Real‐Time Intracellular Measurements of ROS and RNS in Living Cells with Single Core–Shell Nanowire Electrodes

Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living‐cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC‐core–shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano‐core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles.     

Probing microscopic architecture of opaque heterogeneous systems using double-pulsed-field-gradient NMR

Microarchitectural features of opaque porous media and biological tissues are of great importance in many scientific disciplines ranging from chemistry, material sciences, and geology to biology and medicine. Noninvasive characterization of coherently organized pores is rather straightforward since conventional diffusion magnetic resonance methods can detect anisotropy on a macroscopic scale; however, it remains extremely challenging to directly infer on microarchitectural features on the microscopic scale in heterogeneous porous media and biological cells that are comprised of randomly oriented compartments, a scenario widely encountered in Nature. Here, we show that the angular bipolar double-pulsed-field-gradient (bp-d-PFG) methodology is capable of reporting on unique microarchitectural features of highly heterogeneous systems. This was demonstrated on a toluene-in-water emulsion system, quartz sand, and even biological specimens such as yeast cells and isolated gray matter. We find that in the emulsion and yeast cells systems, the angular bp-d-PFG methodology uniquely revealed nearly an image of the pore space, since it conveyed direct microarchitectural information such as compartment shape and size. In two different quartz sand specimens, the angular bp-d-PFG experiments demonstrated the presence of randomly oriented anisotropic compartments. We also obtained unequivocal evidence that diffusion in interconnected interstices is restricted and therefore non-Gaussian. In biological contexts, the angular bp-d-PFG experiments could uniquely differentiate between spherical cells and randomly oriented compartments in gray matter tissue, information that could not be obtained by conventional NMR methods. The angular bp-d-PFG methodology also performs very well even when severe background gradients are present, as is often encountered in realistic systems. We conclude that this method seems to be the method of choice for characterizing the microstructure of porous media and biological cells noninvasively.     

Mapping viscosity in cells using molecular rotors

This article describes an emerging method for quantitative measurement and spatial imaging of microviscosity within individual domains of live cells. The method is based on fluorescence detection from small synthetic molecules termed 'molecular rotors', which are characterised by a strong response of fluorescence lifetimes or spectra to the viscosity of their immediate environment. Alongside this new method, two complementary techniques are discussed, which provide further insights into diffusion controlled processes on a microscopic scale in a biological environment. These are time resolved fluorescence anisotropy and imaging of short-lived excited state of molecular oxygen, termed 'singlet oxygen'. It is possible to utilise all three approaches for the quantitative determination of viscosity in individual organelles of live cells. Finally, it is discussed how the major advantage of molecular rotor imaging, fast signal acquisition, can be used to monitor changing viscosity during dynamic biological processes within cells, such as photoinduced cell death.     

Curvature driven transport of mouse macrophages in a pulsating magnetic garnet film ratchet

Magnetic fields varying on the colloidal length scale are used for the directed transport of magnetically labeled biological cells. The transport is achieved by using the ratchet effect which relies on an asymmetric, symmetry broken periodic potential where nonequilibrium fluctuations or oscillations generate a net cell current. Ferrofluid ingested mouse macrophages were placed on a magnetic garnet film with alternating stripe domain patterns, and a pulsating magnetic potential is provided by superposing an oscillating magnetic field normal to the film. The symmetry of the resulting periodic stripe potential is broken locally by the curvature of the stripes. We show, both experimentally and theoretically, the curvature of such stripes required for inducing directed transport of the macrophages in the ratchet. This may be useful for microfluidic devices such as a digital colloidal shift register for magnetically labeled biological cells.     

Specific Identification of Glycoproteins Bearing the Tn Antigen in Human Cells

Glycoproteins contain a wealth of valuable information regarding the development and disease status of cells. In cancer cells, some glycans (such as the Tn antigen) are highly up‐regulated, but this remains largely unknown for glycoproteins with a particular glycan. Herein, an innovative method combining enzymatic and chemical reactions was first designed to enrich glycoproteins with the Tn antigen. Using synthetic glycopeptides with O‐GalNAc (the Tn antigen) or O‐GlcNAc, we demonstrated that the method is selective for glycopeptides with O‐GalNAc and can distinguish between these two modifications. The diagnostic ions from the tagged O‐GalNAc further confirmed the effectiveness of the method and confidence in the identification of glycopeptides with the Tn antigen by mass spectrometry. Using this method, we identified 96 glycoproteins with the Tn antigen in Jurkat cells. The method can be extensively applied in biological and biomedical research.     

Easy and efficient bonding of biomolecules to an oxide surface of silicon

A new method is described to attach biological molecules to the surface of silicon. Semiconductors such as Si modified with surface-bound capture molecules have enormous potential for use in biosensors for which an ideal detection platform should be inexpensive, recognize targets rapidly with high sensitivity and specificity, and possess superior stability. In this process, a self-assembled film of an organophosphonic acid is bonded to the native or synthesized oxide-coated Si surface as a film of the correspondingphosphonate. The phosphonate film is functionalized to enable covalently coupling biological molecules, ranging in size from small peptides to large multi-subunit proteins, to the Si surface. Surface modification and biomolecule coupling procedures are easily accomplished: all reactions can proceed in air, and most take place under ambient conditions. The biomolecule-modified surfaces are stable under physiological conditions, are selective for adhesion of specific cells types, and are reusable.     

Osmolarity effects on red blood cell elution in sedimentation field-flow fractionation.

Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width.     

Evaluation of a sensitive HPLC method for the determination of Malondialdehyde, and application of the method to different biological materials

Summary Reactive oxygen species (ROS), important mediators of cell and tissue injury during inflammation, are produced by several types of inflammatory cells. The formation of ROS can be monitored by detection of lipid peroxidation products. The extremely broad spectrum of biological effects of aldehydic lipid peroxidation products has necessitated the development of a technique that enables the sensitive routine quantitation of aldehydes formed in biological materials. Malondialdehyde (MDA) is a by-product of enzymatic eicosanoid formation and an end-product of nonenzymatic peroxidation of polyunsaturated fatty acids with three or more bisallylic double bonds. The determination of the thiobarbituric acid derivative of MDA (TBA-MDA) is a widely used method for estimating overall lipid peroxidation. We describe a rapid, isocratic, simple, and sensitive high-performance liquid chromatographic (HPLC) method with spectrofluorimetric detection for measurement of MDA-TBA in human biological samples such as plasma, urine, wound secretions, amniotic fluid, sputum and tissue samples. By use of this method, picomole quantities of MDA can be readily and specifically detected in different biological materials. Coefficients of variation of repeated MDA-TBA assays were 4.4% within run and 6.9% from run to run. Reference values are given for a variety of human body fluids and for rat tissues.     

A polymeric domain that promotes cellular internalization

Polymers have emerged as powerful biological tools; however, their ability to gain access to the intracellular environment is limited. To expand the biological utility of polymer scaffolds, we have synthesized an internalization domain using the ring-opening metathesis polymerization (ROMP). A polymer functionalized with guanidinium groups is effectively internalized by cells and localized in both vesicles and the cytoplasm. Because the synthesis of such materials is modular, we anticipate that compounds of this type can be fashioned that facilitate the delivery of cargo via end-cap derivatization or block copolymer synthesis.     

Molecule‐Membrane Interactions in Biological Cells Studied with Second Harmonic Light Scattering

The nonlinear optical phenomenon second harmonic light scattering (SHS) can be used for detecting molecules at the membrane surfaces of living biological cells. Over the last decade, SHS has been developed for quantitatively monitoring the adsorption and transport of small and medium size molecules (both neutral and ionic) across membranes in living cells. SHS can be operated with both time and spatial resolution and is even capable of isolating molecule‐membrane interactions at specific membrane surfaces in multi‐membrane cells, such as bacteria. In this review, we discuss select examples from our lab employing time‐resolved SHS to study real‐time molecular interactions at the plasma membranes of biological cells. We first demonstrate the utility of this method for determining the transport rates at each membrane/interface in a Gram‐negative bacterial cell. Next, we show how SHS can be used to characterize the molecular mechanism of the century old Gram stain protocol for classifying bacteria. Additionally, we examine how membrane structures and molecular charge and polarity affect adsorption and transport, as well as how antimicrobial compounds alter bacteria membrane permeability. Finally, we discuss adaptation of SHS as an imaging modality to quantify molecular adsorption and transport in sub‐cellular regions of individual living cells.     

Open micro-fluidic system for atomic force microscopy-guided in situ electrochemical probing of a single cell

Ultra-sharp nano-probes and customized atomic force microscopy (AFM) have previously been developed in our laboratory for in situ sub-cellular probing of electrochemical phenomena in living plant cells during their photosynthesis. However, this AFM-based electrochemical probing still has numerous engineering challenges such as immobilization of the live cells, compatibility of the immobilization procedure with AFM manipulation of the probe, maintenance of biological activity of the cells for an extended time while performing the measurements, and minimization of electrochemical noise. Thus, we have developed an open micro-fluidic channel system (OMFC) in which individual cells can be immobilized in micro-traps by capillary flow. This system affords easy AFM access and allows for maintenance of the cells in a well-defined chemical environment, which sustains their biological activity. The use of micro-channels for making the electrochemical measurements significantly reduces parasitic electrical capacitances and allows for current detection in the sub-pico-ampere range at high signal bandwidths. The OMFC was further studied using simulation packages for optimal design conditions. This system was successfully used to measure light-dependent oxidation currents of a few pico-amperes from the green alga Chlamydomonas reinhardtii.     

Mass spectrometry imaging of small molecules from live cells and tissues using nanomaterials

We review recently developed methods for analyzing live cells and tissues in ambient conditions without the use of harsh chemical fixation or physical freezing and drying. The first method is based on laser ablation in atmospheric pressure assisted by atmospheric pressure plasma and nanomaterials such as nanoparticles and graphene to enhance laser ablation. The second method is based on secondary ion mass spectrometry imaging of live cells in solution capped with single-layer graphene to preserve intact and hydrated biological samples even under ultrahigh vacuum for secondary ion mass spectrometry bio-imaging in solution with subcellular spatial resolution. Mass spectrometry imaging of small molecules from live cells and tissues can provide an innovative molecular imaging methodology for several biomedical and material research applications.     

Analytical techniques for single-cell metabolomics: state of the art and trends

Single-cell metabolomics is an emerging field that addresses fundamental biological questions and allows one to observe metabolic phenomena in heterogeneous populations of single cells. In this review, we assess the suitability of different detection techniques and present considerations on sample preparation for single-cell metabolomics. Although targeted analysis of single cells can readily be conducted using fluorescent probes and optical instruments (microscopes, fluorescence detectors), a comprehensive metabolomic approach requires a powerful label-free method, such as mass spectrometry (MS). Mass-spectrometric techniques applied to study small molecules in single cells include electrospray MS, matrix-assisted laser desorption/ionization MS, and secondary ion MS. Sample preparation is an important aspect to be taken into account during further development of methods for single-cell metabolomics.     

Artificial cells in medicine and biotechnology

Since the feasibility of artificial cells was first demonstrated in 1957 [Chang (1, 2)], an increasing number of approaches to their preparation and use have become available. Thus artificial cell membranes can now be formed using a variety of synthetic or biological materials to produce desired variations in their permeability, surface properties, and blood compatibility. Almost any material can be included within artificial cells. These include enzyme systems, cell extracts, biological cells, magnetic materials, isotopes, antigens, antibodies, vaccines, hormones, adsorbents, and others. Since cells are the fundamental units of living organisms, it is not surprising that artificial cells can have a number of possible applications. This is especially so since artificial cells can be “tailor-made” to have very specialized functions. A number of potential applications suggested earlier have now reached a developmental stage appropriate for clinical trial or application. These clinical applications include the use of such cells as a red blood cell substitute, in hemoperfusion, in an artifical kidney or artificial liver, as detoxifiers, in an artificial pancreas, and so on. Artificial red blood cells based on lipid-coated fluorocarbon or crosslinked hemoglobin are being investigated in a number of centers. The principle of the artificial cells is also being used in biotechnology to immobilize enzymes and cells. Developments in biotechnology have also resulted in the use of the principle underlying the artificial cell to help produce interferons and monoclonal antibodies; to create immunosorbents; to develop an artificial pancreas; and to bring enzyme technology usefully into biotechnology and biomedical applications. Artificial cells are also being used as drug delivery systems based on slow release, on magnetic target delivery, on biodegradability, on liposomes, or other approaches. The present status and recent advances will be emphasized in this paper.     

Development of a zinc ion-selective luminescent lanthanide chemosensor for biological applications

Detection of chelatable zinc (Zn(2+)) in biological studies has attracted much attention recently, because chelatable Zn(2+) plays important roles in many biological systems. Lanthanide complexes (Eu(3+), Tb(3+), etc.) have excellent spectroscopic properties for biological applications, such as long luminescence lifetimes of the order of milliseconds, a large Stoke's shift of >200 nm, and high water solubility. Herein, we present the design and synthesis of a novel lanthanide sensor molecule, [Eu-7], for detecting Zn(2+). This europium (Eu(3+)) complex employs a quinolyl ligand as both a chromophore and an acceptor for Zn(2+). Upon addition of Zn(2+) to a solution of [Eu-7], the luminescence of Eu(3+) is strongly enhanced, with high selectivity for Zn(2+) over other biologically relevant metal cations. One of the important advantages of [Eu-7] is that this complex can be excited with longer excitation wavelengths (around 340 nm) as compared with previously reported Zn(2+)-sensitive luminescent lamthanide sensors, whose excitation wavelength is at too high an energy level for biological applications. The usefulness of [Eu-7] for monitoring Zn(2+) changes in living HeLa cells was confirmed. This novel Zn(2+)-selective luminescent lanthanide chemosensor [Eu-7]should be an excellent lead compound for the development of a range of novel luminescent lanthanide chemosensors for biological applications.     

Synthetic Biology—The Synthesis of Biology

Synthetic biology concerns the engineering of man-made living biomachines from standardized components that can perform predefined functions in a (self-)controlled manner. Different research strategies and interdisciplinary efforts are pursued to implement engineering principles to biology. The “top-down” strategy exploits nature's incredible diversity of existing, natural parts to construct synthetic compositions of genetic, metabolic, or signaling networks with predictable and controllable properties. This mainly application-driven approach results in living factories that produce drugs, biofuels, biomaterials, and fine chemicals, and results in living pills that are based on engineered cells with the capacity to autonomously detect and treat disease states in vivo. In contrast, the “bottom-up” strategy seeks to be independent of existing living systems by designing biological systems from scratch and synthesizing artificial biological entities not found in nature. This more knowledge-driven approach investigates the reconstruction of minimal biological systems that are capable of performing basic biological phenomena, such as self-organization, self-replication, and self-sustainability. Moreover, the syntheses of artificial biological units, such as synthetic nucleotides or amino acids, and their implementation into polymers inside living cells currently set the boundaries between natural and artificial biological systems. In particular, the in vitro design, synthesis, and transfer of complete genomes into host cells point to the future of synthetic biology: the creation of designer cells with tailored desirable properties for biomedicine and biotechnology.     

On-chip measurements of cell compressibility via acoustic radiation

Measurements of mechanical properties of biological cells are of great importance because changes in these properties can be strongly associated with the progression of cell differentiation and cell diseases. Although state of the art methods, such as atomic force microscopy, optical tweezers and micropipette aspiration, have been widely used to measure the mechanical properties of biological cells, all these methods involve direct contact with the cell and the measurements could be affected by the contact or any local deformation. In addition, all these methods typically deduced the Young's modulus of the cells based on their measurements. Herein, we report a new method for fast and direct measurement of the compressibility or bulk modulus of various cell lines on a microchip. In this method, the whole cell is exposed to acoustic radiation force without any direct contact. The method exploits the formation of an acoustic standing wave within a straight microchannel. When the polystyrene beads and cells are introduced into the channel, the acoustic radiation force moves them to the acoustic pressure node and the movement speed is dependent on the compressibility. By fitting the experimental and theoretical trajectories of the beads and the cells, the compressibility of the cells can be obtained. We find that the compressibility of various cancer cells (MCF-7: 4.22 ± 0.19 × 10(-10) Pa(-1), HEPG2: 4.28 ± 0.12 × 10(-10) Pa(-1), HT-29: 4.04 ± 0.16 × 10(-10) Pa(-1)) is higher than that of normal breast cells (3.77 ± 0.09 × 10(-10) Pa(-1)) and fibroblast cells (3.78 ± 0.17 × 10(-10) Pa(-1)). This work demonstrates a novel acoustic-based method for on-chip measurements of cell compressibility, complementing existing methods for measuring the mechanical properties of biological cells.     

Mussel‐Inspired Surface‐Imprinted Sensors for Potentiometric Label‐Free Detection of Biological Species

Using sensors to quantify clinically relevant biological species has emerged as a fascinating research field due to their potential to revolutionize clinical diagnosis and therapeutic monitoring. Taking advantage of the wide utility in clinical analysis and low cost of potentiometric ion sensors, we demonstrate a method to use such ion sensors to quantify bioanalytes without chemical labels. This is achieved by combination of chronopotentiometry with a mussel‐inspired surface imprinting technique. The biomimetic sensing method is based on a blocking mechanism by which the recognition reaction between the surface imprinted polymer and a bioanalyte can block the current‐induced ion transfer of an indicator ion, thus causing a potential change. The present method offers high sensitivity and excellent selectivity for detection of biological analytes. As models, trypsin and yeast cells can be measured at levels down to 0.03 U mL⁻¹ and 50 CFU mL⁻¹, respectively.     

Compartmentalized nanocomposite for dynamic nitric oxide release

Nitric oxide (NO) is an important cell-signaling molecule whose role in a variety of cellular processes such as differentiation and apoptosis depends strongly on its concentration and flux levels. This work describes and characterizes a novel nitric oxide releasing nanocomposite, capable of photostimulated NO flux that can by dynamically modulated in within a range of biological levels. This material mimics the common compartmentalization strategies used by living cells to achieve its novel features. The material is constructed by encapsulating a photosensitive nitric oxide donor within lipid vesicles with an average diameter of 150 nm. The vesicles are then doped into the interstitial liquid phase of a solid porous silica matrix, which has previously demonstrated biological compatibility and capabilities as a growth surface for mammalian cells. Stimulation by a light source produces a step increase in NO concentration within seconds. The NO flux at the surface of the material is measured to be 14 pmol-cm(-2) sec(-1) using a NO selective self-referencing amperometric microsensor. The NO concentration profile decreases with distance perpendicular to the surface as expected for diffusion from a surface through an aqueous environment. A pattern of one minute light pulses produced uniform pulses of increased NO concentration of one minute duration. A linear relationship exists between NO surface concentration and photon flux, and this relationship can be used to tune the material response.