Efficient strategies for the ultrasensitive imaging of gene expression in living cells are essential in chemistry and cell biology. Here, we report a novel and efficient enzyme-free dual signal amplification strategy for live cell mRNA imaging by using a smart nucleic acid hairpin-based nanosystem. This nanosystem consists of a ZnO nanoparticle core, an interlayer of polydopamine and an outer layer of four hairpin DNA (hpDNA) probes. Such a core–shell nanosystem facilitates the cellular uptake of molecular hairpin payloads, protects them from nuclease digestion, and delivers them into the cytoplasm by the acid-triggered dissolution of the ZnO core. In the presence of target mRNA, the released hpDNA probes self-assemble via HCR into wire-shaped active DNAzymes that catalyze the generation of a fluorescence signal. The target-initiated HCR events and DNAzyme cascades offer efficient dual amplification and enable the ultrasensitive detection of mRNA with a femtomolar detection limit. Live cell assays show an intense fluorescence response from a tumor-related biomarker survivin mRNA only in tumor cells untreated with a survivin expression repressor YM155, but not in normal cells. The developed nanosystem provides a potential platform for the amplified imaging of low-abundance disease-related biomarkers in live cells.

AbstractPhotodynamic therapy (PDT) has been applied in cancer treatment by converting O2 into reactive singlet oxygen (1O2) to kill cancer cells. However, the effectiveness of PDT is limited by the fact that tumor hypoxia causes an inadequate O2 supply, and the overexpressed glutathione (GSH) in cancer cells consumes reactive oxygen species. Herein, a multifunctional hybrid system is developed for selective and highly efficient PDT as well as gene-silencing therapy using a novel GSH-activatable and O2/Mn2+-evolving nanocomposite (GAOME NC). This system consists of honeycomb MnO2 (hMnO2) nanocarrier loaded with catalase, Ce6, and DNAzyme with folate label, which can specifically deliver payloads into cancer cells. Once endocytosed, hMnO2 carriers are reduced by the overexpressed GSH to Mn2+ ions, resulting in the reduction of GSH level and disintegration of GAOME NC. The released catalases then trigger the breakdown of endogenous H2O2 to generate O2, which is converted by the excited Ce6 into 1O2. The self-sufficiency of O2 and consumption of GSH effectively enhance the PDT efficacy. Moreover, DNAzyme is freed for gene silencing in the presence of self-generated Mn2+ ions as cofactors. The rational synergy of enhanced PDT and gene-silencing therapy remarkably improve the in vitro and in vivo therapeutic efficacy of cancers.

Beta amyloid peptide, tau, and phosphorylated tau are well recognized as promising biomarkers for the diagnosis of Alzheimer’s disease (AD). In this work, we developed a direct, versatile, and ultrasensitive multiplex assay for the quantification of trace amounts of these protein biomarkers for AD in different types of biological fluids including cerebrospinal fluid, serum, saliva, and urine. The detection assay is based on the immunoreaction between the target proteins and their corresponding pair of antibodies followed by fluorescence labelling with a newly developed indolium-based turn-on fluorophore, namely SIM. SIM was tailor-made as a reporter to provide a high signal-to-noise ratio for the detection assay. An exceptionally low limit of detection down to the femto-molar level was achieved in this assay with minute consumption of the sample. This versatile detection assay is capable of reliably quantifying not only the target proteins simultaneously from a CSF sample in an hour but also trace amounts of protein biomarkers in saliva and urine. This assay has a high potential to serve as a practical tool for the diagnosis of AD.

Alzheimer’s disease (AD) is the most prevalent but still incurable neurodegenerative form of dementia. Early diagnosis and intervention are crucial for delaying the onset and progression of the disease. We herein report a novel fluoro-substituted cyanine, F-SLOH, which exhibits good Aβ oligomer selectivity with a high binding affinity, attributed to the synergistic effect of strong π–π stacking and intermolecular CH⋯O and CH⋯F interactions. The selectivity towards the Aβ oligomers in the brain was ascertained by in vitro labelling on tissue sections and in vivo labelling through the systemic administration of F-SLOH in 7 month APP/PS1 double transgenic (Tg) and APP/PS1/Tau triple Tg mouse models. F-SLOH also shows remarkably effective inhibition on Aβ aggregation and highly desirable neuroprotective effects against Aβ-induced toxicities, including the inhibition of ROS production and Ca2+ influx. Its excellent blood–brain barrier (BBB) penetrability and low bio-toxicity further support its tremendous potential as a novel theranostic agent for both early diagnosis and therapy of AD.

A direct and ultrasensitive multiplex assay using an immuno-magnetic platform has been developed for the quantification of trace amounts of circulating cancer-associated antigens in serum. The detection is based on the specific immuno-interactions among the target antigen, detection antibody and capture antibody that is immobilized on the surface of magnetic nanoparticles. The sandwiched immuno-assembly is then labelled with turn-on fluorophores and detected with a fluorescence imaging system. To afford a high signal-to-noise ratio, three turn-on fluorophores with unique optical properties have been designed and synthesized to label the target antigens. The developed assay has achieved a remarkable LOD down to the femto-molar regime without sample pre-treatment. This versatile assay can efficiently differentiate the target antigen from a protein matrix and simultaneously quantify multiple cancer-associated antigens, for instance, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) using only 6 μL of serum sample in an hour. This novel system has a high applicability to serve as a universal and useful tool for early disease diagnostics.

In this work, we demonstrated a new ratiometric method for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly fluorescent bovine serum albumin (BSA) matrix, acting as a pH probe, and pH-insensitive reference dye Alexa 594 enabling ratiometric quantitative pH measurement. The fluorescent BSA matrix was synthesized by cross-linking of the denatured BSA proteins in ethanol with glutaraldehyde. The size of the as-synthesized BSA nanoparticles can be readily manipulated from 30 to 90 nm, which exhibit decent fluorescence at the peak wavelength of 535 nm with a pH response range of 6–8. The potential of this pH sensor for intracellular pH monitoring was demonstrated inside living HeLa cells, whereby a significant change in fluorescence ratio was observed when the pH of the cell was switched from normal to acidic with anticancer drug treatment. The fast response of the nanosensor makes it a very powerful tool in monitoring the processes occurring within the cytosol.Keywords: cellular imaging; fluorescent nanoparticles; pH sensing; protein nanoparticles; ratiometric;

In this work, we developed a simple yet robust single particle scattering intensity measurement method for the quantification of cancer-related biomarkers. The design is based on the plasmonic coupling effect between noble metal nanoparticles. First, the primary and secondary antibodies were conjugated onto the surface of 60 nm gold nanoparticles (AuNPs, act as capture probes) and 50 nm silver nanoparticles (AgNPs, act as signal amplification probes) respectively. In the presence of corresponding antigen, a sandwiched immunocomplex was formed, resulting a significantly enhanced scattering intensity in contrast to that of individual probes. By measuring the intensity change of the particles with a dark-field microscope (DFM), the amount of target protein could be accurately quantified. As a proof of concept experiment, quantification of three types of antigens, including carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and alpha fetoprotein (AFP) by this platform was demonstrated with limit of detection (LOD) of 1.7, 3.3, and 5.9 pM, respectively, with a linear dynamic range of 0 to 300 pM. Furthermore, to elucidate the potential in clinical application, the content of antigens in a serum sample was also quantified directly without additional sample pretreatment. In order to validate the reliability of this method, the measured result was also compared with that obtained by regular enzyme-linked immunosorbent assay (ELISA) kit, showing good consistency between these two data sets. Therefore, owing to the simplicity and accuracy of this method, it could be potentially applied for massive disease screening in clinical assay in the future.

•A FRET-based biosensor was developed for direct quantification of trypsin.•Fast and sensitive screening of pancreatic disease was facilitated.•The direct quantification of trypsin in urine samples was demonstrated.A versatile nanoprobe was developed for trypsin quantification with fluorescence resonance energy transfer (FRET). Here, fluorescence graphene quantum dot is utilized as a donor while a well-designed coumarin derivative, CMR2, as an acceptor. Moreover, bovine serum albumin (BSA), as a protein model, is not only served as a linker for the FRET pair, but also a fluorescence enhancer of the quantum dots and CMR2. In the presence of trypsin, the FRET system would be destroyed when the BSA is digested by trypsin. Thus, the emission peak of the donor is regenerated and the ratio of emission peak of donor/emission peak of acceptor increased. By the ratiometric measurement of these two emission peaks, trypsin content could be determined. The detection limit of trypsin was found to be 0.7 μg/mL, which is 0.008-fold of the average trypsin level in acute pancreatitis patient's urine suggesting a high potential for fast and low cost clinical screening.

Amyloid-β (Aβ) peptide as one of the main components of senile plaques is closely related to the onset and progression of incurable Alzheimer's disease (AD). Numerous efforts have been devoted to develop probes for Aβ species/plaque imaging for AD diagnostics and to develop aggregation inhibitors preventing formation of toxic soluble oligomeric Aβ for therapeutics. Herein, for the first time, a series of novel charged molecules, which can simultaneously perform near infra-red in vivo imaging of Aβ species/plaques in animal model and inhibition of self-aggregation of Aβ monomer from forming toxic oligomers, are reported. Among them, DBA-SLOH showed excellent blood-brain barrier (BBB) permeability and biocompatibility due to the incorporation of lipophilic alkyl chains with moderate length into the charged skeleton. Importantly, DBA-SLOH was found to have a high binding affinity toward Aβ species exhibiting a dramatic fluorescence enhancement upon interacting with Aβ species. Despite a weaker binding with Aβ monomers as compared to Aβ aggregates, DBA-SLOH could effectively prevent the Aβ1-40 and Aβ1-42 peptides from self-aggregation and forming toxic oligomers. This multifunctional fluorescent molecule shows promising potential as a theranostic agent for the diagnosis and therapy of AD.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate human gene expression at the post-transcriptional level. Growing evidence indicates that the expression profile of miRNAs is highly correlated with the occurrence of human diseases including cancers. Playing important roles in complex gene regulation processes, the aberrant expression pattern of various miRNAs is implicated in different types and even stages of cancer. Besides localizing in cells, many of these miRNAs are found circulating around the body in a wide variety of fluids such as urine, serum and saliva. Surprisingly, these extracellular circulating miRNAs are highly stable and resistant to degradation, and therefore, are considered as promising biomarkers for early cancer diagnostic via noninvasive extraction from body fluids. Unfortunately, the abundance of these small RNAs is ultralow in the body fluids, making it challenging to quantify them in complex sample matrixes. Establishing a sensitive, specific yet simple assay for an accurate quantification of circulating miRNAs is therefore desirable. Our group previously reported a sensitive and specific detection assay of miRNAs in single molecule level with the aid of total internal reflection fluorescence microscopy. In this work, we advanced the assay to differentiate the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, a locked nucleic acid-modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 μL of serum and the whole assay can be completed in 1 h. The experimental results agreed well with those previously reported whereas the quantity of miR-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising noninvasive detection tool for early NPC diagnosis, monitoring and staging.

•Direct, specific and highly sensitive yet simple detection assay for circulating miRNA in serum.•Ultrasensitive detection with trace amount of sample consumption.•Self-assembled protein nanofibril acts as an online pre-concentrating platform.MicroRNA (miRNA) has recently emerged as a new and important class of cellular regulators. Strong evidences showed that aberrant expression of miRNA is associated with a broad spectrum of human diseases, such as cancer, diabetes, cardiovascular and psychological disorders. However, the short length and low abundance of miRNA place great challenges for conventional techniques in the miRNA quantification and expression profiling. Here, we report a direct, specific and highly sensitive yet simple detection assay for miRNA without sample amplification. A self-assembled protein nanofibril acted as an online pre-concentrating sensor to detect the target miRNA. Locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1 pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patient’s serums was also demonstrated.

This is the first work that revealed the neuro-protective effect of functionalized quantum dots against the cytotoxicity induced by beta-amyloid peptides. This study gives insight into the future treatment of Alzheimer's disease. It opens many avenues for the development of the next generation nanotechnology for biomedical and therapeutic applications.

We report here the first application of Group 9 metal complexes (i.e.iridium(III) and rhodium(III)) as inhibitors of amyloid fibrillogenesis and as luminescent probes for Aβ1–40peptide. These complexes contained aromatic co-ligands to interact with the hydrophobic residues around the N-terminal domain of the Aβ1–40peptide, as well as solvato co-ligands to allow coordinative bond formation with histidine residues. We demonstrate that these complexes could inhibit Aβ1–40peptide aggregation in vitro, with potency superior to previous metal-based inhibitors reported. Furthermore, we have demonstrated the first example of luminescent detection of Aβ1–40peptides by transition metal complexes.

A one-dimensional nanofibrillar array formed by the co-assembly of native and biotin-functionalized beta-amyloid (Aβ) peptide was developed for biomolecule sensing. With the presence of biotin moiety, a variety of biomolecular probes can be conjugated onto the nanofibrils, thus converting the protein assembly into a miniature biosensor. In this work, DNA probes were immobilized onto the fibril for the detection of cDNA sequences. The as-developed “DNA-nanoarray” achieved a detection limit at subattomole level (183 fM in 10 μL). This highly sensitive, yet simple, assay requires a trace amount of sample consumption (<10 μL) and is pretreatment-free. In addition, we reported the preparation of alternate-segmented amyloid nanofibrils with multifunctionality. The fibrils hereby serve as an encoded template that can be visualized with various fluorescence labeling dyes for barcode recognition purpose, and, hence, multiplex detection of biomolecules was achieved. Regarding that each protein nanofibril represents a single detection platform, a large number of single fibrils simultaneously are monitored with the dual-color TIRFM in a high-throughput manner.

MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRT-PCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis.

One of the primary factors that induce Alzheimer's disease (AD) is the deposition of beta-amyloid (Aβ). The Aβ molecules can self-assemble to form neurotoxic aggregates with various morphologies, such as dimers, oligomers, protofibrils and fibrils. For this aspect, we demonstrated that the amyloid fibrillation can be inhibited by quenching the nucleation and elongation processes with a low concentration of water dispersed N-acetyl-l-cysteine capped quantum dots (NAC-QDs). Based on the concentration dependence of NAC-QDs on the seeded fibril growth, there is a remarkable inhibition effect when the NAC-QDs concentration is increased by 100-fold from 10−9 to 10−7 m. The NAC-QDs concentration required to show inhibition effect is much lower than that of the amyloid peptide concentration (50 μm). The step-like change suggests that the inhibition effect of NAC-QDs displays a threshold response. The inhibition is likely due to the intermolecular attractive interactions such as the hydrogen bonding between NAC-QDs and amyloid fibrils resulting in the blockage of the active elongation sites on the fibrils.

•A label-free detection of DNA–protein interactions at single nanoparticles level.•The λmax shift in LSPR spectrum of individual AuNP was detected.•It is capable of revealing information such as particle–particle variations.We reported a sensitive detection system for measuring DNA–protein interaction at single plasmonic metal nanoparticles level by Localized Scattering Plasmon Resonance (LSPR) spectroscopy. As a proof of concept, DNA molecules were conjugated to gold nanoparticles (AuNPs) through gold–thiol chemistry and the resulted complex was served as single-particle probes of human topoisomerase I (TOPO). By recording the changes in Rayleigh light scattering signal of the individual nanoparticles upon protein binding, DNA–protein interaction was monitored and measured. The λmax shifts in LSPR spectrum of individual AuNP was found to be highly correlated with the amount of TOPO that bound onto. This technique provides a sensitive and high-throughput platform to screen and monitor accurately the specific biomolecular interactions. It is capable of revealing information such as particle–particle variations that might be buried in conventional bulk measurement.

A direct and ultrasensitive multiplex assay using an immuno-magnetic platform has been developed for the quantification of trace amounts of circulating cancer-associated antigens in serum. The detection is based on the specific immuno-interactions among the target antigen, detection antibody and capture antibody that is immobilized on the surface of magnetic nanoparticles. The sandwiched immuno-assembly is then labelled with turn-on fluorophores and detected with a fluorescence imaging system. To afford a high signal-to-noise ratio, three turn-on fluorophores with unique optical properties have been designed and synthesized to label the target antigens. The developed assay has achieved a remarkable LOD down to the femto-molar regime without sample pre-treatment. This versatile assay can efficiently differentiate the target antigen from a protein matrix and simultaneously quantify multiple cancer-associated antigens, for instance, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) using only 6 μL of serum sample in an hour. This novel system has a high applicability to serve as a universal and useful tool for early disease diagnostics.

We report here the first application of Group 9 metal complexes (i.e.iridium(III) and rhodium(III)) as inhibitors of amyloid fibrillogenesis and as luminescent probes for Aβ1–40peptide. These complexes contained aromatic co-ligands to interact with the hydrophobic residues around the N-terminal domain of the Aβ1–40peptide, as well as solvato co-ligands to allow coordinative bond formation with histidine residues. We demonstrate that these complexes could inhibit Aβ1–40peptide aggregation in vitro, with potency superior to previous metal-based inhibitors reported. Furthermore, we have demonstrated the first example of luminescent detection of Aβ1–40peptides by transition metal complexes.

This is the first work that revealed the neuro-protective effect of functionalized quantum dots against the cytotoxicity induced by beta-amyloid peptides. This study gives insight into the future treatment of Alzheimer's disease. It opens many avenues for the development of the next generation nanotechnology for biomedical and therapeutic applications.

Efficient strategies for the ultrasensitive imaging of gene expression in living cells are essential in chemistry and cell biology. Here, we report a novel and efficient enzyme-free dual signal amplification strategy for live cell mRNA imaging by using a smart nucleic acid hairpin-based nanosystem. This nanosystem consists of a ZnO nanoparticle core, an interlayer of polydopamine and an outer layer of four hairpin DNA (hpDNA) probes. Such a core–shell nanosystem facilitates the cellular uptake of molecular hairpin payloads, protects them from nuclease digestion, and delivers them into the cytoplasm by the acid-triggered dissolution of the ZnO core. In the presence of target mRNA, the released hpDNA probes self-assemble via HCR into wire-shaped active DNAzymes that catalyze the generation of a fluorescence signal. The target-initiated HCR events and DNAzyme cascades offer efficient dual amplification and enable the ultrasensitive detection of mRNA with a femtomolar detection limit. Live cell assays show an intense fluorescence response from a tumor-related biomarker survivin mRNA only in tumor cells untreated with a survivin expression repressor YM155, but not in normal cells. The developed nanosystem provides a potential platform for the amplified imaging of low-abundance disease-related biomarkers in live cells.