tumor boundary identification and complete resection of the tumor are essential for glioma removal during brain surgery. We present a method based on visible resonance Raman (VRR) spectroscopy to identify glioma margins and grades. A set of diagnostic spectral biomarkers features are presented based on tissue composition changes revealed by VRR. The Raman spectra include molecular vibrational fingerprints of carotenoids, tryptophan, amide I/II/III, proteins, and lipids. These basic in situ spectral biomarkers are used to identify the tissue from the interface between brain cancer and normal tissue and to evaluate glioma grades. The VRR spectra are also analyzed using principal component analysis for dimension reduction and feature detection and support vector machine for classification. The cross-validated sensitivity, specificity, and accuracy are found to be 100%, 96.3%, and 99.6% to distinguish glioma tissues from normal brain tissues, respectively. The area under the receiver operating characteristic curve for the classification is about 1.0. The accuracies to distinguish normal, low grade (grades I and II), and high grade (grades III and IV) gliomas are found to be 96.3%, 53.7%, and 84.1% for the three groups, respectively, along with a total accuracy of 75.1%. A set of criteria for differentiating normal human brain tissues from normal control tissues is proposed and used to identify brain cancer margins, yielding a diagnostic sensitivity of 100% and specificity of 71%. Our study demonstrates the potential of VRR as a label-free optical molecular histopathology method used for in situ boundary line judgment for brain surgery in the margins.

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.

An ultrasensitive flow cytometric immunoassay based on bead surface-initiated template-free DNA extension.

Proteins lack the duplication mechanism like nucleic acids, so the connection of immunoassays with effective nucleic acid amplification techniques has become a powerful way for the detection of trace protein biomarkers in biological fluids. However, such immunoassays generally suffer from rather stringent DNA sequence design and complicated operations. Herein, we propose a simple but highly sensitive flow cytometric immunoassay (FCI) by employing on-bead terminal deoxynucleotidyl transferase (TdT)-initiated template-free DNA extension as an effective signal amplification pathway (TdT-FCI), and gold nanoparticles (AuNPs) co-functionalized with both the detection antibody and a 3'-OH oligonucleotide (ODN) as the transducer to bridge the immunoassay and subsequent TdT-mediated DNA amplification. The target antigen can sandwich with the capture antibody immobilized on the magnetic beads (MBs) and the detection antibody on the AuNPs to bring a lot of ODNs onto the surface of MBs. Each ODN on the MBs can be effectively elongated by TdT in a template-free manner to produce a long poly(T) tail, which will then bind to many 6-carboxyfluorescein (FAM)-labeled poly(A)25. Since each AuNP can carry multiple ODNs and each extended ODN can ultimately capture numerous FAM-poly(A)25, efficiently amplified fluorophore accumulation on the MBs can be achieved. The fluorescent MBs can be individually interrogated with a flow cytometer and thus quantitative analysis of the target antigen can be realized. Coupled with the powerful flow cytometry analysis, the simple but efficient TdT-based signal amplification mechanism has pushed the detection limit of prostate specific antigen (PSA) down to a low level of 0.5 pg mL-1. Furthermore, based on an elegant bead size-encoding principle, we have further advanced the TdT-FCI for multiplexed antigen detection in a single reaction. Sharing the unique merits of simple design and operation, efficient signal amplification, powerful signal readout and the capability for multiplexed analysis, this TdT-FCI provides a versatile tool for detecting trace antigen biomarkers towards clinical diagnosis as well as prognosis.

A Clamp-Based One-Step Droplet Digital Reverse Transcription PCR (ddRT-PCR) for Precise Quantitation of Messenger RNA Mutation in Single Cells.

Precise detection of the low copy numbers of messenger RNA (mRNA) mutation in single cells is of great significance but still remains challenging. Herein, by integrating the outstanding features of a rationally designed peptide nucleic acid (PNA) clamp for highly selective discrimination of single-nucleotide variation, and droplet digital PCR for ultrasensitive and precise quantification, we have developed a robust one-step droplet digital reverse transcription PCR (ddRT-PCR) method which enables precise mRNA mutation detection in single cells with ultrahigh specificity to clearly discern as low as 0.01% mutated mRNA in a high background of wild-type mRNA. Because of its outstanding single-molecule level sensitivity and ultrahigh specificity, this ddRT-PCR method holds great promise for studying cellular heterogeneity at the single cell level, as well as for the precise quantification of mutant mRNAs in complex plasma or serum for liquid biopsy.

Identification of a selective DNA ligase for accurate recognition and ultrasensitive quantification of N6-methyladenosine in RNA at one-nucleotide resolution.

N6-Methyladenosine (m6A) is the most frequent post-transcriptional modification in RNA, and it plays a critical role in biological processes. The functions of m6A remain largely unexplored due to a lack of highly sensitive methods to quantitatively determine the m6A modification fraction at a precise location. Here, we first reveal that T3 DNA ligase has significant selectivity towards the m6A modification. On the basis of the new finding, we establish an ultrasensitive quantitation assay for accurately determining m6A at one-nucleotide resolution in RNA. With the proposed assay, as low as 4 fM RNA containing m6A can be determined and the selectivity is up to 54.1-fold to discriminate m6A against unmodified adenosine (A). The sensitivity has been improved about 106-fold so the proposed method can be successfully employed to accurately determine m6A in real biological samples, even in low abundance RNA.

A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction.

Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.