Semiarbitrary qPCR for Sensitive Detection of Circulating miRNA via Terminal Deoxynucleotidyl Transferase-Assisted RNA-Primed DNA Polymerization.

Circulating microRNAs (miRNAs) have recently emerged as noninvasive disease biomarkers. Quantitative detection of circulating miRNAs could offer significant information for clinical diagnosis due to its significance in the development of biological processes. In response to the current challenges of circulating miRNA detection, we introduce a sensitive, selective, and versatile circulating miRNA detection strategy using terminal deoxynucleotidyl transferase (TdT)-catalyzed RNA-primed DNA polymerization (TCRDP) coupled with semiarbitrary qPCR (SAPCR). Semiarbitrary qPCR was first developed here to detect long fragment targets with only a short-known sequence or to detect a short fragment target after extension with terminal transferase. Besides, the subsequent results show that TdT has a preference for RNA, particularly for extending RNAs with purine-rich and unstructured ends. Consequently, utilizing this assay, we have successfully applied it to the quantitative analysis of circulating miR-122 in animal models, a sensitive and informative biomarker for drug-induced liver injury, and as low as 200 zmol of the target is detected with desirable specificity and sensitivity, indicating that the TCRDP-SAPCR can offer a promising platform for nucleic acids analysis.

Development of highly sensitive dual-enhanced fluorescence quenching immunochromatographic test strips based on Pt nanoprobes.

The fluorescence-quenching method is crucial in vitro analysis, particularly for immunochromatographic test strips (ICTs) using noble metal nanoparticles as probes. However, ICTs still fall short in meeting the requirements for the detection of traces biomarkers due to the noble metal nanoparticles can only quench fluorescence of the dyes within a confined distance. Interestingly, noble metal nanoparticles, such as Pt NPs cannot only perform fluorescence-quenching ability based on the Förster resonance energy transfer (FRET), but also show perfect oxidase-like catalytic performance on many kinds of substrates, such as 3,3',5,5' -tetramethylbenzidine (TMB). We observed that the oxTMB (the oxidation products of TMB) exhibited notable effectiveness in quenching Cy5 fluorescence by the strong inner filter effect (IFE), which obviously improved the fluorescence-quenching efficiency with extremely low background signal. Through the dual-enhanced fluorescence quenching mechanism, the fluorescence quenching constant (Kn) was 661.24-fold that of only Pt NPs on the NC membrane. To validate the feasibility of this technique, we employed two types of biomarkers, namely microRNA (miR-15a-5p) and the signature protein (PSA). The sensitivity of miR-15a-5p was 9.286 × 10-18 mol/L and 17.5-fold more than that based on Pt NPs. As for the PSA, the LOD (0.6265 pg/mL) was 15.5-fold enhancement more sensitive after catalysis. Overall, the dual-enhanced fluorescence quenching rFICTs could act as a practical detection for biomarker in real samples.

NQO1-activated multifunctional theranostic probe for imaging-guided mitochondria-targeted photodynamic therapy and boosting immunogenic cell death.

NAD(P)H: quinine oxidoreductase (NQO1) is overexpressed in many types of cancer cells, and have been used as a biomarker for cancer diagnosis and targeted therapy. The development of activatable theranostic agents is highly desirable for precise cancer diagnosis and therapy. Herein, a NQO1-activated near-infrared multifunctional theranostic probe I-HCy-Q is successfully developed for imaging guided photodynamic therapy. The NIR fluorescence (λex/em = 685/703 nm) and capacity of reactive oxygen species generation are sensitive controllable by the level of NQO1, the linear detection range of NQO1 and limit of detection are 0.05-1.5 µg/mL and 5.66 ng/mL, respectively. On the one hand, I-HCy-Q can monitor the activity of NQO1 and distinguish the NQO1 positive cancer cells; on the other hand, the capacity of mitochondria-targeted photodynamic therapy makes I-HCy-Q an effective inducer of apoptosis and immunogenic cell death. Attribute to its complementary advantages, I-HCy-Q holds potential for the imaging and treatment of tumors in complex organisms.

Mechanism of action of platinum nanoparticles implying from antioxidant to metabolic programming in light-induced retinal degeneration model.

Photoreceptors (PRs) degeneration is central to visual impairment and loss in most blind retinal diseases, including age-related macular disease (AMD) and diabetic retinopathy (DR). PRs are susceptible to oxidative stress owing to their unique metabolic features. Accumulating evidence has demonstrated that the targeting oxidative stress is a promising treatment strategy for PR degeneration. Herein, we introduced potent antioxidative platinum nanoparticles (Pt NPs) to treat PRs degeneration in this study. The Pt NPs exhibited multi-enzymatic antioxidant activity and protected PRs from H2O2-induced oxidative damage in vitro assays. Based on the same mechanism, the intravitreal injection of Pt NPs significantly reduced cell apoptosis, maintained retinal structure and preserved retinal function in a mouse model of light-induced retinal degeneration (LIRD). Most importantly, the results of RNA sequencing showed that the transcription of antioxidative genes was upregulated, and metabolic reprogramming occurred in the LIRD-retina after treatment with Pt NPs, both of which benefited retinal survival from oxidative damage. The results indicated that Pt NPs were indeed potent therapeutic candidates for PRs degeneration in blind retinal diseases.

pH-Responsive Mucus-Penetrating Nanoparticles for Enhanced Cellular Internalization by Local Administration in Vaginal Tissue.

Low mucus penetration ability and cellular uptake seriously limit the effectiveness of local vaginal drug administration because of the rapid foreign particulate and pathogen removal property of the mucus layer. Our previous work proved that nanoparticles with a highly dense polyethylene glycol (PEG) coating can penetrate mucus rapidly (mucus-penetrating nanoparticles, MPPs) and improve drug distribution and retention at mucosal surfaces. However, the "stealth-effect" of the PEG coating also restricts cellular uptake of MPPs. In this work, we designed pH-responsive mucus-penetrating nanoparticles (pMPPs) with hydrazone bonds as the linker to conjugate a dense PEG surface coating, which enabled the pMPPs to rapidly penetrate through the mucus layer. More importantly, the acidic environment of the vaginal mucus induces slow shedding of the PEG layer, leading to a positive charge exposure to facilitate cellular uptake. Overall, pMPPs demonstrate potential as an effective delivery platform for the prophylactic and therapeutic treatment of female reproductive diseases.

A lateral flow strip biosensor platform based on cascade nucleic acid amplification technology for ultrasensitive detection of OSCC-associated salivary MicroRNA.

Oral squamous cell carcinoma (OSCC) is the well-known malignancy and poses a serious threat to human health with high morbidity and mortality. Early detection and treatment can improve the recovery rate and reduce complications of OSCC. Therefore, we designed a lateral flow strip biosensor platform (HRCA-strip) based on the cascade nucleic acid amplification technology (HRCA) for colorimetric analysis of OSCC-associated has-microRNA 31-5p (miRNA 31). In this work, the target miRNA 31 mediated the formation of the sandwich complex structure on the surface of magnetic beads (MBs). Then, the sandwich complex structure could activate cascade amplification reaction between hybridization chain reaction (HCR) and rolling-circle amplification (RCA) to generate numerous G-quadruplex structures. The G-quadruplex structures combined with hemin to form hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (H/G-HRP mimic enzyme) which were enriched on the T-line and catalyzed the oxidation of chromogenic substrates to generate colorimetric signal on the strip. The HRCA-strip platform could achieve highly sensitive and specific miRNA 31 detection with the limit of detection (LOD) as low as 3.21 fM. Moreover, the designed HRCA-strip platform also enabled portable detection of miRNA 31 in clinical sample which might show good potential for early clinical diagnosis of OSCC.

An amplified fluorescent biosensor for Ag+ detection through the hybridization chain reactions.

Ag is widely distributed in nature and it is used in almost all areas of human life. However, due to the widespread use of Ag materials, Ag+ pollution seriously threatens the human health and environment. The traditional detection methods for Ag+ suffer from disadvantages including high operational cost, complicated operating unit and instrument, and high requirements for professionals. Thus, in this study, a new type of Ag+ detection biosensor based on the hybridization signal amplification was designed to overcome these problems. Combining cytosine-Ag+-cytosine mismatch structure with the hybridization chain reaction, this biosensor converted the conventional detection signal into the nucleic acid amplification signal, which realized efficient, rapid, sensitive, and specific detection of Ag+. The limit-of-detection of this sensor reached 0.69 pM, which is much less than the maximum concentration (0.1 mg L-1, 927 nM) suggested for drinking water by the World Health Organization, and the maximum concentration (0.05 mg L-1, 464 nM) suggested by the United States Environmental Protection Agency. This method provides a promising new platform for detecting Ag+ concentrations at ultralow levels.

MicroRNA-Responsive DNA-Programmed Nanomedicine with Controllability of Cascaded Events for Cancer Therapy Enhancement.

Chemotherapy is a prime tool for cancer clinical therapy. The effectiveness has been improved considerably with the assistance of nanotechnology. However, it still meets the challenge of unsatisfied therapeutic effects caused by multidrug resistance and uncontrollable drug release. For further enhancement of the treatment performance, we develop a kind of microRNA-responsive nanomedicine that uses the biomarker microRNA-21 as a trigger of cascaded killing effects on cancer cells, including chemotherapy and gene silencing. The nanomedicine consists of a gold nanoparticle core and a DNA layer. Strand migrations within the layer can accurately control the events of anticancer drug doxorubicin release and multidrug-resistant-associated protein 1 downregulation, yielding an alleviation of multidrug resistance and enhanced killing on cancer cells. This work demonstrates a microRNA-responsive nanomedicine in combination with chemotherapy and gene silencing, which paves the way to the advancement of DNA-based nanomedicine for cancer theranostics.

A Novel Targeted and High-Efficiency Nanosystem for Combinational Therapy for Alzheimer's Disease.

Alzheimer's disease (AD) remains the most prevalent neurodegenerative disease, and no effective treatment is available yet. Metal-ion-triggered aggregates of amyloid-beta (Aß) peptide and acetylcholine imbalance are reported to be possible factors in AD pathogenesis. Thus, a combination therapy that can not only inhibit and reduce Aß aggregation but also simultaneously regulate acetylcholine imbalance that can serve as a potential treatment for AD is needed. Here, clioquinol (metal-ion chelating agent) and donepezil (acetylcholinesterase (AChE) inhibitor) co-encapsulated human serum albumin (HSA) nanoparticles (dcHGT NPs) are designed, which are modified with transcriptional activator protein (TAT) and monosialotetrahexosylganglioside (GM1). The GM1 lipid and TAT peptide endow this drug delivery nanosystem with high brain entry efficiency and long-term retention capabilities through intranasal administration. It is found that dcHGT NPs can significantly inhibit and eliminate Aß aggregation, relieve acetylcholine-related inflammation in microglial cells, and protect primary neurons from Aß oligomer-induced neurotoxicity in vitro. The alleviation of Aß-related inflammation and AChE-inhibited effect further synergistically adjust acetylcholine imbalance. It is further demonstrated that dcHGT NPs reduce Aß deposition, ameliorate neuron morphological changes, rescue memory deficits, and greatly improve acetylcholine regulation ability in vivo. This multifunctional synergetic nanosystem can be a new candidate to achieve highly efficient combination therapy for AD.

Paper-Based Strip for Ultrasensitive Detection of OSCC-Associated Salivary MicroRNA via CRISPR/Cas12a Coupling with IS-Primer Amplification Reaction.

As the most common malignancy in humans, oral squamous cell carcinoma (OSCC) not only harms the people's health but also undermines their confidence after facial surgery. Early detection and treatment can effectively reduce these damages. The unique collateral trans-cleavage nuclease activity of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was utilized to realize the detection of nucleic acid with high sensitivity. So, in this work, we designed a point-of-care testing (POCT) platform for the detection of OSCC-associated salivary hsa-miRNA 31-5p (miR-31) via the cascade signal amplification of "invading stacking primer" (IS-primer) amplification reaction (ISAR), CRISPR/Cas12a, and dual-mode paper-based strip (dm-Strip). To amplify the detection signal of trace miR-31, the cascade signal amplification of CRISPR/Cas12a system coupling with ISAR was designed in a one-pot reaction at a constant temperature. The target miR-31 could activate the ISAR to generate numerous DNAs, which would further trigger the trans-cleavage effect of Cas12a to catalyze the nonspecific single-stranded DNA (ssDNA) cleavage. This ssDNA was labeled with digoxin and biotin at the 5' and 3' termini (digoxin/ssDNA/biotin), respectively, which led to generate the naked-eye signal and fluorescent signal of the designed dm-Strip. The whole detection time was 90 min with limit-of-detection (LOD) down to aM level. This ISAR/Cas12a-based dm-Strip (ISAR/Cas12a-dmStrip) allowed for the portable and ultrasensitive detection of miRNA, an important step in early diagnosis of OSCC and biomedical research.

High fluorescence quenching probe-based reverse fluorescence enhancement LFTS coupling with IS-primer amplification reaction for the rapid and sensitive Parkinson Disease-associated MicroRNA detection.

Parkinson Disease (PD) is the second-most common neurodegenerative disorder in the population. Recent researches indicated that hsa-microRNA 5010-3p (miR-5010) and hsa-microRNA 331-5p (miR-331) were significantly important for the detection of PD. So, in this work, a kind of high fluorescence quenching probe-based reverse fluorescence enhancement lateral flow test strip (rLFTS) was constructed to realize the synchronous detection of miR-5010 and miR-331. The formation of black hole quencher 2 (BHQ2) coating gold nanoparticles (AuNPs) effectively enhanced the fluorescence quenching property of the probes so as to significantly improve the detection sensitivity. This rLFTS also coupled with "invading stacking primer" (IS-primer) isothermal amplification reaction (ISAR) to accomplish rapid, sensitive, specific, and synchronous detection of PD-associated microRNA (miRNA). The whole detection time was shorter (35 min), and the limit-of-detection (LOD) reached to fM level. For the high accuracy diagnosis of PD, the synchronous determination of miR-5010 and miR-331 was successfully realized on one rLFTS by labeling fluorescent molecules to different T-line. This rLFTS also allowed for miRNA detection in total microRNA extracts from whole blood samples of PD patients, which performed important value in PD diagnosis and biomedical research.

Construction of a Novel Biosensor Based on the Self-assembly of Dual-Enzyme Cascade Amplification-Induced Copper Nanoparticles for Ultrasensitive Detection of MicroRNA153.

MicroRNAs (miRNAs) have received extensive attention because of their potential as biomarkers for cancer diagnosis and monitoring, and their effective detection is very significant. Here, a specific, one-pot, rapid, femtomolar sensitive miRNAs detection biosensor was developed based on the target-triggered three-way junction (3-WJ) and terminal deoxynucleotide transferase (TDT)/Nt.BspQI in combination with activated copper nanoparticles (CuNPs) self-assembly. To this end, a 3-WJ hairpin probe and helper probe were designed to selectively identify the target miRNA, so as to form a stable 3-WJ structure that further triggered the double-enzyme cycling to produce poly T to activate the self-assembly of CuNPs. Based on the simplicity of CuNPs generation, the poly T template fluorescence CuNPs can detect the minimum detection limit of 1 fm within 1.75 h. In addition, the applicability of this method in complex samples was demonstrated by analyzing the whole-blood RNA extraction from Parkinson patients, consisting of the results of commercial miRNA kits. The developed strategy performs powerful implications for miRNA detection, which may be beneficial for the effective diagnostic assays and biological research of Parkinson's disease.

Nonenzyme Cascaded Amplification Biosensor Based on Effective Aggregation Luminescence Caused by Disintegration of Silver Nanoparticles.

Sensitive and portable quantification of biomarkers has particular significance in the monitoring and treatment of clinical diseases. Conventional immunoassays were accustomed to introducing or incorporating enzymes for signal amplification, which commonly suffered from poor stability and inferior tolerance. Herein, we constructed a novel nonenzyme amplification methodology based on fluorogenic Ag+-tetrazolate aggregation coupled with silver corrosion sensitization for biomarker determination. A significant cascade enhancement strategy was achieved by the valid aggregation luminescence caused by the potent disintegration of silver nanoparticles. Furthermore, efficient magnetic separation was also combined and performed for the rapidity and simplicity of operation. As the target, the detection limit of prostate-specific antigen was 15.66 pg/mL in our designed biosensor. Besides, a good linear relationship was obtained. The designed biosensor demonstrated good specificity and was successfully applied to clinical serum sample detection.

A novel analytical principle using AP site-mediated T7 RNA polymerase transcription regulation for sensing uracil-DNA glycosylase activity.

Uracil DNA glycosylase (UDG) is a highly conserved damage repair glycosylase; the abnormal expression of DNA glycosylase has important research value in many human diseases. Therefore, highly sensitive and specific detection of UDG activity is crucial to biomedical research and clinical diagnosis. In this work, we propose an AP site-mediated T7 RNA polymerase transcription regulation analytical principle for uracil-DNA glycosylase activity analysis. T7 RNA polymerase is highly promoter-specific and only transcribes DNA downstream of the T7 promoter. We have found that modifying the T7 promoter sequence with an AP site can regulate T7 RNA polymerase transcription ability according to different modification sites. In the binding region of the promoter, AP sites greatly inhibit transcription. Moreover, AP sites in the initiation region of the promoter enhance transcription activity. Based on this research, we designed a new transcription substrate template by replacing deoxythymidine (dT) in the T7 RNA polymerase promoter sequence with one tetrahydrofuran abasic site mimic (THF) and one deoxyuridine (dU). The THF site was labeled in the transcription-enhanced region to improve transcription background, and the dU site was labeled in the transcription inhibition region to sense the UDG enzyme. In our strategy, this template can be transcribed into RNAs by T7 RNA polymerase with great multicycle amplifications. When UDG is present, dU is excised to form an AP site. The AP site damages the interaction between T7 RNA polymerase and the T7 promoter, resulting in weak transcription activity. The detection limit of this strategy is as low as 2.5 × 10-4 U mL-1, and it has good selectivity for UDG. In addition, this strategy can also detect UDG activity in complex HeLa cell lysate samples. Therefore, our developed sensor might become a promising technique for UDG activity assay.

Correction: An NIR-responsive mesoporous silica nanosystem for synergetic photothermal-immunoenhancement therapy of hepatocellular carcinoma.

Correction for 'An NIR-responsive mesoporous silica nanosystem for synergetic photothermal-immunoenhancement therapy of hepatocellular carcinoma' by Han Yang et al., J. Mater. Chem. B, 2020, 8, 251-259.

An NIR-responsive mesoporous silica nanosystem for synergetic photothermal-immunoenhancement therapy of hepatocellular carcinoma.

To create a more precise, efficient imaging and therapeutic strategy is a big challenge for the current treatment of hepatocellular carcinoma (HCC). Photothermal therapy (PTT) has attracted enormous attention due to its non-invasive property and precise spatial and temporal control. Here, we developed a strategy to realize superior imaging performance and treatment, utilizing an indocyanine green (ICG) and sorafenib (S) co-loaded mesoporous silica nanosystem for synergetic PTT/immuno-enhanced therapy. We proved that (ICG+S)@mSiO2 could be easily endocytosed by H22 cells, carried out outstanding real-time fluorescence imaging, and enhanced cytotoxicity abilities by near-infrared radiation (NIR) in vitro. Moreover, (ICG+S)@mSiO2 also had excellent fluorescence imaging ability, displayed a remarkable photothermal tumor killing effect and immune enhancement capability under 808 nm irradiation in an H22 tumor-bearing mice model, without apparent adverse effects in other organs. This study provides a new strategy for the development of a PTT/immuno-enhanced synergistic theranostic nanosystem of HCC.

A fluorescent signal "removal" sensor via duplex-specific nuclease-aided cleavage for miRNA detection in flow cytometry.

Considered as the next-generation biomarkers, microRNAs play an important role in the early diagnosis of cancers. Here, we designed a fluorescent signal "removal" sensor for one-step, sensitive and specific detection of multiple microRNAs by flow cytometry (FCM). In this work, single-stranded DNA (ssDNA), working as the interlinkage, immobilized the fluorescent nanosphere (FS) onto the SiO2 microspheres surface to form the SiO2-ssDNA-FS probes. When target miRNAs integrated with SiO2-ssDNA-FS probes, the duplex-specific nuclease (DSN) could cleave the ssDNA selectively and release FS with numerous cycles to enhance the fluorescent signal attenuation of SiO2-ssDNA-FS, so as to remarkably improve the analysis sensitivity. It achieved a simple, accurate and quantitative microRNA-21 detection for clinical blood samples. Parallel multi-target detection of microRNA-21 and Let-7d was also realized by different color labeled FS. Moreover, our designed sensor was suitable for other targets' detection with the corresponding probes.

Reusable Bioluminescent Sensor for Ultrasensitive MicroRNA Detection Based on a Target-Introducing "Fuel-Loading" Mechanism.

As a kind of important potential biomarkers, the expression level of some microRNAs (miRNAs) is closely related to cancer development and progression. Herein, a reusable ultra-sensitive "fuel-loadings" bioluminescent sensor was constructed to detect the trace miRNA based on the cascading signal amplification, which combined the target-introducing "fuel-loading" mechanism and cyclic bioluminescence assay. In this sensor, magnetic beads labeled with hairpin DNA probes (hDNA) could specifically hybridize with the target miRNA and isolate these targets from samples. Then, the target-introducing "fuel loading" mechanism worked because the poly(A) polymerase can catalyze the template-independent sequential addition of adenosine monophosphate (AMP) to the 3' ends of the miRNA targets to produce long poly(A) tails. The long poly(A) tails provided lots of 5'AMPs (cleaved by Exonuclease T), which further as fuels were converted into adenosine-triphosphate (ATP) to generate an enhanced bioluminescent signal by cyclic AMP pyrophosphorylation-ATP dephosphorylation. The "fuel-loadings" bioluminescent sensor realized a high sensitivity with a limit-of-detection of about 22.6 aM for miRNA 21. Moreover, this "fuel-loadings" bioluminescent sensor not only achieved regenerable and reusable measurement in the same microwell to decrease the analysis costs, but also could directly detect miRNA 21 in the serum without complicated extraction procedures. It showed excellent coherence with quantitative reverse transcription polymerase chain reaction for miRNA 21 detection of cancer patients' samples, indicating clinical translation potential for miRNA detection.

Nanoparticle-based diagnostic and therapeutic systems for brain tumors.

Brain tumors, especially the most prevalent and aggressive glioblastoma, remain among the deadliest of all types of cancer due to inefficient theranostic options. They have a limited therapeutic window because of physiological barriers such as the blood-brain barrier (BBB), blood cerebrospinal fluid (CSF) barrier and interstitial fluid (ISF) that restrict the penetration of imaging probes and therapeutic drugs. In order to achieve more accurate brain tumor diagnosis and better therapeutic effects, many strategies have been explored; among them multifunctional nanoparticles offer a novel and potential opportunity depending on their size effects, and their optical, magnetic, photodynamic and other properties. After modification, nanoparticles can cross the BBB and specifically accumulate in the tumor site, thereby achieving accurate tumor imaging and drug release. This review is focused on various types of nanoparticles that are being used for improving nano-carriers of diagnostic and therapeutic agents into brain tumors and also provides a concise summary of various multifunctional theranostic strategies, particularly in clinical applications. In this manner, we provide evidence for the key role of nanoparticle based diagnosis and therapy systems in brain tumors.

Astragaloside III Enhances Anti-Tumor Response of NK Cells by Elevating NKG2D and IFN-γ.

Natural killer (NK) cells play an irreplaceable role in the development of colon cancer, in which antitumor function of NK cells was impaired. Astragaloside III is a natural compound from Astragalus that has been shown to have immunomodulatory effects in various systems. However, few studies have evaluated the antitumor effects of Astragaloside III through stimulating systemic immunity and regulating NK cells. In this study, flow cytometry, immunohistochemical analysis, and immunofunctional assays were performed to elucidate the functions of Astragaloside III in restoring antitumor function of NK cells. We demonstrated that Astragaloside III significantly elevated the expression of natural killer group 2D (NKG2D), Fas, and interferon-γ (IFN-γ) production in NK cells, leading to increased tumor-killing ability. Experiments in cell co-culture assays and CT26-bearing mice model further confirmed that Astragaloside III could effectively impede tumor growth by increasing infiltration of NK cells into tumor and upregulating the antitumor response of NK cells. We further revealed that Astragaloside III increased IFN-γ secretion of NK cells by enhancing the expression of transcription factor T-bet. In conclusion, the effective anti-tumor function of Astragaloside III was achieved through up-regulation of the immune response of NK cells and elevation of NKG2D, Fas, and IFN-γ production.