Plasmonic antennas and zero mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy towards physiological concentrations

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as F\"orster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero mode waveguides and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometre scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET and FCS. Single molecule spectroscopy techniques greatly benefit from zero mode waveguides and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.Comment: WIREs Nanomed Nanobiotechnol 201

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Molecular spectroscopic markers of DNA damage

Every cell in a living organism is constantly exposed to physical and chemical factors which damage the molecular structure of proteins, lipids, and nucleic acids. Cellular DNA lesions are the most dangerous because the genetic information, critical for the identity and function of each eukaryotic cell, is stored in the DNA. In this review, we describe spectroscopic markers of DNA damage, which can be detected by infrared, Raman, surface-enhanced Raman, and tip-enhanced Raman spectroscopies, using data acquired from DNA solutions and mammalian cells. Various physical and chemical DNA damaging factors are taken into consideration, including ionizing and non-ionizing radiation, chemicals, and chemotherapeutic compounds. All major spectral markers of DNA damage are presented in several tables, to give the reader a possibility of fast identification of the spectral signature related to a particular type of DNA damage

A multiplex self-referencing detection of pathogens using surface enhanced raman scattering nanoprobes with a nano-DEP microfluidic concentrator

In this dissertation, I successfully developed the multiplex self-referencing SERS pathogen (E.coli O157: H7) detection biosensor platform which offers high sensitivity (10^1 CFU/mL) and strain level discrimination by measuring the superimposed SERS signatures with multiple characteristic peaks. To harvest the effective Raman molecular probes, I developed methods to fabricate anisotropic metallic nanoparticles to serve as SERS enhancers, and more importantly, I developed surface modification methodology to add functionality to the SERS enhancers so that they can bind specifically to their pathogen targets for highly accurate and sensitive detection. Gold nanorods (GNRs) and gold/silver nanocages are successfully fabricated with high particle yield. Three highly effective linker molecules (4-Aminothiophenol (4-ATP), 3-Amino-1,2,4-triazole-5-thiol (ATT), and 3-Mercaptopropionic acid (3-MPA)) are identified and designed to conjugate on gold nanostructures, and then different monoclonal antibody molecules are designed to bond to the different linkers through diazo-histine binding (4-ATP and ATT) and EDC/NHS bonding (3-MPA-antibody). In addition, this platform demonstrated excellent separation and concentration capacities by using DEP microfluidic devices and further improves the sensitivity to 10^0 CFU/mL. The integration of microfluidic devices with SERS detection has yielded simple and miniaturized instrumentation that is suitable for the detection and characterization of small volume of chemical and biological analytes with high sensitivity and specificity. For data analysis, various preprocessing methods are used for spectral background removal, baseline correction, smoothing, and normalization. Principle Component Analysis (PCA) is applied to reduce the variable dimensions. A Support Vector Machine (SVM) discriminant analysis model based on statistical multivariate model is being developed for superimposed spectra classification. The validation of spectral classification model (target binding VS no target binding) is evaluated by the accuracy percentage, which is above 95%

Review on carbon-derived, solid-state, micro and nano sensors for electrochemical sensing applications

The aim of this review is to summarize the most relevant contributions in the development of electrochemical sensors based on carbon materials in the recent years. There have been increasing numbers of reports on the first application of carbon derived materials for the preparation of an electrochemical sensor. These include carbon nanotubes, diamond like carbon films and diamond film-based sensors demonstrating that the particular structure of these carbon material and their unique properties make them a very attractive material for the design of electrochemical biosensors and gas sensors. Carbon nanotubes (CNT) have become one of the most extensively studied nanostructures because of their unique properties. CNT can enhance the electrochemical reactivity of important biomolecules and can promote the electron-transfer reactions of proteins (including those where the redox center is embedded deep within the glycoprotein shell). In addition to enhanced electrochemical reactivity, CNT-modified electrodes have been shown useful to be coated with biomolecules (e.g., nucleic acids) and to alleviate surface fouling effects (such as those involved in the NADH oxidation process). The remarkable sensitivity of CNT conductivity with the surface adsorbates permits the use of CNT as highly sensitive nanoscale sensors. These properties make CNT extremely attractive for a wide range of electrochemical sensors ranging from amperometric enzyme electrodes to DNA hybridization biosensors. Recently, a CNT sensor based fast diagnosis method using non-treated blood assay has been developed for specific detection of hepatitis B virus (HBV) (human liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma caused by hepatitis B virus). The linear detection limits for HBV plasma is in the range 0.5–3.0 μL−1 and for anti- HBVs 0.035–0.242 mg/mL in a 0.1 M NH4H2PO4 electrolyte solution. These detection limits enables early detection of HBV infection in suspected serum samples. Therefore, non-treated blood serum can be directly applied for real-time sensitive detection in medical diagnosis as well as in direct in vivo monitoring. Synthetic diamond has been recognized as an extremely attractive material for both (bio-) chemical sensing and as an interface to biological systems. Synthetic diamond have outstanding electrochemical properties, superior chemical inertness and biocompatibility. Recent advances in the synthesis of highly conducting nanocrystalline-diamond thin films and nano wires have lead to an entirely new class of electrochemical biosensors and bio-inorganic interfaces. In addition, it also combines with development of new chemical approaches to covalently attach biomolecules on the diamond surface also contributed to the advancement of diamond-based biosensors. The feasibility of a capacitive field-effect EDIS (electrolyte-diamond-insulatorsemiconductor) platform for multi-parameter sensing is demonstrated with an O-terminated nanocrystalline-diamond (NCD) film as transducer material for the detection of pH and penicillin concentration. This has also been extended for the label-free electrical monitoring of adsorption and binding of charged macromolecules. One more recent study demonstrated a novel bio-sensing platform, which is introduced by combination of a) geometrically controlled DNA bonding using vertically aligned diamond nano-wires and b) the superior electrochemical sensing properties of diamond as transducer material. Diamond nanowires can be a new approach towards next generation electrochemical gene sensor platforms. This review highlights the advantages of these carbon materials to promote different electron transfer reactions specially those related to biomolecules. Different strategies have been applied for constructing carbon material-based electrochemical sensors, their analytical performance and future prospects are discussed

Surface Plasmon Resonance for Biosensing

The rise of photonics technologies has driven an extremely fast evolution in biosensing applications. Such rapid progress has created a gap of understanding and insight capability in the general public about advanced sensing systems that have been made progressively available by these new technologies. Thus, there is currently a clear need for moving the meaning of some keywords, such as plasmonic, into the daily vocabulary of a general audience with a reasonable degree of education. The selection of the scientific works reported in this book is carefully balanced between reviews and research papers and has the purpose of presenting a set of applications and case studies sufficiently broad enough to enlighten the reader attention toward the great potential of plasmonic biosensing and the great impact that can be expected in the near future for supporting disease screening and stratification

Plasmonic Nanoagents in Biophysics and Biomedicine

The significant rise in implementation and applications of plasmonic nanosystems in biophysics, biochemistry, and medicine has culminated in the emergence of refined plasmonic enabling reagents, or “nanoagents”. These are defined as tools that allow researchers to not only investigate, but also actively manipulate biological processes and complex biosystems, such as living cells, on the nanoscale. This development is based on a combination of sensing capabilities, photothermal control, and optical force manipulation offered by metallic nanoparticles. The article reviews the trajectory that plasmonic nanoagents have taken in recent years and highlights seminal recent examples of their application, such as optical sensing both in vitro and in vivo, optical control of biomolecular interactions and protein function, the manipulation of lipid membrane properties, and the possibility of guiding cellular behavior.Fil: Huergo, María Ana Cristina. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Schuknecht, Francis. Ludwig Maximilians Universitat; AlemaniaFil: Zhang, Jinhua. Ludwig Maximilians Universitat; AlemaniaFil: Lohmüller, Theobald. Ludwig Maximilians Universitat; Alemani

Monitoring cell cultures in real time in a biochip

Drug screening is a very important procedure in the approval of drugs for cancer treatment. This process is generally carried out using in vitro or in vivo models that aren’t very efficient due to the non-reproducibility of the cellular and/or tissue microenvironment and ethical issues due to the use of animal models. Additionally, drug approval is a process that could last 10 to 15 years, too much time when therapy is required with urgency. Microfluidic structures can address such issues, decreasing the time per assay, as well as decreasing the quantity of reagents used and the volume of waste generated, thus decreasing the costs. Also, due to the generation of concentration gradients inside a microfluidic device, it mimics the microenvironment characteristic of conventional cell culture. In this work, a reproducible cell culture of HCT-116, a human colon cancer cell line, is successfully grown inside a microfluidic device for a posterior exposure to anti-cancer drugs. The cell viability, detected through staining the DNA with fluorophores, is on average 90%. To monitor the cell death via exposure to drugs, a specific cell death biomarker, adenylate kinase (AK), is detected inside a microfluidic device using a photomultiplier and a fluorescence microscope in a chip-based immunoassay. AK concentrations near the concentrations of the enzyme released by dead cells were detected with the immunoassay by concentrating the AK in packed agarose beads inside de microfluidic structure