The role of “disaggregation” in optical probe development

10.1039/c3cs60368gChemical Society Reviews4382402-241

Spectroscopy-Based Biosensors

Biosensors are analytical devices capable of providing quantitative or semi-quantitative information by using a biological recognition element and a transducer. Depending upon the nature of the recognition element, different surface sensitive techniques can be applied to monitor these molecular interactions. In order to increase sensitivities and to lower detection limits down to even individual molecules, nanomaterials are promising candidates. This is possible due to the potential to immobilize more bioreceptor units at reduced volumes and their ability to act as transduction elements by themselves. Among such nanomaterials, gold nanoparticles, quantum dots, polymer nanoparticles, carbon nanotubes, nanodiamonds, and graphene are intensively studied. Biosensors provide rapid, real-time, accurate, and reliable information about the analyte under investigation and have been envisioned in a wide range of analytical applications, including medicine, food safety, bioprocessing, environmental/industrial monitoring, and electronics. A variety of biosensors, such as optical, spectroscopic, molecular, thermal, and piezoelectric, have been studied and applied in countless fields. In this book, examples of spectroscopic and optical biosensors and immunoassays are presented. Furthermore, two comprehensive reviews on optical biosensors are include

Fluorescence modulation of an aggregation-induced emission active ligand via rigidification in a coordination polymer and its application in singlet oxygen sensing

A new Zn(II)-based coordination polymer (CP) having the formula [Zn(L)(2,2'-bpy)] (1) was synthesized using ZnCl2, 3,3'-(anthracene-9,10-diyl)diacrylic acid ligand (H2L), and 2,2'-bipyridine (2,2'-bpy) in DMF under solvothermal conditions. Here, the anthracene-based dicarboxylic acid ligand shows aggregation-induced emission (AIE) activity in an ethanol/hexane medium. Single-crystal X-diffraction analysis reveals that the one-dimensional (1D) zigzag chainlike structure of 1 is assembled from tetrahedrally coordinated Zn2+ ions interconnected by 2,2'-bpy and ditopic anthracene-based ligand molecules. The crystal structure analysis reveals that the ditopic anthracene-based flexible ligand adopts a twisted conformation in the CP crystal compared to its free state. Because of the twisted conformation of the ATE active ligand in the CP crystal, a large (similar to 80 nm) hypsochromic shift was observed in the emission spectrum with a drastic color change compared to the free state of ligand. The origin of these rare fluorescence properties is ascribed to the twisted diacrylic acid ligand conformation and rigidity in the CP crystal. An unprecedented response was observed toward singlet oxygen (O-1(2)) by 1 via a fluorescence turn-off mechanism. The presence of the anthracene moiety is the main influential factor for O-1(2) sensing, which undergoes [4 + 2] cycloaddition reaction with O-1(2), producing a nonemissive 9,10-endoperoxide product. The unique photoluminescence properties along with tunable fluorescence responses indicate that incorporating an AIE active anthracene core into the CP crystal is a beneficial strategy to develop new fluorescent materials with significant sensing ability

Design of Luminescent Magnetic Nanostructures for Sensor, Drug Delivery and Bioimaging Applications

The present dissertation entitled, “Design of Luminescent Magnetic Nanostructures for Sensor, Drug delivery and Bioimaging Applications” is an embodiment of the investigations intended at developing simple inexpensive synthetic methods for producing luminescent carbon quantum dot and multifunctional magnetic luminescent nanostructures applicable for sensor and biomedical application. The in vitro applications of synthesized materials have also been investigated. The thesis is divided into two parts. The first part of the thesis includes the low cost synthetic route for fabrication of carbon quantum dots (CD) and their composites for sensing and bioimaging applications. Highly photoluminescent CDs with a quantum yield of 26% have been synthesized in one step by hydrothermal treatment of orange juice (Citrus nobilis deliciosa). Due to high photostability and low toxicity these CDs have been demonstrated as excellent probes in cellular imaging. These synthesized CD has been also used for the development of reusable novel magnetic silica/CD based hybrid nanostructure for monitoring and separation of fluoride ion through fluorescence sensing and external magnetic field respectively. The assay is based on the binding of fluoride ion into magnetic receptor substituting already bound CD. This method is highly sensitive, fast and selective for fluoride ion in aqueous solution having a linear response range of 1 to 20 μM (R2=0.992). The practical utility of the method is well tested with tap water and also extended for fluoride detection in cellular environment. Furthermore an easily separable sensitive CD based fluorescence glucose sensor comprising of CD deposited mesoporous silica nocomposite (m-SiO2-CD) and 3-aminophenylboronic acid (APBA) has been prepared. The observed fluorescence recovery of quenched APBA adsorbed m-SiO2-CD on addition of glucose is due to the formation of glucoboronate ester which could lift out the APBA from close vicinity of fluorophore. The boronic acid modified (m-SiO2-CD-APBA) fluorescent probe is also explored for targeted imaging of colon cancer cell overexpressed with sialyl Lewis A (sLea) receptors. To improve the luminescence properties of the CD, nitrogen, sulphur co-doped carbon quantum dot (NSCD) with an improved fluorescent quantum yield of 69% have been synthesized from single molecular precursor. The synthesized NSCD exhibits high selectivity and sensitivity towards mercury ion in aqueous environment. Due to high photostability, low toxicity and low detection limit as 0.05 nM, these NSCDs are demonstrated as excellent probes for the detection of Hg2+ in living cells.The second part of this thesis demonstrates the design and fabrication of multifunctional fluorescent magnetic nanostructures which are of special interest in cancer diagnostic and therapy. Multifunctional luminescent magnetic Fe3O4@mesoporous silica-YPO4:Tb core-shell nanoparticle has been prepared for the storage as well as controlled targeted release of 5-fluorouracil (5-FU). The hydrophobic anticancer drug 5-FU has been successfully loaded on the fluorescent magnetic nanoparticles via formation of 5-FU/β-cyclodextrin inclusion complex which favors more sustained release at lower pH owing to stability of inclusion complex. These findings show that the developed multifunctional nanocomposite can be potentially used in magnetically guided delivery of 5-FU. Furthermore, hierarchical theranostic hollow magnetic mesoporous spherical particles with fluorescent carbon encapsulated within mesoporous framework have been prepared by hydrothermal carbonization approach. These fluorescent magnetic nanoparticles have been conjugated with hydrophobic drug camptothecin and a molecular marker folic acid using appropriate surface chemistry to ensure the targeted specific delivery of the camptothecin. The drug conjugated hybrid nanoparticles inhibit cell growth through induction of apoptosis as demonstrated in HeLa cells. In addition to this, the particles show MR contrast behaviour by affecting the proton relaxation with transverse relaxivity (r2) 150.03 mM-1S-1

Development Of Molecular Biosensors And Multifunctional Graphene-Based Nanomaterials

In the first project, a simple, rapid, and reversible fluorescent DNA INHIBIT logic gate has been developed for sensing mercury (Hg2+) and iodide (I-) ions based on a molecular beacon (MB). In this logic gate, a mercury ion was introduced as the first input into the MB logic gate system to assist in the hybridization of the MB with an assistant DNA probe through the thymine–Hg2+–thymine interaction, which eventually restored the fluorescence of MB as the output. With this signal-on process, mercury ions can be detected with a limit of detection as low as 7.9 nM. Furthermore, when iodide ions were added to the Hg2+/MB system as the second input, the fluorescence intensity decreased because Hg2+ in the thymine–Hg2+–thymine complex was grabbed by I- due to a stronger binding force. Iodide ions can be detected with a limit of detection of 42 nM. Meanwhile, we studied the feasibility and basic performance of the DNA INHIBIT logic gate, optimized the logic gate conditions, and investigated its sensitivity and selectivity. The results showed that the MB based logic gate is highly selective and sensitive for the detection of Hg2+ and I- over other interfering cations and anions. In the second project, an ultrasensitive and rapid turn-on fluorescence assay has been developed for the detection of 3’-5’ exonuclease activity of exonuclease III (Exo III) using molecular beacons (MBs). This method has a linear detection range from 0.04 to 8.00 U mL-1 with a limit of detection of 0.01 U mL-1. In order to improve the selectivity of the method, a dual-MB system has been developed to distinguish between different exonucleases. With the introduction of two differently designed MBs which respond to different exonucleases, the T5 exonuclease, Exo III and RecJf exonucleases can be easily distinguished from each other. Furthermore, fetal bovine serum and fresh mouse serum were used as complex samples to investigate the feasibility of the dual-MB system for the detection of the enzymatic activity of Exo III. As a result, the dual-MB system showed a similar calibration curve for the detection of Exo III as in the ideal buffer solution. The designed MB probe could be a potential sensor for the detection of Exo III in biological samples. In the third project, A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 5´-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled λ exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the fluorescence intensity of the SYBR Green I decreased because less affinity with ssDNA than dsDNA. The decrease extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of λ exonuclease and reaction time, the activity of T4 PNK was monitored by the fluorescence measurement, with the limit of detection of 0.11 U/mL and good linear correlation between 0.25-1.00 U/mL (R2=0.9896). In this assay, no label was needed for the fluorescence detection. Moreover, the inhibition behaviours of the T4 PNK’s inhibitors were evaluated by this assay. The result indicated a potential of using this assay for monitoring of phosphorylation-related process. In the fourth project, a facile bottom-up method for the synthesis of highly fluorescent graphene quantum dots (GQDs) has been developed using a one-step pyrolysis of a natural amino acid, L-glutamic acid, with the assistance of a simple heating mantle device. The developed GQDs showed strong blue, green and red luminescence under irradiation with ultra-violet, blue and green light, respectively. Moreover, the GQDs emitted near-infrared (NIR) fluorescence in the range 800–850 nm with an excitation-dependent manner. This NIR fluorescence has a large Stokes shift of 455 nm, providing a significant advantage for the sensitive determination and imaging of biological targets. The fluorescence properties of the GQDs, such as the quantum yields, fluorescence life times, and photostability, were measured and the fluorescence quantum yield was as high as 54.5%. The morphology and composites of the GQDs were characterized using TEM, SEM, EDS, and FT-IR. The feasibility of using the GQDs as a fluorescent biomarker was investigated through in vitro and in vivo fluorescence imaging. The results showed that the GQDs could be a promising candidate for bioimaging. Most importantly, compared to the traditional quantum dots (QDs), the GQDs are chemically inert. Thus, the potential toxicity of the intrinsic heavy metal in the traditional QDs would not be a concern for GQDs. In addition, the GQDs possessed an intrinsic peroxidase-like catalytic activity that was similar to graphene sheets and carbon nanotubes. Coupled with 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), the GQDs can be used for the sensitive detection of hydrogen peroxide with a limit of detection of 20 mM. In the fifth project, a general, environmental-friendly, one-pot method for the fabrication of reduced graphene oxide (RGO)/metal (oxide) (e.g. RGO/Au, RGO/Cu2O, and RGO/Ag) nanocomposties was developed using glucose as the reducing agent and stabilizer. The RGO/metal (oxide) nanocomposites were characterized using STEM, FE-SEM, EDS, UV-vis absorption spectroscopy, XRD, FT-IR and Raman spectroscopy. The reducing agent, glucose, not only reduced GO effectively to RGO, but it also reduced the metal precursors to form metal (oxide) nanoparticles on the surface of RGO. Moreover, the RGO/metal (oxide) nanocomposites were stabilized by gluconic acid on the surface of RGO. Finally, the developed nanomaterials were successfully applied to simultaneous electrochemical analysis of L-ascorbic acid (L-AA), dopamine (DA) and uric acid sing RGO/Au nanocomposite as an electrode catalyst. In the sixth project, a reduced graphene oxide/silver nanoparticle (RGO/Ag) nanocomposite using glucose as the environmental-friendly reducing agent was developed. The antibacterial activity of RGO/Ag nanocomposite was carefully investigated using Escherichia coli (E. coli) and Klebsiella pneumoniae (Kp) as bacterial models. We found that, compared with AgNPs, graphene oxide (GO) and RGO, RGO/Ag nanocomposite had higher antibacterial efficiency. Furthermore, under the near-infrared (NIR) irradiation, RGO/Ag nanocomposite demonstrated enhanced synergetic antibacterial activity through the photothermal effect. Almost 100 % of E. coli and Kp were killed by the treatment of 15 µg/mL and 20 µg/mL, respectively, with NIR irradiation. Moreover, the membrane integrity assay and ROS species assay demonstrated that RGO/Ag nanocomposite under NIR irradiation caused the cell membranes disruption and generation of ROS species, providing other possible mechanisms for their high antibacterial activity besides photothermal effect. In the seventh project, a rigid distance spacer, silica shell, was used between GO and dyes in this work to elucidate the quenching ability of GO. First, an organic dye was doped in silica nanoparticles, followed by the modification of another layer of silica shell with a different thickness. Due to the electrostatic interaction between GO and positively charged silica nanoparticles, GO wrapped the silica nanoparticles when they were mixed together. Therefore, the distance between GO and organic dyes was adjusted by the thickness of the silica shell. The quenching efficiency of GO to two different organic dyes, including Tetramethylrhodamine (TAMRA) and Tris(bipyridine)ruthenium(II) chloride (Rubpy), was measured at various distances. This quenching ability investigation of GO to dyes with distance-dependent manner would provide a guideline for the design of the fluorescent functional composite using GO in the future. In the eighth project, we characterized the antibacterial activity of GO in both cell culture and animal models. Klebsiella pneumoniae (Kp) is one of the most common multidrug resistant (MDR) pathogens in causing persistent nosocomial infections and is very difficult to eradicate once established in the host. First, we demonstrated that GO exerted direct killing of Kp in agar dishes and afforded the protection of alveolar macrophages (AM) from Kp infection in culture. We then evaluated the mortality, tissue damage, polymorphonuclear neutrophil (PMN) penetration, and bacterial dissemination in Kp-infected mice. Our results revealed that GO can counteract the invasive ability of Kp in vivo, resulting in lessened tissue injury, significant but subdued inflammatory response, and prolonged mouse survival. These findings indicate that GO may be an alternative agent for controlling MDR pathogens in clinics

G-quadruplex DNA aptamers and their ligands: Structure, function and application

Highly specific and tight-binding nucleic acid aptamers have been selected against a variety of molecular targets for over 20 years. A significant proportion of these oligonucleotides display G-quadruplex structures, particularly for DNA aptamers, that enable molecular recognition of their ligands. G-quadruplex structures couple a common scaffold to varying loop motifs that act in target recognition. Here, we review DNA G-quadruplex aptamers and their ligands from a structural and functional perspective. We compare the diversity of DNA G-quadruplex aptamers selected against multiple ligand targets, and consider structure with a particular focus on dissecting the thrombin binding aptamer - thrombin interaction. Therapeutic and analytical applications of DNA G-quadruplex aptamers are also discussed. Understanding DNA G-quadruplex aptamers carries implications not only for therapeutics and diagnostics, but also in the natural biochemistry of guanine-rich nucleic acids. © 2012 Bentham Science Publishers.postprin

Cellulose-Based Biosensing Platforms

Cellulose empowers measurement science and technology with a simple, low-cost, and highly transformative analytical platform. This book helps the reader to understand and build an overview of the state of the art in cellulose-based (bio)sensing, particularly in terms of the design, fabrication, and advantageous analytical performance. In addition, wearable, clinical, and environmental applications of cellulose-based (bio)sensors are reported, where novel (nano)materials, architectures, signal enhancement strategies, as well as real-time connectivity and portability play a critical role

A repertoire of biomedical applications of noble metal nanoparticles

Noble metals comprise any of several metallic chemical elements that are outstandingly resistant to corrosion and oxidation, even at elevated temperatures. This group is not strictly defined, but the tentative list includes ruthenium, rhodium, palladium, silver, osmium, iridium, platinum and gold, in order of atomic number. The emerging properties of noble metal nanoparticles are attracting huge interest from the translational scientific community and have led to an unprecedented expansion of research and exploration of applications in biotechnology and biomedicine. Noble metal nanomaterials can be synthesised both by top-down and bottom up approaches, as well as via organism-assisted routes, and subsequently modified appropriately for the field of use. Nanoscale analogues of gold, silver, platinum, and palladium in particular, have gained primary importance owing to their excellent intrinsic properties and diversity of applications; they offer unique functional attributes, which are quite unlike the bulk material. Modulation of noble metal nanoparticles in terms of size, shape and surface functionalisation has endowed them with unusual capabilities and manipulation at the chemical level, which can lead to changes in their electrical, chemical, optical, spectral and other intrinsic properties. Such flexibility in multi-functionalisation delivers ‘Ockham's razor’ to applied biomedical science. In this feature article, we highlight recent advances in the adaptation of noble metal nanomaterials and their biomedical applications in therapeutics, diagnostics and sensing

Plasmon-Enhanced Optical Sensing by Engineering Metallic Nanostructures

The world’s booming population projected to reach 10 billion by 2050 causes enormous stresses on environmental safety, food supply, and healthcare, which in return threatens human civilizations. One of the most promising solutions lies at innovating point-of-care (POC) sensing technologies to conduct detection of environmental hazards, monitoring of food safety, and early diagnosis of diseases in a timely and accurate manner. The discovery of surface-enhanced spectroscopy in the 1970s has significantly stimulated research on light-matter interaction which gives rise to enhanced optical phenomena such as surface-enhanced Raman scattering (SERS), plasmon-enhanced fluorescence (PEF), and particularly, they have found enormous applications in optical sensing. To fully exploit surface-enhanced spectroscopy to advance sensing technologies, it requires innovations in the sensor design as well as the plasmonic metallic nanostructures, which is exactly the focus of this dissertation. Owing to their strong capabilities of revealing molecular fingerprints and conducting single molecule analysis, both SERS and PEF have received extensive research interests. Since SERS directly correlates with the local electromagnetic (EM) field enhancement, it is featured by the simplicity in signal amplification. However, high SERS spectral resolution cannot be achieved without a tightly focused laser beam, which compromises the design of SERS-based POC sensing platforms. In contrast, the emission nature of fluorescence makes PEF easily coupled with POC readers, but optimal PEF requires a delicate control of the separation distance between the fluorophore and the nanostructure to minimize fluorescence quenching. SERS and PEF are essentially two complementary techniques and both hold great promise for POC sensing technologies. In the dissertation, in the first place, two label-free SERS sensors have been developed aiming to reduce the number of elements used in a sensor, which could potentially minimize interference, reduce the cost, and enhance the performance. In this regard, a label-free SERS sensor for mercury ions (Hg2+) detection has been developed based on functionalized gold nanoparticles, which employs a small molecule 4-mercaptobenzoic acid to capture mercury ions. A coordination bond formed only in the presence of mercury ions produces a new SERS peak at 374 cm-1, allowing unique detection of mercury ions. The other label-free SERS sensor has been developed for nitrite (NO2-) detection following the mechanism of Griess reaction based on the plasmonic coupling between gold nanostars and silver nanopyramid arrays. A newly formed azo compound produces at least three characteristic SERS peaks at 1140 cm-1, 1389 cm-1, and 1434 cm-1, which allow a highly specific detection of nitrite. While label-free SERS sensing has proved effective to enhance the performance, the need for a tightly focused laser beam hinders SERS from being easily coupled with POC readers for rapid signal readout. To address this limitation of SERS, on-chip PEF sensors have been developed, which can be inserted into POC readers for rapid signal readout. Optimizing PEF usually requires a delicate control of the separation distance between the fluorophore and the nanostructure to balance the excitation and emission enhancement which have different distance dependence. In addition to the separation distance, scattering has been found to be strongly correlated with quantum efficiency enhancement, which has been established as another tuning parameter in optimizing PEF. By making PEF work in the near-infrared (NIR) biological transparency window, the strength of PEF is further manifested by its compatibility with biological matrix featured as low background interference and high penetration depth. As a proof of concept, a NIR fluorescent biosensor has been developed for detection of traumatic brain injury biomarker in the blood plasma. The selection of a gold nanopyramid array pattern as the sensing platform not only generates intense localized EM field for the excitation enhancement, but also allows all the tests to be conducted using a POC fluorescence reader. While noble metals such as gold and silver are often used in developing sensing technologies as they support strong localized surface plasmon resonance (LSPR), it remains an open question as whether they could be replaced by alternative inexpensive metals such as copper and aluminum without compromising the performance. The discovery of a strong and sharp LSPR on copper nanoparticles when the shape is made cubic strongly suggests this possibility. By means of a numeric and theoretical study, it is found that the observed LSPR on copper nanocubes originates from the corner mode which survives damping as it is spectrally separated from the interband transitions. Compared to the dipole mode of a gold nanosphere of the same volume, a copper nanocube displays a comparable extinction coefficient but a local EM field enhancement 7.2 times larger. Furthermore, a film-coupled copper nanocube system has been designed for plasmon-enhanced NIR fluorescence. Because of the coupling between the copper nanocube and the underlying film, a plasmonic cavity mode is generated and featured as a spectrally tunable LSPR and an intense local EM field. By tailoring the resonance to the NIR wavelength region, the film-coupled copper nanocube system has been demonstrated to support a large NIR fluorescence enhancement owing to the strong excitation enhancement and the quantum efficiency enhancement