Generalized Ratiometric Indicator Based Surface-Enhanced Raman Spectroscopy for the Detection of Cd²⁺ in Environmental Water Samples

The concept of generalized ratiometric indicator based surface-enhanced Raman spectroscopy was first introduced and successfully implemented in the detection of Cd²⁺ in environmental water samples using Au nanoparticles (AuNPs) modified by trithiocyanuric acid (TMT). Without the use of any internal standard, the proposed method achieved accurate concentration predictions for Cd²⁺ in environmental water samples with recoveries in the ranges of 91.8–108.1%, comparable to the corresponding values obtained by atomic absorption spectroscopy. The limit of detection and limit of quantification were estimated to be 2.9 and 8.7 nM, respectively. More importantly, other species present in water samples which cannot react with TMT and have weaker binding ability to AuNPs than TMT do not interfere with the quantification of Cd²⁺. Therefore, it is expected that the combination of the generalized ratiometric indicator based surface-enhanced Raman spectroscopy with the proposed AuNP–TMT probing system can be a competitive alternative for the primary screening of Cd²⁺ pollution

Cooperative Amplification-Based Electrochemical Sensor for the Zeptomole Detection of Nucleic Acids

In this work, we developed a multiple-amplification-based electrochemical sensor for ultrasensitive detection of nucleic acids using a disease-related sequence of the p53 gene as the model target. A capture probe (CP) with a hairpin structure is immobilized on the electrode surface via thiol–gold bonding, while its stem is designed to contain a restriction site for <i>Eco</i>RI. In the absence of target DNA, the probe keeps a closed conformation and forms a cleavable region. After treatment with <i>Eco</i>RI, the target binding portion (loop) plus the biotin tag can be peeled off, suppressing the background current. In contrast, the CP is opened by the target hybridization, deforming the restriction site and forcing the biotin tag away from the electrode. On the basis of the biotin–streptavidin complexation, gold nanoparticles (GNPs) modified with a large number of ferrocene-signaling probes (Fc-SPs) are captured by the resulting interface, producing an amplified electrochemical signal due to the GNP-based enrichment of redox-active moieties. Furthermore, Fc tags can be dragged in close proximity to the electrode surface via hybridization between the signaling probes and the CP residues after <i>Eco</i>RI treatment, facilitating interfacial electron transfer and further enhancing the signal. With combination of these factors, the present system is demonstrated to achieve an ultrahigh sensitivity of zeptomole level and a wide dynamic response range of over 7 orders of magnitude

Core–Shell–Shell Multifunctional Nanoplatform for Intracellular Tumor-Related mRNAs Imaging and Near-Infrared Light Triggered Photodynamic–Photothermal Synergistic Therapy

A multifunctional nanoplatform, which generally integrates biosensing, imaging diagnosis, and therapeutic functions into a single nanoconstruct, has great important significance for biomedicine and nanoscience. Here, we developed a core–shell–shell multifunctional polydopamine (PDA) modified upconversion nanoplatform for intracellular tumor-related mRNAs detection and near-infrared (NIR) light triggered photodynamic and photothermal synergistic therapy (PDT–PTT). The nanoplatform was constructed by loading a silica shell on the hydrophobic upconversion nanoparticles (UCNPs) with hydrophilic photosensitizer methylene blue (MB) entrapped in it, and then modifying PDA shells through an in situ self-polymerization process, thus yielding a core–shell–shell nanoconstruct UCNP@SiO₂–MB@PDA. By taking advantages of preferential binding properties of PDA for single-stranded DNA over double-stranded DNA and the excellent quenching property of PDA, a UCNP@SiO₂–MB@PDA–hairpin DNA (hpDNA) nanoprobe was developed through adsorption of fluorescently labeled hpDNA on PDA shells for sensing intracellular tumor-related mRNAs and discriminating cancer cells from normal cells. In addition, the fluorescence resonance energy transfer from the upconversion fluorescence (UCF) emission at 655 nm of the UCNPs to the photosensitizer MB molecules could be employed for PDT. Moreover, due to the strong NIR absorption and high photothermal conversion efficiency of PDA, the UCF emission at 800 nm of the UCNPs could be used for PTT. We demonstrated that the UCNP@SiO₂–MB@PDA irradiated with NIR light had considerable PDT–PTT effect. These results revealed that the developed multifunctional nanoplatform provided promising applications in future oncotherapy by integrating cancer diagnosis and synergistic therapy

Peptide-Templated Gold Nanocluster Beacon as a Sensitive, Label-Free Sensor for Protein Post-translational Modification Enzymes

Protein post-translational modifications (PTMs), which are chemical modifications and most often regulated by enzymes, play key roles in functional proteomics. Detection of PTM enzymes, thus, is critical in the study of cell functioning and development of diagnostic and therapeutic tools. Herein, we develop a simple peptide-templated method to direct rapid synthesis of highly fluorescent gold nanoclusters (AuNCs) and interrogate the effect of enzymatic modifications on their luminescence. A new finding is that enzymes are able to exert chemical modifications on the peptide-templated AuNCs and quench their fluorescence, which furnishes the development of a real-time and label-free sensing strategy for PTM enzymes. Two PTM enzymes, histone deacetylase 1 and protein kinase A, have been employed to demonstrate the feasibility of this enzyme-responsive fluorescent nanocluster beacon. The results reveal that the AuNCs’ fluorescence can be dynamically decreased with increasing concentration of the enzymes, and subpicomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a useful, label-free biosensor platform for the detection of protein-modifying enzymes and their inhibitors in biomedical applications

In Situ Imaging of Individual mRNA Mutation in Single Cells Using Ligation-Mediated Branched Hybridization Chain Reaction (Ligation-bHCR)

Ultrasensitive and specific in situ imaging of gene expression is essential for molecular medicine and clinical theranostics. We develop a novel fluorescence in situ hybridization (FISH) strategy based on a new branched hybridization chain reaction (bHCR) for efficient signal amplification in the FISH assay and a ligase-mediated discrimination for specific mutation detection. To our knowledge, this is the first time that HCR has been realized for mutation detection in the FISH assay. In vitro assay shows that the ligation-bHCR strategy affords high specificity in discriminating single-nucleotide variation in mRNA, and it generates a highly branched polymeric product that confers more efficient amplification or better sensitivity than HCR. Imaging analysis reveals that ligation-bHCR generates highly bright spot-like signals for localization of individual mRNA molecules, and spot signals of different colors are highly specific in genotyping point mutation of individual mRNA. Moreover, this strategy is shown to have the potential for quantitative imaging of the expression of mRNA at the single-cell level. Therefore, this strategy may provide a new promising paradigm in developing highly sensitive and specific FISH methods for various diagnostic and research applications

Graphitic Carbon Nitride Nanosheets-Based Ratiometric Fluorescent Probe for Highly Sensitive Detection of H₂O₂ and Glucose

Graphitic carbon nitride (g-C₃N₄) nanosheets, an emerging graphene-like carbon-based nanomaterial with high fluorescence and large specific surface areas, hold great potential for biosensor applications. Current g-C₃N₄ nanosheets based fluorescent biosensors majorly rely on single fluorescent intensity reading through fluorescence quenching interactions between the nanosheets and metal ions. Here we report for the first time the development of a novel g-C₃N₄ nanosheets-based ratiometric fluorescence sensing strategy for highly sensitive detection of H₂O₂ and glucose. With <i>o</i>-phenylenediamine (OPD) oxidized by H₂O₂ in the presence of horseradish peroxidase (HRP), the oxidization product can assemble on the g-C₃N₄ nanosheets through hydrogen bonding and π–π stacking, which effectively quenches the fluorescence of g-C₃N₄ while delivering a new emission peak. The ratiometric signal variations enable robust and sensitive detection of H₂O₂. On the basis of the glucose converting into H₂O₂ through the catalysis of glucose oxidase, the g-C₃N₄-based ratiometric fluorescence sensing platform is also exploited for glucose assay. The developed strategy is demonstrated to give a detection limit of 50 nM for H₂O₂ and 0.4 μM for glucose, at the same time, it has been successfully used for glucose levels detection in human serum. This strategy may provide a cost-efficient, robust, and high-throughput platform for detecting various species involving H₂O₂-generation reactions for biomedical applications

Quantitative Fluorescence Spectroscopy in Turbid Media: A Practical Solution to the Problem of Scattering and Absorption

The presence of practically unavoidable scatterers and background absorbers in turbid media such as biological tissue or cell suspensions can significantly distort the shape and intensity of fluorescence spectra of fluorophores and, hence, greatly hinder the in situ quantitative determination of fluorophores in turbid media. In this contribution, a quantitative fluorescence model (QFM) was proposed to explicitly model the effects of the scattering and absorption on fluorescence measurements. On the basis of the proposed model, a calibration strategy was developed to remove the detrimental effects of scattering and absorption and, hence, realize accurate quantitative analysis of fluorophores in turbid media. A proof-of-concept model system, the determination of free Ca²⁺ in turbid media using Fura-2, was utilized to evaluate the performance of the proposed method. Experimental results showed that QFM can provide quite precise concentration predictions for free Ca²⁺ in turbid media with an average relative error of about 7%, probably the best results ever achieved for turbid media without the use of advanced optical technologies. QFM has not only good performance but also simplicity of implementation. It does not require characterization of the light scattering properties of turbid media, provided that the light scattering and absorption properties of the test samples are reasonably close to those of the calibration samples. QFM can be developed and extended in many application areas such as ratiometric fluorescent sensors for quantitative live cell imaging

Surface Enhanced Laser Desorption Ionization of Phospholipids on Gold Nanoparticles for Mass Spectrometric Immunoassay

High-throughput and sensitive detection of proteins are essential for clinical diagnostics and biomarker discovery. We develop a novel high-throughput, multiplexed, sensitive mass spectrometric (MS) immunoassay method, which utilizes antibody-modified phospholipid bilayer coated gold nanoparticles (PBL-AuNPs) as the detection label and antibody-immobilized magnetic beads as the capture reagent. This method enables magnetic enrichment of the PBL-AuNPs label specific to target protein, allowing sensitive surface enhanced laser desorption ionization (SELDI)-TOF MS detection of the protein via its specific label. AuNPs act as not only the support but also the matrix for the phospholipids in SELDI TOF MS detection. Moreover, with phospholipids with varying molecular weights as the encoded MS reporters, this method allows multiplexed detection of multiple proteins. With the use of a predefined phospholipids internal standard, this method also affords excellent reproducibility in protein quantification. We have demonstrated this method using the assays of two tumor biomarkers, and the results reveal that it provides a sensitive platform for multiplexed protein detection with detection limits in the picomolar ranges. This method may provide a useful platform for high-throughput and sensitive detection of protein biomarkers for clinical diagnostics

Branched Hybridization Chain Reaction Circuit for Ultrasensitive Localizable Imaging of mRNA in Living Cells

Hybridization chain reaction (HCR) circuits are valuable approaches to monitor low-abundance mRNA, and current HCR is still subjected to issues such as limited amplification efficiency, compromised localization resolution, or complicated designs. We report a novel branched HCR (bHCR) circuit for efficient signal-amplified imaging of mRNA in living cells. The bHCR can be realized using a simplified design by hierarchically coupling two HCR circuits with two split initiator fragments of the secondary HCR circuit incorporated in the probes for the primary HCR circuit. The bHCR circuit enables one to generate a hyperbranched assembly seeded from a single target initiator, affording the potential for localizing single target molecules in live cells. In vitro assays show that bHCR offers very high amplification efficiency and specificity in single mismatch discrimination with a detection limit of 500 fM. Live cell studies reveal that bHCR displays intense fluorescence spots indicating mRNA localization in living cells with improved contrast. The bHCR method can provide a useful platform for low-abundance biomarker detection and imaging for cell biology and diagnostics

Phospholipid-Modified Upconversion Nanoprobe for Ratiometric Fluorescence Detection and Imaging of Phospholipase D in Cell Lysate and in Living Cells

Phospholipase D (PLD) is a critical component of intracellular signal transduction and has been implicated in many important biological processes. It has been observed that there are abnormalities in PLD expression in many human cancers, and PLD is thus recognized as a potential diagnostic biomarker as well as a target for drug discovery. We report for the first time a phospholipid-modified nanoprobe for ratiometric upconversion fluorescence (UCF) sensing and bioimaging of PLD activity. The nanoprobe can be synthesized by a facile one-step self-assembly of a phospholipid monolayer composed of poly­(ethylene glycol) (PEG)­ylated phospholipid and rhodamine B-labeled phospholipid on the surface of upconversion nanoparticles (UCNPs) NaYF₄: 20%Yb, 2%Er. The fluorescence resonance energy transfer (FRET) process from the UCF emission at 540 nm of the UCNPs to the absorbance of the rhodamine B occurs in the nanoprobe. The PLD-mediated hydrolysis of the phosphodiester bond makes rhodamine B apart from the UCNP surface, leading to the inhibition of FRET. Using the unaffected UCF emission at 655 nm as an internal standard, the nanoprobe can be used for ratiometric UCF detection of PLD activity with high sensitivity and selectivity. The PLD activity in cell lysates is also determined by the nanoprobe, confirming that PLD activity in a breast cancer cell is at least 7-fold higher than in normal cell. Moreover, the nanoprobe has been successfully applied to monitoring PLD activity in living cells by UCF bioimaging. The results reveal that the nanoprobe provides a simple, sensitive, and robust platform for point-of-care diagnostics and drug screening in biomedical applications