Intrarenal pH-Responsive Self-Assembly of Luminescent Gold Nanoparticles for Diagnosis of Early Kidney Injury.

Metabolic acidosis-induced kidney injury (MAKI) is asymptomatic and lack of clinical biomarkers in early stage, but rapidly progresses to severe renal fibrosis and ultimately results in end-stage kidney failure. Therefore, developing rapid and noninvasive strategies direct responsive to renal tubular acidic microenvironment rather than delayed biomarkers are essential for timely renoprotective interventions. Herein, we develop pH-responsive luminescent gold nanoparticles (p-AuNPs) in the second near-infrared emission co-coated with 2,3-dimethylaleic anhydride conjugated ß-mercaptoethylamine and cationic 2-diethylaminoethanethiol hydrochloride, which showed sensitive pH-induced charge reversal and intrarenal self-assembly for highly sensitive and long-time (~24 h) imaging of different stages of MAKI. By integrating advantages of pH-induced intrarenal self-assembly and enhanced interactions between pH-triggered positively charged p-AuNPs and renal tubular cells, the early- and late-stage MAKI could be differentiated rapidly within 10 min post-injection (p.i.) with contrast index (CI) of 3.5 and 4.3, respectively. The corresponding maximum CI could reach 5.1 and 9.2 at 12 h p.i., respectively. Furthermore, p-AuNPs were demonstrated to effectively real-time monitor progressive recovery of kidney injury in MAKI mice after therapy, and also exhibit outstanding capabilities for drug screening. This pH-responsive strategy showed great promise for feedback on kidney dysfunction progression, opening new possibilities for early-stage diagnosis of pH-related diseases.

Inhibition of TGF-ß1/Smad3 signaling by compound 5aa: A potential treatment for idiopathic pulmonary fibrosis.

The incidence of idiopathic pulmonary fibrosis (IPF) has been steadily increasing each year, posing significant challenges in its treatment. In this study, we conducted the design and synthesis of 23 new inhibitors that specifically target the TGF-ß1/Smad3 pathway. Initially, we employed a cell model of TGF-ß-induced pulmonary fibrosis, using cell survival rate and HYP expression as indicators to identify the potent ingredient 5aa, which demonstrated significant anti-pulmonary fibrosis activity. Subsequently, we induced mice with bleomycin (BLM) to establish an experimental animal model of pulmonary fibrosis, and evaluated the pharmacodynamics of 5aa in vivo against pulmonary fibrosis. The alterations in HYP and collagen levels in BLM-induced pulmonary fibrosis mice were analyzed using ELISA and immunohistochemistry techniques. The results indicated that compound 5aa effectively suppressed the fibrotic response induced by TGF-ß1, inhibited the expression of the fibrotic marker α-SMA, and hindered the EMT process in NIH3T3 cells. Additionally, oral administration of 5aa demonstrated significant therapeutic effects in a mouse model of IPF, comparable to the established drug Nintedanib. Moreover, compound 5aa exhibited higher bioavailability in vivo compared to Nintedanib. These collective outcomes suggest that 5aa holds promise as a potential inhibitor of TGF-ß1/Smad3 signaling for the treatment of IPF.

The new N2, N4-diphenylpyridine-2,4-diamine deuterated derivatives as EGFR inhibitors to overcome C797S-mediated resistance.

A series of new deuterated and non-deuterated N2, N4-diphenylpyridine - 2,4-diamine derivatives were synthesized and evaluated as EGFR C797S-mediated resistance inhibitors. Most of these compounds exhibited potent antiproliferative activity against Baf3-EGFR L858R/T790M/C797S and Baf3-EGFR Del19/T790M/C797S cancel cell lines, with IC50 values in the nanomolar concentration range. Among them, compound 14l represented the most active compound with IC50 values of 8-11 nM. Interestingly, metabolic stability assay with rat liver microsomes indicated that the half-life of the deuterated derivative 14o was significantly increased compared to that of 14l. In xenograft mice models, 14o inhibited tumor growth with excellent inhibitory rate of 75.1 % at the dosage of 40 mg/kg, comparing 73.2 % of the TGI with its non-deuterated compound 14l, at a dosage of 80 mg/kg. Mechanism studies revealed that 14o was a potent EGFR L858R/T790M/C797S and EGFR Del19/T790M/C797S kinase inhibitor, which could downregulate the protein phosphorylation of EGFR and m-TOR signaling pathways, arrest cell cycle at G2/M phase by affecting the expression of CDC25C, and promote cell apoptosis by regulating the expression of cleaved caspase-3. In summary, 14o could serve as a promising deuterated compound for the development of highly efficient anticancer agents.

Luminescent Gold Nanoparticles with Discrete DNA Valences for Precisely Controlled Transport at the Subcellular Level.

Ultrasmall luminescent gold nanoparticles (AuNPs) with excellent capabilities to cross biological barriers offer great promise in designing intelligent model nanomedicines for investigating structure-property relationships at the subcellular level. However, the strict surface controllability of ultrasmall AuNPs is challenging because of their small size. Herein, we report a facile in situ method for precisely controlling DNA aptamer valences on the surface of luminescent AuNPs with emission in the second near-infrared window using a phosphorothioate-modified DNA aptamer, AS1411, as a template. The discrete DNA aptamer number of AS1411-functionalized AuNPs (AS1411-AuNPs, ≈1.8 nm) with emission at 1030 nm was controlled in one aptamer (V1), two aptamers (V2), and four aptamers (V4). It was then discovered that not only the tumor-targeting efficiencies but also the subcellular transport of AS1411-AuNPs were precisely dependent on valences. A slight increase in valence from V1 to V2 increased tumor-targeting efficiencies and resulted in higher nucleus accumulation, whereas a further increase in valence (e.g., V4) significantly increased tumor-targeting efficiencies and led to higher cytomembrane accumulation. These results provide a basis for the strict surface control of nanomedicines in the precise regulation of in vivo transport at the subcellular level and their translation into clinical practice in the future.

A novel c-Met/TRK inhibitor 1D228 efficiently inhibits tumor growth by targeting angiogenesis and tumor cell proliferation.

Multiple tumors are synergistically promoted by c-Met and TRK, and blocking their cross-signalling pathway may give better effects. In this study, we developed a tyrosine kinase inhibitor 1D228, which exhibited excellent anti-tumor activity by targeting c-Met and TRK. Models in vitro, 1D228 showed a significant better inhibition on cancer cell proliferation and migration than the positive drug Tepotinib. Models in vivo, 1D228 showed robust anti-tumor effect on gastric and liver tumor growth with 94.8% and 93.4% of the TGI, respectively, comparing 67.61% and 63.9% of Tepotinib. Importantly, compared with the combination of Larotrectinib and Tepotinib, 1D228 monotherapy in MKN45 xenograft tumor models showed stronger antitumor activity and lower toxicity. Mechanistic studies showed that 1D228 can largely inhibit the phosphorylation of TRKB and c-Met. Interestingly, both kinases, TRKs and c-Met, have been found to be co-expressed at high levels in patients with gastric cancer through IHC. Furthermore, bioinformatics analysis has revealed that both genes are abnormally co-expressed in multiple types of cancer. Cell cycle analysis found that 1D228 induced G0/G1 arrest by inhibiting cyclin D1. Additionally, vascular endothelial cells also showed a pronounced response to 1D228 due to its expression of TRKB and c-Met. 1D228 suppressed the migration and tube formation of endothelial cells, which are the key functions of tumor angiogenesis. Taken together, compound 1D228 may be a promising candidate for the next generation of c-Met and TRK inhibitors for cancer treatment, and offers a novel potential treatment strategy for cancer patients with abnormal expressions of c-Met or NTRK, or simultaneous of them.

Discovery of potent and effective inhibitors containing sulfoxide structures targeting EML4-ALK rearrangement and EGFR mutant non-small cell lung cancer.

For non-small cell lung cancer patients with dual mutations in EGFR and ALK, there are currently no effective therapies. Consequently, novel EGFR/ALK dual-target inhibitors are urgently needed for the treatment of NSCLC. Here, we designed a series of highly effective small molecule dual inhibitors of ALK and EGFR. The biological evaluation highlighted that most of these new compounds could effectively inhibit both ALK and EGFR in enzymatic and cellular assays. Compound (+)-8l was investigated for its antitumor properties, and it was found that (+)-8l blocked the phosphorylation of EGFR and ALK induced by ligands and inhibited phosphorylation-ERK and phosphorylation-AKT induced by ligands. Furthermore, (+)-8l also induces apoptosis and G0/G1 cell cycle arrest in cancer cells and inhibits proliferation, migration, and invasion. Notably, (+)-8l significantly suppressed tumor growth in the H1975 cell-inoculated xenograft model (20 mg/kg/d, TGI: 96.11%), PC9 cell-inoculated xenograft model (20 mg/kg/d, TGI: 96.61%) and EML4 ALK-Baf3 cell-inoculated xenograft model (30 mg/kg/d, TGI: 80.86%). These results highlight the differentiated potential of (+)-8l to inhibit ALK rearrangement and EGFR mutation in NSCLC.

A 12-Port MIMO Antenna System for 5G/WLAN Applications.

In this paper, a 12-port MIMO antenna system for 5G/WLAN applications is proposed. The proposed antenna system consists of two types of antenna modules: an L-shaped antenna module covering the C-band (3.4-3.6 GHz) for 5G mobile applications and a folded monopole module for the 5G/WLAN mobile application band (4.5-5.9 GHz). Each two antennas form a pair, six pairs in total, forming a 12 × 12 MIMO antenna array, and the elements between the antenna pairs can achieve an isolation of 11 dB or more without additional decoupling structures. Experimental results show that the antenna can cover the 3.3-3.6 GHz and 4.5-5.9 GHz bands with an overall efficiency greater than 75% and an envelope correlation coefficient less than 0.04. Finally, the one-hand holding mode and two-hand holding mode are discussed to demonstrate their stability in practical applications, and the results show that they still exhibit good radiation and MIMO performance when operating in both modes.

Novel Dual-Target Kinase Inhibitors of EGFR and ALK Were Designed, Synthesized, and Induced Cell Apoptosis in Non-Small Cell Lung Cancer.

ALK-positive NSCLC coexisting with EGFR mutations is a frequently occurring clinical phenomenon. Targeting ALK and EGFR simultaneously may be an effective way to treat these cancer patients. In this study, we designed and synthesized ten new dual-target EGFR/ALK inhibitors. Among them, the optimal compound 9j exhibited good activity with IC50 values of 0.07829 ± 0.03 µM and 0.08183 ± 0.02 µM against H1975 (EGFR T790M/L858R) and H2228 (EML4-ALK) cells, respectively. Immunofluorescence assays indicated that the compound could simultaneously inhibit the expression of phosphorylated EGFR and ALK proteins. A kinase assay demonstrated that compound 9j could inhibit both EGFR and ALK kinases; thus, exerting an antitumor effect. Additionally, compound 9j induced apoptosis in a dose-dependent manner and inhibited the invasion and migration of tumor cells. All of these results indicate that 9j is worthy of further study.

Novel third-generation pyrimidines-based EGFR tyrosine kinase inhibitors targeting EGFR T790M mutation in advanced non-small cell lung cancer.

The critical T790M secondary mutation in epidermal growth-factor receptor (EGFR) mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors. Herein, we identified 12 new 2,4-diaryl pyrimidine derivatives containing thiophene fragments as new selective third-generation EGFR inhibitors. Among them, Compound 6a showed good inhibitory activity against EGFR mutant cells with an IC50 value of 0.0022 ± 0.001 µM and was approximately 1730-fold less potent against EGFR WT cells (IC50: 4.499 ± 0.057 µM). Moreover, it strongly affected EGFR-mediated signaling pathways, attenuated tumor proliferation via the intrinsic mitochondrial apoptotic pathway, arrested the cell cycle at G0/G1 phase, and induced apoptosis in H1975 cells. It also displayed appropriate pharmacokinetic (PK) parameters with an oral bioavailability value of 33.57%. Additionally, in vivo studies confirmed that 6a suppressed tumor growth in an H1975 xenograft model (25 mg/kg/d, TGI: 90.24%). Overall, these results suggest that 6a could be a promising lead compound for overcoming the clinical EGFR T790M resistance of patients with non-small-cell lung cancers (NSCLCs).

Point-of-Care Assay of Alkaline Phosphatase Enzymatic Activity Using a Thermometer or Temperature Discoloration Sticker as Readout.

Alkaline phosphatase (ALP) is a significant biomarker in clinical diagnostics, and the abnormal level of ALP enzyme in serum is closely related to various diseases such as bone or liver cancer, bone metastases, and extrahepatic biliary obstruction. Herein a simple and portable photothermal biosensor was developed for sensitive detection of ALP enzyme based on the formation of polydopamine (PDA) nanoparticles using a thermometer or temperature discoloration sticker as readout. A MnO2 nanosheet was first prepared using a novel one-pot strategy which was operationally simple and not overly time-consuming. Then dopamine (DA) was quickly polymerized into PDA nanoparticles in the presence of the MnO2 nanosheet. When the model analyte ALP was present, the substrate 2-phospho-l-ascorbic acid trisodium salt (AAP) was catalytically hydrolyzed into l-ascorbic acid (AA), resulting in the inhibition of the formation of the PDA nanoparticles owing to the fact that the MnO2 nanosheet was reduced to Mn2+ by the generated AA. Thus, a portable biosensor based on the photothermal properties of PDA nanoparticles for ALP enzyme detection was established with a detection limit as low as 0.1 U/L (thermometer) and 1 U/L (temperature discoloration sticker). In addition, it also showed excellent sensing performance for the ALP assay in human serum. Such a simple, label-free, cost-effective, and sensitive assay could exhibit real potential application for ALP detection and early diagnosis, especially in developing countries or remote regions.

Direct quantification of cancerous exosomes via surface plasmon resonance with dual gold nanoparticle-assisted signal amplification.

Sensitive detection of cancerous exosomes is critical to early diseases diagnosis and prognosis. Herein, a sensitive aptasensor was demonstrated for exosomes detection by surface plasmon resonance (SPR) with dual gold nanoparticle (AuNP)-assisted signal amplification. Dual nanoparticle amplification was achieved by controlled hybridization attachment of AuNPs resulting from electronic coupling between the Au film and AuNPs, as well as coupling effects in plasmonic nanostructures. By blocking the Au film surface with 11-Mercapto-1 -undecanol (MCU), nonspecific adsorption of AuNPs onto the SPR chip surface was suppressed and regeneration of the SPR sensor was realized. This method was highly sensitive and we have achieved the limit of detection (LOD) down to 5 × 103 exosomes/mL, which showed a 104-fold improvement in LOD compared to commercial ELISA. Moreover, the SPR sensor had the capability to differentiate the exosomes secreted by MCF-7 breast cancer cells and MCF-10A normal breast cells. Furthermore, the SPR sensor could effectively detect the exosomes in 30% fetal bovine serum. The work provides a sensitive and efficient quantification approach to detect cancerous exosomes and offers an avenue toward future diagnosis and comprehensive studies of exosomes.

Aptamer as a Tool for Investigating the Effects of Electric Field on Aß40 Monomer and Aggregates Using Single-Molecule Force Spectroscopy.

The effects of electric field (EF) on amyloid proteins have been given increasing attention in regulation of amyloid-ß (Aß) aggregation and therapeutic methods because of prevalence of Alzheimer's disease. Herein, for the first time, aptamer was used as a tool for investigating the effects of EF on Aß40 monomer and Aß40 aggregates (Aß40 oligomer and Aß40 fibril) by single-molecule force spectroscopy. Interestingly, EF had different effects on Aß40 monomer and Aß40 aggregates, which might be due to the interactions of aptamer with EF-treated Aß40 monomer and Aß40 aggregates. With application of EF to Aß40 oligomer, specific interaction and binding probability of aptamer-Aß40 oligomer slightly increased. However, the interactions of aptamer with Aß40 monomer and Aß40 fibril changed greatly when EF was respectively applied to Aß40 monomer and Aß40 fibril. Meanwhile, the interaction of aptamer with Aß40 monomer changed from nonspecific binding to specific binding. And the rupture force distribution of aptamer-Aß40 fibril changed from bimodal distribution to unimodal distribution. Hence, it was presumed that Aß40 oligomer almost maintained oligomeric structure, while Aß40 fibril and Aß40 monomer changed into Aß40 oligomer when EF was applied. Subsequently, this presumption was mainly verified by atomic force microscopy imaging and circular dichroism. This work testifies that aptamer may be a valuable tool to investigate effects of EF on Aß40 monomer and Aß40 aggregates. Moreover, the established method provides a new perspective to study aggregation of amyloid proteins.

Low-Fouling Surface Plasmon Resonance Sensor for Highly Sensitive Detection of MicroRNA in a Complex Matrix Based on the DNA Tetrahedron.

Antifouling surfaces that could reduce nonspecific adsorption from a complex matrix are a great challenge in surface plasmon resonance (SPR) sensors. An antifouling surface made by the covalent attachment of DNA tetrahedron probes (DTPs) onto gold surfaces demonstrated superior antifouling property against protein and cell. DTP-modified Au (DTPs-Au) film for two single protein samples (1 mg/mL myoglobin, 48 mg/mL HSA) and five complex matrices (100% serum, 100% plasma, 9.85 × 108 red cells/mL, 5% whole blood, and cell lysate) had low or ultralow adsorption amounts (≤8.0 ng/cm2). More interestingly, DTPs-Au film could also avoid Au deposition on the surface in the process of the catalytic growth of gold nanoparticles (AuNPs). Thus, a low-fouling and sensitive SPR sensor for miRNA detection in a complex matrix was developed by integrating DTPs-Au film with the catalytic growth of AuNPs. Exploiting the amplification of catalytic growth of AuNPs, the detection limit was 0.8 fM toward target let-7a. Moreover, the SPR sensor revealed excellent selectivity and could distinguish let-7a from homologous family. More importantly, the SPR sensor could be feasible for determining miRNA in 100% human serum and cancer cell lysates, and the results of detecting miRNA from cancer cells were in excellent accord with the results obtained using qRT-PCR. This assay may have great potential as an miRNA quantification method in complex samples.

Investigation of the interactions between aptamer and misfolded proteins: From monomer and oligomer to fibril by single-molecule force spectroscopy.

Increasing knowledge on the understanding interactions of aptamer with misfolded proteins (including monomer, oligomer, and amyloid fibril) is crucial for development of aggregation inhibitors and diagnosis of amyloid diseases. Herein, the interactions of lysozyme monomer-, oligomer-, and amyloid fibril-aptamer were investigated using single-molecule force spectroscopy. The results revealed that the aptamer screened against lysozyme monomer could also bind to oligomer and amyloid fibril, in spite of the recognition at a lower binding probability. It may be attributed to the inherent structural differences of misfolded proteins and the flexible conformation of aptamer. In addition, dynamic force spectra showed that there were similar dissociation paths in the dissociation process of lysozyme monomer-, oligomer-, and amyloid fibril-aptamer complexes. It showed that the dissociation only passed 1 energy barrier from the binding state to the detachment. However, the dynamic parameters suggested that the oligomer- and amyloid fibril-aptamer were more stable than lysozyme monomer-aptamer. The phenomena may result from the exposure of aptamer-recognized sequences on the surface and the electrostatic interactions. This work demonstrated that single-molecule force spectroscopy could be a powerful tool to study the binding behavior of the aptamer with misfolded proteins at single-molecule level, providing abundant information for researches and comprehensive applications of aptamer probes in diagnosis of amyloid diseases.

High sensitivity surface plasmon resonance biosensor for detection of microRNA based on gold nanoparticles-decorated molybdenum sulfide.

A high sensitive surface plasmon resonance (SPR) biosensor was proposed based on the gold nanoparticles-decorated molybdenum sulfide (AuNPs-MoS2) for the first time. This SPR platform using the AuNPs-MoS2 nanocomposites as signal labels for the sensitive and facile measurement of microRNA (miRNA) was executed in only two steps. At first, the thiol-modified DNA oligonucleotide probes, including a sequence complementary to the target miRNA-141, were fixed on the Au film to identify the segment sequence of target miRNA-141. Then, the assistant DNA-linked AuNPs-MoS2 nanocomposites were used to combine with the other section of the miRNA-141. Performance of SPR biosensor was enhanced by taking advantage of the AuNPs-MoS2 nanocomposites, thus this newly presented sensing assay exhibited high sensitivity toward miRNA with a detection limit of 0.5 fM. Furthermore, the method showed high specificity, resulting in distinguishing differences among miRNA-200 family members. Especially, the assay could also be used to detect human miRNA from cancer cells, and the results were in excellent accord with the ones obtained using qRT-PCR. What is more, this presented method could be feasible for determining miRNA in 10% human serum. This assay may provide a great potential as a miRNA quantification method in complex samples, and exert significant effect on biomedical research and clinical early diagnosis.

High sensitivity surface plasmon resonance biosensor for detection of microRNA and small molecule based on graphene oxide-gold nanoparticles composites.

A versatile and sensitive surface plasmon resonance (SPR) biosensor based on two layers of graphene oxide-gold nanoparticles (GO-AuNPs) composites was designed for the detection of microRNA (miRNA) and small molecule adenosine. The bottom layer, which acted as a functionalized substrate on the sensor chip, provided a high specific surface area convenient for the immobilization of capture DNA molecules. The upper layer served as a signal-amplification element. By employing these two layers of GO-AuNPs composites, the dual amplification strategy was achieved so that a measurement of miRNA-141 with a detection limit of 0.1fM was obtained. Moreover, the developed SPR biosensor showed decent selectivity toward miRNA-200 family members. Especially, the SPR biosensor demonstrated its applicability for the detection of miRNA-141 in cancer cell extractions, and the results obtained were consistent with those obtained by qRT-PCR. Interestingly, small molecule adenosine could also be detected using this SPR biosensor in combination with a split aptamer. Considering the superior sensitivity, selectivity and generality, this work promised much potential for the detection of various biomolecules.

Evaluating the Effect of Lidocaine on the Interactions of C-reactive Protein with Its Aptamer and Antibody by Dynamic Force Spectroscopy.

Effects of medicine on the biomolecular interaction have been given extensive attention in biochemistry and biomedicine because of the complexity of the environment in vivo and the increasing opportunity of exposure to medicine. Herein, the effect of lidocaine on the interactions of C-reactive protein (CRP) with its aptamer and antibody under different temperature was investigated through dynamic force spectroscopy (DFS). The results revealed that lidocaine could reduce the binding probabilities and binding affinities of the CRP-aptamer and the CRP-antibody. An interesting discovery was that lidocaine had differential influences on the dynamic force spectra of the CRP-aptamer and the CRP-antibody. The energy landscape of the CRP-aptamer turned from two activation barriers to one after the treatment of lidocaine, while the one activation barrier in energy landscape of the CRP-antibody almost remained unchanged. In addition, similar results were obtained for 25 and 37 °C. In accordance with the result of molecular docking, the reduction of binding probabilities might be due to the binding of lidocaine on CRP. Additionally, the alteration of the dissociation pathway of the CRP-aptamer and the change of binding affinities might be caused by the conformational change of CRP, which was verified through synchronous fluorescence spectroscopy. Furthermore, differential effects of lidocaine on the interactions of CRP-aptamer and CRP-antibody might be attributed to the different dissociation processes and binding sites of the CRP-aptamer and the CRP-antibody and different structures of the aptamer and the antibody. This work indicated that DFS provided information for further research and comprehensive applications of biomolecular interaction, especially in the design of biosensors in complex systems.

Surface plasmon resonance biosensor for sensitive detection of microRNA and cancer cell using multiple signal amplification strategy.

A sensitive and versatile surface plasmon resonance (SPR) biosensor was proposed for the detection of microRNA (miRNA) and cancer cell based on multiple signal amplification strategy. Thiol-modified hairpin probe, including a sequence complementary to the target miRNA, was first immobilized on the Au film. In the presence of target miRNA, the stem-loop structure of hairpin probe was unfolded, and then DNA-linked Au nanoparticles (AuNPs) were hybridized with the terminus of the unfolded hairpin probe. Subsequently, DNA-linked AuNPs initiated the formation of DNA supersandwich structure through the addition of two report DNA sequences. Owing to the electronic coupling between localized plasmon of the AuNPs and the surface plasmon wave, as well as the enhancement of the refractive index of the medium over the Au film induced by DNA supersandwich structure, the SPR response was significantly enhanced. Next, numerous positively charged silver nanoparticles (AgNPs) were absorbed onto the long-range DNA surpersandwich equably, resulting in a further increase of SPR response. Due to the enzyme-free multiple signal amplification strategy, as low as ca. 0.6 fM miRNA-21 could be detected. In addition, this biosensor showed high selectivity toward single-base mismatch. More importantly, this SPR biosensor was also used for cancer cell detection coupled with the cell-specific aptamer modified magnetic nanoparticles. Given that the biosensor avoided enzyme introduction, the limitation of the enzyme was overcome. The versatile biosensor has great potential for the broad applications in the field of clinical analysis.

An isothermal electrochemical biosensor for the sensitive detection of microRNA based on a catalytic hairpin assembly and supersandwich amplification.

A novel isothermal electrochemical biosensor was proposed for the sensitive detection of microRNA (miRNA) based on the ingenious combination of the target-catalyzed hairpin assembly (CHA) and supersandwich amplification strategies. Since miRNA-221 has been reported to be overexpressed in cancers and has been a potentially useful biomarker for the diagnosis of the related diseases, miRNA-221 was chosen as a model target miRNA. The target miRNA-221 triggered a toehold strand displacement assembly of the two hairpin substrates, which led to the cyclicality of the target miRNA and the CHA products. Subsequently, the CHA products hybridized with a capture probe on the electrode and the exposed stem of the CHA products was further used to propagate the supersandwich. After this, the signal probe was modified with horseradish peroxidase (HRP) to form a supersandwich multiplex HRP-DNA label, which could achieve an amplified electrochemical signal. Using the isothermal dual signal amplification strategies, miRNA-221 as low as 0.6 pM (3σ) could be detected. In addition, this biosensor showed high selectivity and could discriminate miRNA-221 from the homologous miRNAs. Note that human miRNA from cancer cells could also be detected and the results were in excellent agreement with those obtained using qRT-PCR. Given that the biosensor avoided the introduction of nanoparticles, the limitation of using the nanoparticles was overcome. The proposed biosensor has great potential for broad applications in the field of clinical analysis.