Jiahui Zhao

Fluorescence Light-Up Biosensor for MicroRNA Based on the Distance-Dependent Photoinduced Electron Transfer

It is demonstrated that miRNAs exhibit significant regulatory roles in a series of biological processes and associated with diverse human diseases. Herein, we report a convenient fluorescent biosensor for the quantitative determination of miR-21, a key miRNA related to cardio-cerebrovascular diseases. Our proposal involves not only the rational design of single stranded DNA as the probe, successively including a C-rich sequence as the synthetic template of DNA/Ag nanoclusters (DNA/AgNCs), a complementary (Com) sequence to hybridize with the miR-21, and a G-rich sequence to form a complex of G-quadruplex/hemin but also the distance-dependent property of photoinduced electron transfer (PET) between the preformed DNA/AgNCs (electron donor) and G-quadruplex/hemin complex (electron acceptor). In the presence of the target miR-21, the initial flexible single strand Com in the probe turns to the rigid Com/RNA heteroduplexes, and then the PET could be interrupted owing to the extended distance between the electron donor and acceptor, accompanying with the fluorescence quenching and recovery of DNA/AgNCs. Therefore, a fluorescence light-up biosensor for miR-21 could be developed through the monitoring of the degree of fluorescence recovery of DNA/AgNCs. Preferential to other previous PET-based detection methods, we construct the biosensor by utilizing the distance dependent property for the first time and only need to adjust the sequences of Com in different miRNAs assays.

In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual-Readout Assay of Tyrosinase Activity

As a well-known copper-containing oxidase, tyrosinase has been anticipated to serve as the biomarker of skin diseases. We describe here an exquisite label-free fluorescent and colorimetric dual-readout assay of its activity, inspired by the specific oxidation ability of monophenolamine substrates to catecholamines and a unique fluorogenic reaction between resorcinol and catecholamines. By employing commercially available tyramine as the model substrate (dopamine as the product), it is found that the tyrosinase-incubated tyramine solution exhibits obvious pale yellow with intense blue fluorescence in the presence of resorcinol and O2, where the absorbance and fluorescence intensity are directly related to the concentration of added tyrosinase (i.e., the amount of conversion of tyramine to dopamine). The overall process of sensing tyrosinase activity takes less than 100 min at ambient temperature and pressure conditions with exceedingly simple operation procedure, explicit response mechanism, and formation of fluorophore with high quantum yield from scratch. Furthermore, such a convenient, rapid, cost-effective, and highly sensitive dual-readout assay exhibits promising prospect for the tyrosinase activity in extensive bioassays and clinic research as well as in screening potential tyrosinase inhibitors.

A Fluorescence Immunoassay Based on the Phosphate-Triggered Fluorescence Turn-on Detection of Alkaline Phosphatase

A simple and cost-effective fluorescence immunoassay for the sensitive quantitation of disease biomarker α-fetoprotein (AFP) has been developed based on the phosphate-triggered fluorescence turn-on detection of alkaline phosphatase (ALP), with the reversible binding between calcein and Ce3+ as a signaling element. In this immunoassay, fluorescent calcein is readily quenched by Ce3+ via a coordination process. The ALP-catalyzed hydrolysis of p-nitrophenyl phosphate leads to the formation of p-nitrophenol and inorganic orthophosphate, and the newly formed orthophosphate could potently combine with Ce3+ due to the higher affinity, thus, recovering the fluorescence of calcein. The corresponding fluorescence signal triggered by phosphate is related to ALP activities labeled on antibody, and thus could be applied to detect target antigen in an enzyme-linked immunosorbent assay (ELISA) platform. The fluorescence intensity correlated well to the AFP concentration ranges of 0.2-1.0 and 1.0-4.0 ng/mL, with a detection limit of 0.041 ng/mL. The proposed fluorescence ELISA possesses convincing recognition mechanism and exhibits excellent assay performance in the evaluation of the AFP level in serologic test, which unambiguously reveals great application potential in the clinic diagnosis of disease biomarkers.

FRET Effect between Fluorescent Polydopamine Nanoparticles and MnO2 Nanosheets and Its Application for Sensitive Sensing of Alkaline Phosphatase

As an essential and universal hydrolase, alkaline phosphatase (ALP) has been identified as a crucial indicator of various diseases. Herein, we, for the first time, expanded the application of fluorescent polydopamine (F-PDA) nanoparticles to nanoquencher-based biosensing system, as well as discovered the reversible quenching effect of manganese dioxide (MnO2) nanosheets on the fluorescence of F-PDA nanoparticles and intensively confirmed the quenching mechanism of Förster resonance energy transfer by using transmission electron microscopy, UV-vis, Fourier transform infrared spectroscopy, and fluorescence lifetime experiments. By means of the ALP-triggered generation of ascorbic acid (AA) from the substrate ascorbic acid 2-phosphate, the AA-triggered reduction of MnO2 nanosheets to Mn2+, as well as the clear quenching mechanism of F-PDA nanoparticles by MnO2 nanosheets, we have developed a label-free, low-cost, visual, and facile synthetic fluorescent biosensor for convenient assay of ALP activity. The fluorescent bioassay shows a good linear relationship from 1 to 80 mU/mL (R2 = 0.999), with a low detection limit of 0.34 mU/mL, and the excellent applicability in human serum samples demonstrates potential applications in clinical diagnosis and biomedical research.

Photo-Induced Electron Transfer-Based Versatile Platform with G-Quadruplex/Hemin Complex as Quencher for Construction of DNA Logic Circuits

G-quadruplex has been developed as an innovator for analytical chemistry and biomedicine due to its vibrant binding activity, structural polymorphism, and critical roles in biological regulation. Herein, a simple but versatile platform was obtained by integrating split G-quadruplex and fluorophore into a molecular beacon, where the photoinduced electron transfer could occur when the fluorophore approached the preformed G-quadruplex/hemin complexes. Such design subtly combined the G4 disruption-induced fluorescent turn-on strategy and the photoinduced electron transfer property into one platform for constructing the logic circuits. On the basis of such a universal platform, a series of binary logic gates (OR, INHIBIT, AND, and XOR), a combinatorial gate (INHIBIT-OR), and even a complex logic operation for discrimination of multiples of three from natural numbers less than ten have been successfully achieved only by employing such platform as work unit and single-strand DNAs as inputs. The set-reset function of this platform could be realized by alternatively introducing blocking and releasing strands. In addition, this platform could operate in a biological matrix stably and precisely. Therefore, such a universal platform lays the foundation for complicating the logic systems, realizing the biocomputing and also points out a new direction for target detection.

Inner Filter Effect-Based Sensor for Horseradish Peroxidase and Its Application to Fluorescence Immunoassay

Being an important model peroxidase, horseradish peroxidase (HRP) has been thoroughly understood, and the detection of HRP is not only directly related to peroxidase-triggered catalytic process, but also linked to the development of HRP-based enzyme-linked immunosorbent assay (ELISA). Herein, we have reported an unconventional fluorescent sensor for convenient assay of HRP activity based on the HRP-catalyzed specific conversion of p-phenylenediamine (PPD) into chromogenic PPDox with H2O2 as the oxidizing agent, accompanied by the fluorescence quenching effect on fluorescein. By combining UV-vis absorption spectrum, isothermal titration calorimetry, and fluorescence lifetime analysis, we have confirmed the inner filter effect as a main quenching mechanism in our proposed fluorescent assay. According to the intrinsic sensitivity of fluorescent sensor and high selectivity, our PPD/fluorescein-based sensing system can be utilized for real-time monitoring of the HRP activity in real biological samples. Furthermore, the unambiguous response mechanism and excellent sensing performance encourage us to extend such HRP assay into the HRP-based fluorescent ELISA, which has a broad prospect of application in fluorescent diagnosis of hepatocellular carcinoma (HCC) by sensing alpha-fetoprotein, the well-known serologic HCC marker.

Polydopamine Nanoparticles as Efficient Scavengers for Reactive Oxygen Species in Periodontal Disease

Antioxidative therapy has been considered an efficient strategy for the treatment of a series of excessive reactive oxygen species (ROS)-triggered diseases, including oxidative-stress-induced periodontal disease. However, current natural enzymes and nanozymes often show their high specificity toward given ROS and have insufficient antioxidative effects against multiple ROS generated in the diseases process. Meanwhile, multienzyme-based antioxidant defense systems are usually confined by the complicated synthesis as well as potential unwanted residue and toxicity. Various supports are highly needed to immobilize natural enzymes and antioxidants during the biorelated usages due to their low operational stability and difficulty of reuse. To overcome these limitations, we develop a high-performance platform by using biodegradable polydopamine nanoparticles (PDA NPs) as smart ROS scavengers in oxidative stress-induced periodontal disease. Although PDA-based materials are well-known to eliminate ROS both in vitro and in vivo, their antioxidative performance in periodontal disease and relative mechanisms have yet to be well-explored. In this study, PDA NPs can act as ROS scavengers in dental specialties with ideal outcomes. Spectroscopic and in vitro experiments provide strong evidence for the roles of PDA NPs in scavenging multiple ROS and suppressing ROS-induced inflammation reactions. In addition to the above investigations, the results from a murine periodontitis model clearly demonstrate the feasibility of PDA NPs as robust antioxidants with which to remove ROS and decrease periodontal inflammation without any side effects. Taken together, the results from our present study will provide valuable insight into the development of safe and efficient antioxidant defense platforms for further biomedical uses.

An Electric Potential Modulated Cascade of Catalyzed Hairpin Assembly and Rolling Chain Amplification for MicroRNA Detection

MicroRNAs serve as a new type of biomarker for multifarious diseases due to its critical roles in post transcriptional gene regulation. Herein, we firstly integrate the catalyzed hairpin assembly (CHA) and rolling circle amplification (RCA) into an electrochemical biosensor for sensitive and specific detection of miR-21. Meanwhile, an electric potential was employed to modulate the efficiency of CHA occurred on the electrode, which offer a simple but effective method to surmount the accessibility problem of probes. The biosensor achieved an ultrasensitive determination of miR-21 with a low limit of detection of 13.5 fM and a linear range from 15 fM to 250 pM. This research encourages us to challenge the hyphenated multiple amplification strategies and provides a stable and effective method for the detection of diseases-related miRNAs in peripheral biofluids, as well as paves a road for the future clinical diagnostics and treatment of disease.

Alkaline Phosphatase Assay Based on the Chromogenic Interaction of Diethanolamine with 4-Aminophenol

Diethanolamine (DEA) has been extensively utilized as an alkaline buffer in current assays of alkaline phosphatase (ALP) activity in the past decades. While playing the role of a buffer, the chemical reactivity of DEA has been widely ignored in such assays. Herein, we report an interesting chromogenic interaction between DEA and 4-aminophenol (AP) in the presence of H2O for the first time, which inspires us to develop a novel DEA-participated ALP activity assay by using 4-aminophenyl phosphate (APP) as a substrate. This APP/DEA-based colorimetric approach has been proved to be comparable and even superior to the conventional p-nitrophenyl phosphate (pNPP)-based one, especially in the low ALP activity region, due to its higher sensitivity. The clear response mechanism and excellent sensing performance ensure that it can be further applied to determining ALP activity in real biological samples, screening potential ALP inhibitors in vitro, establishing ALP-enabled ELISA, and even fluorophore-assisted fluorescent ALP activity assay. It is demonstrated that this strategy not only possesses a good feasibility, but also exhibits a promising outlook for a series of ALP-related and -extended detections.

An Enzyme Cascade-Triggered Fluorogenic and Chromogenic Reaction Applied in Enzyme Activity Assay and Immunoassay

An enzyme cascade-triggered reaction with novel signal generation mechanism is beneficial for the development and insight of the enzyme cascade, which is extensively used for signal transduction in potential applications. Inspired by the fluorogenic and chromogenic reaction between dopamine and resorcinol, and the specific catalytic properties of alkaline phosphatase (ALP) and tyrosinase, we designed and synthesized an unconventional substrate of ALP, named p-aminoethyl-phenyl phosphate disodium salt (PAPP). As expected, the ALP and tyrosinase-incubated PAPP solution exhibited pale yellow with intense blue fluorescence upon addition of resorcinol, owing to the ALP-catalyzed transformation of PAPP into an intermediate tyramine, and the tyrosinase-catalyzed hydroxylation of tyramine to dopamine, as well as the specific reaction between dopamine and resorcinol. Therefore, an enzyme cascade system has been developed herein based on the ALP and tyrosinase coupled enzymes-triggered fluorogenic and chromogenic reaction. According to the direct relationship between the activity of ALP/tyrosinase and absorbance/fluorescence intensity of the resultant solution, the proposed enzyme cascade-triggered reaction was utilized for assaying ALP and tyrosinase activity with fluorometric and colorimetric dual-readout signals. Furthermore, such enzyme cascade catalysis process was integrated into the ALP-based cascade enzyme-linked immunosorbent assay with dual-readout signals, resulting in the sensitive detection of cardiac troponin I in diluted serum.