Fluorescent nanoparticles for intracellular sensing: A review

Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.     

Triple Amplification Strategy for the Improved Efficiency of a Microplate-Based Assay for the Chemiluminescent Detection of DNA

Significant research efforts are currently focused on advancing DNA detection methodology. Various nanoparticles (NPs), which are currently the most widely used solid-phase carriers for nucleic acid assays, have a number of essential drawbacks. Microtiter plates provide a simple and economical alternative to the NPs. This article reports the development of a sandwich assay for DNA detection using a microtiter plate as the solid carrier. A capture oligonucleotide modified with fluorescein was bound to the anti-fluorescein antibody adsorbed on the polystyrene microplate surface. A hepatitis B virus DNA fragment was used as the model analyte. To improve the assay sensitivity, the biotinylated reporter oligonucleotide and streptavidin-horseradish polyperoxidase (polyHRP) conjugate were used to provide an amplified detection system. Additional amplification was achieved because the peroxidase activity was measured by a chemiluminescent method using the 3-(10′-phenothiazinyl)propane-1-sulfonate/N-morpholinopyridine pair as an enhancing reagent. The detection limit of the developed assay was 0.9?pM with a linear dynamic range from 0.9 to 100?pM. This method may be used as a platform for the development of sensitive DNA assays.     

Probing hydroxyl radicals and their imaging in living cells by use of FAM-DNA-Au nanoparticles

The incorporation of gold nanoparticles (Au NPs) as quencher modules in fluorescent probes for DNA damage caused by intracellular hydroxyl radicals (HO*) is reported. Au NPs of 15 nm diameter were decorated with DNA oligomers terminating in thiol functions in their 3' positions and possessing 5' fluorophore modifications. The Au NPs, which have high extinction coefficients, functioned as excellent fluorescent quenchers in the fluorophore-Au NP composites. FRET is switched off as a factor of HO*-induced strand breakage in the single-stranded DNAs, restoring the fluorescence of the quenched fluorophores, which can be followed by spectrofluorimetry. In vitro assays with HO*-generating Fenton reagent demonstrated increases in fluorescence intensity with a linear range from 8.0 nM to 1.0 microM and a detection limit as low as 2.4 nM. Confocal microscopic imaging of macrophages and HepG2 revealed that the probe is cell-permeable and intracellular HO*-responsive. The unique combination of good selectivity and high sensitivity establishes the potential value of the probe for facilitating investigations of HO*-mediated cellular homeostasis and injury.     

Semiconductor quantum dots for bioanalysis

Semiconductor nanoparticles, or quantum dots (QDs), have unique photophysical properties, such as size-controlled fluorescence, have high fluorescence quantum yields, and stability against photobleaching. These properties enable the use of QDs as optical labels for the multiplexed analysis of immunocomplexes or DNA hybridization processes. Semiconductor QDs are also used to probe biocatalytic transformations. The time-dependent replication or telomerization of nucleic acids, the oxidation of phenol derivatives by tyrosinase, or the hydrolytic cleavage of peptides by proteases are probed by using fluorescence resonance energy transfer or photoinduced electron transfer. The photoexcitation of QD-biomolecule hybrids associated with electrodes enables the photoelectrochemical transduction of biorecognition events or biocatalytic transformations. Examples are the generation of photocurrents by duplex DNA assemblies bridging CdS NPs to electrodes, and by the formation of photocurrents as a result of biocatalyzed transformations. Semiconductor nanoparticles are also used as labels for the electrochemical detection of DNA or proteins: Semiconductor NPs functionalized with nucleic acids or proteins bind to biorecognition complexes, and the subsequent dissolution of the NPs allows the voltammetric detection of the related ions, and the tracing of the recognition events.     

Ultrahighly Sensitive Homogeneous Detection of DNA and MicroRNA by Using Single‐Silver‐Nanoparticle Counting

DNA and RNA analysis is of high importance for clinical diagnoses, forensic analysis, and basic studies in the biological and biomedical fields. In this paper, we report the ultrahighly sensitive homogeneous detection of DNA and microRNA by using a novel single‐silver‐nanoparticle counting (SSNPC) technique. The principle of SSNPC is based on the photon‐burst counting of single silver nanoparticles (Ag NPs) in a highly focused laser beam (about 0.5 fL detection volume) due to Brownian motion and the strong resonance Rayleigh scattering of single Ag NPs. We first investigated the performance of the SSNPC system and then developed an ultrasensitive homogeneous detection method for DNA and microRNA based on this single‐nanoparticle technique. Sandwich nucleic acid hybridization models were utilized in the assays. In the hybridization process, when two Ag‐NP–oligonucleotide conjugates were mixed in a sample containing DNA (or microRNA) targets, the binding of the targets caused the Ag NPs to form dimers (or oligomers), which led to a reduction in the photon‐burst counts. The SSNPC method was used to measure the change in the photon‐burst counts. The relationship between the change of the photon‐burst counts and the target concentration showed a good linearity. This method was used for the assay of sequence‐specific DNA fragments and microRNAs. The detection limits were at about the 1 fM level, which is 2–5 orders of magnitude more sensitive than current homogeneous methods.     

Surface‐Bound Cucurbit[8]uril Catenanes on Magnetic Nanoparticles Exhibiting Molecular Recognition

We demonstrate the preparation of surface‐bound cucurbit[8]uril (CB[8]) catenanes on silica nanoparticles (NPs), where CB[8] was employed as a tethered supramolecular “handcuff” to selectively capture target guest molecules. In this catenane, CB[8] was threaded onto a methyl viologen (MV²⁺) axle and immobilized onto silica NPs. The formation of CB[8] catenanes on NPs were confirmed by UV/Vis titration experiments and lithographic characterization, demonstrating a high density of CB[8] on the silica NPs surface, 0.56 nm^?2. This CB[8] catenane system exhibits specific molecular recognition towards certain aromatic molecules such as perylene bis(diimide), naphthol and aromatic amino acids, and thus it can act as a nanoscale molecular receptor for target guests. Furthermore, we also demonstrate its use as an efficient and recyclable nano‐platform for peptide separation. By embedding magnetic NPs inside silica NPs, separation could be achieved by simply applying an external magnetic field. Moreover, the peptides captured by the catenanes could be released by reversible single‐electron reduction of MV²⁺. The entire process demonstrated high recoverability.     

Electrochemical and Electrochemiluminescence Determination of Cancer Cells Based on Aptamers and Magnetic Beads

The electrochemical and electrochemiluminescence (ECL) detection of cell lines of Burkitt’s lymphoma (Ramos) by using magnetic beads as the separation tool and high‐affinity DNA aptamers for signal recognition is reported. Au nanoparticles (NPs) bifunctionalized with aptamers and CdS NPs were used for electrochemical signal amplification. The anodic stripping voltammetry technology employed for the analysis of cadmium ions dissolved from CdS NPs on the aggregates provided a means to quantify the amount of the target cells. This electrochemical method could respond down to 67 cancer cells per mL with a linear calibration range from 1.0×10² to 1.0×10⁵ cells mL^?1, which shows very high sensitivity. In addition, the assay was able to differentiate between target and control cells based on the aptamer used in the assay, indicating the wide applicability of the assay for diseased cell detection. ECL detection was also performed by functionalizing the signal DNA, which was complementary to the aptamer of the Ramos cells, with tris(2,2‐bipyridyl) ruthenium. The ECL intensity of the signal DNA, replaced by the target cells from the ECL probes, directly reflected the quantity of the amount of the cells. With the use of the developed ECL probe, a limit of detection as low as 89 Ramos cells per mL could be achieved. The proposed methods based on electrochemical and ECL should have wide applications in the diagnosis of cancers due to their high sensitivity, simplicity, and low cost.     

Fluorescent pH nanosensors: Design strategies and applications

Accurate measuring of pH is of great significance for various research areas ranging from environmental to chemical and biological sciences. In the past decades, there has been growing interest in the use of nanoparticles (NPs) for pH measurement, especially for intracellular pH sensing and imaging. In this regard, a number of different NP-based fluorescent pH sensors have been developed, which can be classified into three major categories including (I) fluorescent NPs with direct or indirect responses to pH, (II) nonfluorescent NPs that are used only as scaffold and carriers for pH-sensitive fluorescent dyes, and (III) nonfluorescent NPs whose pH-responsive structural change is converted into the fluorescence signal of their conjugated dyes. This review is a complete coverage of all NPs used so far for fluorescence pH sensing. The authors of this review invite readers to find all design strategies for employing semiconductor quantum dot, nanoclusters, carbon-based dots, polymer dots, upconversion NPs, fluorescent metal-organic frameworks, metallic NPs, silica NPs, polymer NPs, micellar NPs, nanogels, and protein NPs for this purpose.     

Fabrication of silica-coated magnetic nanoparticles with highly photoluminescent lanthanide probes

Bi-functional nanoparticles (NPs) that consist of silica-coated magnetic cores and luminescent lanthanide (Ln) ions anchored on the silica surface via organic linker molecules are reported. Compared to individual Ln ions, the hybrid NPs show a drastically enhanced photoluminescence due to the efficient ligand-to-metal energy transfer in the Ln-loaded NPs: the new bi-functional NPs could be used in a variety of biological applications involving magnetic separation and optical detection.     

Nanoparticle-based electrochemical detection in conventional and miniaturized systems and their bioanalytical applications: a review

With recent advances in nanotechnology making more easily available the novel chemical and physical properties of metal nanoparticles (NPs), these have become extremely suitable for creating new sensing assays. Many kinds of NPs, including metal, metal-oxide, semiconductor and even composite-metal NPs, have been used for constructing electrochemical sensors. This article reviews the progress of NP-based electrochemical detection in recent applications, especially in bioanalysis, and summarizes the main functions of NPs in conventional and miniaturized systems. All references cited here generally show one or more of the following characteristics: a low detection limit, good signal amplification and simultaneous-detection capabilities.     

A label-free, quadruplex-based functional molecular beacon (LFG4-MB) for fluorescence turn-on detection of DNA and nuclease

We demonstrate a novel concept for the construction of a label-free, quadruplex-based functional molecular beacon (LFG4-MB) by using G-quadruplex motif as a substitute for Watson-Crick base pairing in the MB stem and a specific G-quadruplex binder, N-methyl mesoporphyrin IX (NMM) as a reporter. It shows high sensitivity in assays for UDG activity/inhibition and detection of DNA sequence based on the unique fluorescence increase that occurs as a result of the strong interaction between NMM and the folded quadruplex upon removal of uracil by UDG or displacement of block sequence by target DNA. The LFG4-MB is simple in design, fast in operation and could be easily transposed to other biological relevant target analysis by simply changing the recognition portion. The LFG4-MB does not require any chemical modification for DNA, which offers the advantages of simplicity and cost efficiency and obviates the possible interference with the affinity and specificity of the MB as well as the kinetic behavior of the catalysts caused by the bulky fluorescent groups. More importantly, the LFG4-MB offers great extent of freedom to tune the experimental conditions for the general applicability in bioanalysis.     

DNA interaction probed by evanescent wave cavity ring-down absorption spectroscopy via functionalized gold nanoparticles

Evanescent wave cavity ring-down absorption spectroscopy (EW-CRDS) is employed to study interaction and binding kinetics of DNA strands by using gold nanoparticles (Au NPs) as sensitive reporters. These Au NPs are connected to target DNA of study that hybridizes with the complementary DNA fixed on the silica surface. By the absorbance of Au NPs, the interaction between two DNA strands may be examined to yield an adsorption equilibrium constant of 2.2 × 10¹⁰ M⁻¹ using Langmuir fit. The binding efficiency that is affected by ion concentration, buffer pH and temperature is also examined. This approach is then applied to the label-free detection of the DNA mutation diseases using the sandwich hybridization assay. For monitoring a gene associated with sickle-cell anemia, the detection limit and the adsorption equilibrium constant is determined to be 1.2 pM and (3.7 ± 0.8) × 10¹⁰ M⁻¹, distinct difference from the perfectly matched DNA sequence that yields the corresponding 0.5 pM and (1.1 ± 0.2) × 10¹¹ M⁻¹. The EW-CRDS method appears to have great potential for the investigation of the kinetics of a wide range of biological reactions.     

Study the damage of DNA molecules induced by three kinds of aqueous nanoparticles

In this paper, the interaction of DNA molecules with aqueous CdTe quantum dots (CdTe QDs), CdTe/SiO₂ composite nanoparticles (CdTe/SiO₂ NPs), and Mn-doped ZnSe quantum dots (Mn:ZnSe d-dots) was studied with ethidium bromide as a probe. The purpose of this work was to study the damage of DNA molecules induced by these three kinds of water-soluble nanoparticles. It was found that ionic strength, pH value and UV irradiation influenced the PL emission properties of CdTe QDs, CdTe/SiO₂ NPs and Mn:ZnSe d-dots, and also influenced the interaction of DNA molecules with them. Among the three kinds of nanoparticles, DNA molecules were most easily damaged by CdTe QDs whether in the dark or under UV irradiation. The CdTe/SiO₂ NPs led to much less DNA damage when compared with CdTe QDs, as a silica overcoating layer could isolate the QDs from the external environment. Mn:ZnSe d-dots as a new class of non-cadmium doped QDs demonstrated almost no damage for DNA molecules, which have great potentials as fluorescent labels in the applications of biomedical assays, imaging of cells and tissues, even in vivo investigations.     

毒剂的生化分析技术

本文论述了酶法分析、核酸探针、免疫分析法特别是生物传感器在毒剂检测中的应用，提出了毒剂生化分析的发展方向。     

Nanoparticle labelling-based magnetic immunoassay on chip combined with electrothermal vaporization-inductively coupled plasma mass spectrometry for the determination of carcinoembryonic antigen in human serum

A sensitive and selective on chip magnetic immunoassay method, based on a sandwich-type immunoreaction with PbS nanoparticle (NPs) labels in combination with electrothermal vaporization-inductively coupled plasma mass spectrometry (ETV-ICP-MS), was proposed for the determination of carcinoembryonic antigen (CEA). We designed and fabricated a microfluidic chip for magnetic immunoassay, and the prepared iminodiacetic acid modified silica coated magnetic nanoparticles (IDA-SCMNPs) were packed into the central microchannel to form a solid phase column by self-assembly under the magnetic field. After completion of the immunoreaction involving a primary antibody, CEA and a secondary antibody labeled with PbS NPs on a magnetic solid phase packed-column, ETV-ICP-MS was used to determine the concentration of Pb that was released from the captured PbS NPs using an acid-dissolution step. The concentrations of CEA can be correlated with that of Pb. The established method demonstrated a limit of detection of 0.058 μg L(-1) for CEA, with a relative standard deviation (RSD) of 6.7% (c = 10 μg L(-1), n = 7). A linearity ranging from 0.2 μg L(-1) to 50 μg L(-1) and a 2-fold enrichment factor (from 60 μL sample solution to 30 μL eluent) were achieved. The proposed method was further validated by analyzing CEA in human serum. The results were in good agreement with those obtained by chemiluminescent immunoassay, which is currently used as a clinical method. Overall, this method offers the advantages of high speed, high sensitivity, high selectivity, low sample/reagents consumption, high integrity and versatility. Moreover, it can be easily applied to other biological and medical assays.     

A facile route to synthesize silver nanoparticles in polyelectrolyte capsules

We are reporting a novel green approach to incorporate silver nanoparticles (NPs) selectively in the polyelectrolyte capsule shell for remote opening of polyelectrolyte capsules. This approach involves in situ reduction of silver nitrate to silver NPs using PEG as a reducing agent (polyol reduction method). These nanostructured capsules were prepared via layer by layer (LbL) assembly of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on silica template followed by the synthesis of silver NPs and subsequently the dissolution of the silica core. The size of silver nanoparticles synthesized was 60±20 nm which increased to 100±20 nm when the concentration of AgNO(3) increased from 25 mM to 50 mM. The incorporated silver NPs induced rupture and deformation of the capsules under laser irradiation. This method has advantages over other conventional methods involving chemical agents that are associated with cytotoxicity in biological applications such as drug delivery and catalysis.     

A highly efficient and versatile microchip capillary electrophoresis method for DNA separation using gold nanoparticle as a tag

A highly efficient and versatile method for DNA separation using Au nanoparticles (Au NPs) as a tag based on microchip capillary electrophoresis (MCE) was developed. The thiol-modified DNA-binding Au NPs were utilized as a tag. Target DNA was sandwiched between Au NPs and probe DNA labeled with horseradish peroxidase (HRP). In electrophoresis separation, the difference in electrophoretic mobility between free probe and probe-target complex was magnified by Au NPs, which enabled the resulting mixture to be separated with high efficiency by microchip capillary electrophoresis. Horseradish peroxidase was used as a catalytic label to achieve sensitive electrochemical DNA detection via fast catalytic reactions. With this protocol, 27-mer DNA fragments with different sequences were separated with high speed and high resolution. The proposed method was critical to achieve improved DNA separations in hybridization analyses.     

π-Conjugated Copper Phthalocyanine Nanoparticles as Highly Sensitive Sensor for Colorimetric Detection of Biomarkers

Though numerous nanomaterials with enzyme-like activities have been utilized as probes and sensors for detecting biological molecules, it is still challenging to construct highly sensitive detectors for biomarkers using polymeric materials. Benefiting from the π-d delocalization effect of electrons, excellent metal-chelating property, high electron transferability, and good chemical stability of π-conjugated phthalocyanine, the design of the copper phthalocyanine-based conjugated polymer nanoparticles (Cu-PcCP NPs) as a colorimetric sensor for a variety of biomarkers is reported. The Cu-PcCP NPs are synthesized through a simple microwave-assisted polymerization, and their chemical structures are thoroughly characterized. The colorimetric results of Cu-PcCP NPs demonstrate excellent peroxidase-like detecting activity and also great substrate selectivity than most of the reported Cu-based nanomaterials. The Cu-PcCP NPs can achieve a detection limit of 4.88 μM for the H₂O₂, 4.27 μM for the L-cysteine, and 21.10 μM for the glucose via a cascade catalytic system, which shows comparable detecting sensitivity as that of many earlier reported enzyme-like nanomaterials. Moreover, Cu-PcCP NPs present remarkable resistance to harsh conditions, including high temperature, low pH, and excessive salts. These highly specific π-conjugated copper-phthalocyanine nanoparticles not only overcome the current limitation of polymeric material-based sensors but also provide a new direction for designing next-generation enzyme-like nanomaterial-based colorimetric biosensors.     

Controlled nano-particle dyeing of cotton can ensure low cytotoxicity risk with multi-functional property enhancement

The Nanoparticle (NP) and their oxides are being progressively used and expected to be more frequently used in textiles. Nanoparticle (NP) has higher toxicological risk than larger particles because of their physicochemical properties, chemical reactivity and biological activity. In fact, the stability of nano-oxide particles in the medium is always challenging as they lack functional groups to leverage upon textile materials directly. Thus, in many finishing processes, cross-linkers and/or adhesives are applied together with NP at the cost of inferior comfort, feel and fastness which tends to be toxic and prone to release NPs under common laundering, physical stress and sweat. This study shows that the diffusion of NPs into the fibre polymer matrix via dyeing technique could be much durable, safer in terms of cytotoxicity levels and easy to process for tailoring desired functional attributes. We studied the possibility of a simple application technique via dyeing of vinyl sulphone based reactive dye with four kinds of NPs followed by their cytotoxicity test using cell line A431.1% silica dyed sample have shown highest (198.5%) increase in tensile strength followed by 2% silica and 2% CNT whereas a decrease in elongation is highest in the case of CNT 2% (5.31%) and significantly enhancing the moisture management properties in case of CNT and silica. The study showed promising results in dyeing with TiO2, CNT (Carbon nanotubes), Silica and Alumina NPs in enhancing the mechanical, moisture management, and surface frictional properties to ensure comfortable and safe wear.     

Magnetic field induced aggregation of nanoparticles for sensitive molecular detection

A molecular detection method utilizing the magnetically induced aggregation of silver nanoparticle (NP)-embedded silica NPs for SERS activation is described. Here, silver embedded magnetic NPs (Ag-M-dots) composed of a magnetic core and silica shells, on whose surface silver NPs were formed, were used. Because the magnetic field induced aggregated Ag-M-dots exhibit a strong SERS signal compared to the dispersed Ag-M-dots, the system allows for the detection of adsorbed Raman label compound even at the 100 fM level. Adenine was tested as a model biocompound and its Raman spectrum could be observed at concentrations as low as 1 pM. The experimental results were supported by the theoretical calculations.