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Magnetophoretic lab on a chip technologies are rapidly evolving into integrated systems for the identification of biomarkers and cells with ultra-high sensitivity. We demonstrate the highly efficient detection of the Human herpes simplex virus type 1 (HSV) UL27 gene through the programmed assembly of superparamagnetic (SPM) nanoparticles based on oligonucleotide hybridization. The state of assembly of the SPM nanoparticles was determined by optical signature of the synchronized motion on the beads on a micromagnetic array (MMA). This technique has been used to identify

This protocol describes the design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy (VH) and variable light (VL) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria. Typically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. The protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. The protocol is accessible for research groups who may not be specialized in this area, and should permit the straightforward reverse engineering of functional, recombinant antigen-binding molecules from hybridoma cells secreting functional IgGs within 50 working days. Furthermore, convenient strategies for purification of antibody fragments are described.

In this article, we review lab on a chip (LOC) devices that have been developed for processing magnetically labelled biological analytes, e.g., proteins, nucleic acids, viruses and cells, based on micromagnetic structures and a time-varying magnetic field. We describe the methods that have been developed for fabricating micromagnetic arrays and the bioprocessing operations that have been demonstrated using superparamagnetic (SPM) beads, i.e., programmed transport, switching, separation of specific analytes, and pumping and mixing of fluids in microchannels. The primary advantage of micromagnet devices is that they make it possible to develop systems that control individual SPM beads, enabling high-efficiency separation and analysis. These devices do not require hydrodynamic control and lend themselves to parallel processing of large arrays of SPM beads with modest levels of power consumption. Micromagnet devices are well suited for bioanalytical applications that require high-resolution separation, e.g., detection of rare cell types such as circulating tumour cells, or biosensor applications that require multiple magnetic bioprocessing operations on a single chip.

The nutritional and health benefits of soy protein have been extensively studied over recent decades. The Bowman-Birk inhibitor (BBI), derived from soybeans, is a double-headed inhibitor of chymotrypsin and trypsin with anticarcinogenic and anti-inflammatory properties, which have been demonstrated in vitro and in vivo. However, the lack of analytical and purification methodologies complicates its potential for further functional and clinical investigations. This paper reports the construction of anti-BBI antibody fragments based on the principle of protein design. Recombinant antibody (scFv and diabody) molecules targeting soybean BBI were produced and characterized in vitro (K(D)~1.10(-9) M), and the antibody-binding site (epitope) was identified as part of the trypsin-specific reactive loop. Finally, an extremely fast purification strategy for BBI from soybean extracts, based on superparamagnetic particles coated with antibody fragments, was developed. To the best of the authors' knowledge, this is the first report on the design and characterization of recombinant anti-BBI antibodies and their potential application in soybean processing.

A new mode of transport is described that was capable of high-resolution separation of superparamagnetic materials from complex mixtures based on their size. Laminar flow and a rotating external magnetic field were applied to superparamagnetic beads assembled on a semiperiodic micromagnet array. Beads at the edge of the micromagnet array oscillated in-phase with the external magnetic field with an amplitude that decreased with increasing frequency, omega, until they reached an immobilization frequency, omega(nu) where the beads stopped moving. Laminar flow along the edge of the array could be tuned to sweep the beads for which omega < omega(i) downstream at a velocity that increased with size while leaving beads for which omega > omega(i) undisturbed. Flow-enhanced nonlinear magnetophoresis (F-NLM) promises to enable multiple superparamagnetc bead types to be used in the fractionation of cells and implementation of diagnostic assays.

The formation of a range of precursor ion charge states from a single concentrated and purified charge state, followed by activation of each charge state, is introduced as a means to obtain more protein structural information than is available from dissociation of a single charge state alone. This approach is illustrated using off-resonance collisional activation of the [M + 8H]8+ to [M + 6H]6+ precursor ions of the bacteriophage MS2 viral coat protein following concentration and purification of the [M + 8H]8+ charge state. This range of charge states was selected on the basis of an ion trap collisional activation study of the effects of precursor ion charge state on the dissociation of the [M + 12H]12+ to [M + 5H]5+ ions. Gas-phase ion/ion proton-transfer reactions and the ion parking technique were applied to purify and concentrate selected precursor ion charge states as well as to simplify the product ion spectra. The high-charge-state ions fragment preferentially at the N-terminal side of proline residues while the product ion spectra of the lowest charge states investigated are dominated by C-terminal aspartic acid cleavages. Maximum structural information is obtained by fragmentation of the intermediate-charge states.

Several developing technologies rely upon the activity of surface-bound biomolecules to control the functionality of an interface. The specificity of these biomolecules can be compromised by mechanical stresses present during their preparation or use. Moreover, many new biomechanical devices are also sensitive to mechanical stress. This paper is concerned with the direct assessment of the mechanical limits beyond which biomolecules are rendered inactive. These mechanical limits are evaluated by a combined approach involving an atomic force microscope (AFM), an optical microscope, and standard colorimetric techniques. The AFM is used to systematically stress micrometer-sized domains of biomolecule-laden surfaces; the results of which can be directly observed with an optical microscope following histochemical staining of the surface with biologically specific dyes. Using this method, we examined the limiting conditions of two benchmark systems, i.e., streptavidin and DNA. Because of the breadth of histochem...

We present the design and calibration of a scanning force microscope which can be used to study friction, adhesion, and contact potential differences between the cantilever tip and surface. The microscope uses a modular design where the laser, cantilever/sample holder, reflecting mirror, and detector are mounted directly on an optical table. The laser, reflecting mirror, and detector are mounted on translation and rotation stages. With this design the components can be rearranged to calibrate the Z piezo motion as a function of applied voltage. Using the detector micrometers, the detector response (voltage‐to‐distance relationship) can be determined after each series of measurements. The cantilever/sample holder is constructed such that the components are material matched and thermally compensated from a common reference point. This design feature minimizes thermal drift of the instrument. The instrument can be used in a contact scanning mode where both normal and lateral deflections of the cantilever are...

Variability in the coverage or usable lifetime of active molecules at the apex of an atomic force microscope (AFM) tip is a key limitation to biomolecular force measurements with AFM. Microfabricated tip arrays make it possible to measure molecular forces between large arrays of biological molecules with AFM. The forces are measured between a probeless microfabricated cantilever and a microfabricated array containing approximately 105 addressable probes with variable radii. We measure intermolecular forces between the model ligand–receptor pair streptavidin–biotin, to demonstrate that these tip arrays can circumvent these coverage and lifetime obstacles. Further development of these arrays promises to provide a means for measuring millions of different intermolecular interactions, paving the way for AFM to be realistically applied to screen combinatorial libraries.

Two new microscopic techniques make it possible to obtain images of biologically interesting molecules directly in air, vacuum, or under water. Scanning tunneling microscopy and atomic force microscopy both have the capacity to visualize atoms on the surface of rigid structures and provide details of molecular structure for lipids, proteins, carbohydrates, and nucleic acids. In addition to providing visualizations of individual molecules, these scanning probe techniques allow direct imaging of complexes between molecules or between molecules and higher-order subcellular structures such as membranes and cytoskeletal components. Both microscopes can be operated under a variety of ambient conditions ranging from high vacuum to above atmospheric pressure. Specimens need not be dry; both techniques have been used to image molecules in aqueous media under nearly physiological conditions. It is proposed that as these techniques mature they will allow direct observation of many molecular interactions under physiological conditions or even in vivo while they are occurring within the cell.

Neuronal growth cones are motile sensory structures at the tip of axons, transducing guidance information into directional movements towards target cells. The morphology and dynamics of neuronal growth cones have been well characterized with optical techniques; however, very little quantitative information is available on the three-dimensional structure and mechanical properties of distinct subregions. In the present study, we imaged the large Aplysia growth cones after chemical fixation with the atomic force microscope (AFM) and directly compared our data with images acquired by light microscopy methods. Constant force imaging in contact mode in combination with force-distant measurements revealed an average height of 200 nm for the peripheral (P) domain, 800 nm for the transition (T) zone, and 1200 nm for the central (C) domain, respectively. The AFM images show that the filopodial F-actin bundles are stiffer than surrounding F-actin networks. Enlarged filopodia tips are 60 nm higher than the corresponding shafts. Measurements of the mechanical properties of the specific growth cone regions with the AFM revealed that the T zone is stiffer than the P and the C domain. Direct comparison of AFM and optical data acquired by differential interference contrast and fluorescence microscopy revealed a good correlation between these imaging methods. However, the AFM provides height and volume information at higher resolution than fluorescence methods frequently used to estimate the volume of cellular compartments. These findings suggest that AFM measurements on live growth cones will provide a quantitative understanding of how proteins can move between different growth cone regions.

Many of the questions in biochemistry and cell biology are concerned with the relationships of proteins and other macromolecules in complex arrays which are responsible for carrying out metabolic sequences. The simplistic notion that the enzymes we isolate in soluble form from the cytoplasm were also soluble in vivo is being replaced by the concept that these enzymes occur in organized systems within the cell. In this newer view, the cytoplasm is organized and the “soluble enzymes” are in fact fixed in the cellular space and the only soluble components of the cell are small metabolites, inorganic ions etc. Further support for the concept of metabolic organization is provided by the evidence of metabolic channeling. It has been shown that for some metabolic pathways, the intermediates are not in free diffusion equilibrium with the bulk liquid in the cell but are passed along, more or less directly, from one enzyme to the next.

We are developing a sensor capable of detecting biological species such as cells, proteins, toxins, and DNA at concentrations as low as 10−18 M. The force amplified biological sensor will take advantage of the high sensitivity of force microscope cantilevers to detect the presence of as little as one superparamagnetic particle bound to a cantilever by a sandwich immunoassay technique. The device, which will ultimately be small enough for hand‐held use, will perform an assay in about 10 min. Lock‐in detection and use of a reference cantilever will provide a high degree of vibration immunity. An array of ten or more cantilevers will provide greater sensitivity and the capability to detect multiple species simultaneously. The force amplified biological sensor also offers the potential of distinguishing and studying chemical species via its ability to measure binding forces.

The muscle glycogenolytic complex is responsible for providing access to the reserve carbohydrate energy stores in skeletal muscle during times of vigorous exercise. The complex is a set of enzymes and regulatory factors that are bound to the carbohydrate storage polymer, glycogen. These components provide the ordered synthesis and utilization of that stored form of glucose. Glycogen and the enzyme proteins, phosphorylase and phosphorylase kinase, have been imaged by atomic force microscopy (AFM) or scanning tunneling microscopy (STM). The images of all three generally correlated well with the known features of those molecules, as measured by traditional physicochemical methods. The exception for all three polymers is that the measured height by STM is in error. In each case, the molecules appear to be only about 30% of their true thickness, as measured by height above the graphite surface. It is clear that both AFM and STM will play important roles in biomedical investigation of macromolecular structures...

Enzymes can be immobilized at a solid–liquid interface and observed with a scanning tunneling microscope (STM) using the inherent charge of proteins and an external potential applied to the STM substrate. Phosphorylase kinase, and dimers and oligomers of phosphorylase b have been observed at the interface of aqueous solutions and highly oriented pyrolytic graphite (HOPG). The lateral dimensions of phosphorylase kinase determined by STM at the solid–liquid interface are from 74%–78% of the dimensions determined by STM at the solid–air interface [Biochem. 28, 4939 (1989); Biophys. J. (in press)]. The phosphorylase b lateral dimensions of both enzymes are between 1.9 and 2.5 nm greater than the dimensions determined by x‐ray crystallography. The vertical dimensions determined by STM at the solid–liquid and solid–air interfaces are in reasonable agreement with each other. Mixtures of the two enzymes show aggregates in which the complexes of the two enzymes are identifiable. This technique will make it possibl...

The events of the last year have highlighted the complexity of implementing large-scale molecular diagnostic testing for novel pathogens. The purpose of this study was to determine the chemical influences of sample collection media and storage on the stability and detection of viral nucleic acids by qRT-PCR. We studied the mechanism(s) through which viral transport media (VTM) and number of freeze-thaw cycles influenced the analytical sensitivity of qRT-PCR detection of SARS-CoV-2. Our goal is to reinforce testing capabilities and identify weaknesses that could arise in resource-limited environments that do not have well-controlled cold chains. The sensitivity of qRT-PCR analysis was studied in four VTM for synthetic single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) simulants of the SARS-CoV-2 genome. The sensitivity and reproducibility of qRT-PCR for the synthetic ssRNA and dsDNA were found to be highly sensitive to VTM with the best results observed for ssRNA in HBSS and PBS-G. Surprisingly, the presence of epithelial cellular material with the ssRNA increased the sensitivity of the qRT-PCR assay. Repeated freeze-thaw cycling decreased the sensitivity of the qRT-PCR with two noted exceptions. The choice of VTM is critically important to defining the sensitivity of COVID-19 molecular diagnostics assays and this study suggests they can impact upon the stability of the SARS-CoV-2 viral genome. This becomes increasingly important if the virus structure is destabilised before analysis, which can occur due to poor storage conditions. This study suggests that COVID-19 testing performed with glycerol-containing PBS will produce a high level of stability and sensitivity. These results are in agreement with clinical studies reported for patient-derived samples.

SCANNING tunnelling microscopy (STM) has been used to map the surface topography of inorganic materials at the atomic level, and is potentially one of the most powerful techniques for probing biomolecular structure1–3. Recent STM studies of calf thymus DNA4,5and poly(rA) · poly(rU)5 have shown that the helical pitch and periodic alternation of major and minor grooves can be visualized and reliably measured. Here we present the first STM images of poly(dG-me5d) · poly(dG-me5dC) in the Z-form. Both the general appearance of the fibres and measurements of helical parameters are in good agreement with models derived from X-ray diffraction6–4.

The scanning tunneling microscope (STM) has been used to measure properties of poly(rA).poly(rU) and DNA, such as helical pitch, half-period oscillations that were interpreted as the alternation between the major and minor grooves, and interhelical spacing. Average pitches were measured by two-dimensional Fourier transforms and by topographic profiles along the fiber axes. Values were typically 7 percent less than standard dimensions of A-form RNA and B-form DNA fibers. This result is compatible with the mild dehydration that occurred under air-drying conditions. More extensive dehydration typically led to 19 percent shrinkage. Analysis of specific regions allowed local variations in helical pitch as small as 1 angstrom to be detected, thus demonstrating that the STM can visualize functionally significant modulations of nucleic acid structure.

Dramatic contrast variation of STM images has been investigated as a function of bias on a bis[1,3-di(p-n-tetradecyloxyphenyl)propane-1,3-dionato] copper(II) (abbreviated as C 1 4 O-Cu) adlayer physisorbed on HOPG with STM. It was found that STM images changed with bias change of 100 mV or less. Submolecular features of the molecular core and interdigitated alkyl chain were clearly revealed. A preliminary theoretical calculation was carried out to simulate the image variation. The simulated results were in agreement with STM observation.

STM is one of the most promising techniques available for visualizing the fine details of biomolecular structure. It has been used to map the surface topography of inorganic materials in atomic dimensions, and thus has the resolving power not only to determine the conformation of small molecules but to distinguish site-specific features within a molecule. That level of detail is of critical importance in understanding the relationship between form and function in biological systems. The size, shape, and accessibility of molecular structures can be determined much more accurately by STM than by electron microscopy since no staining, shadowing or labeling with heavy metals is required, and there is no exposure to damaging radiation by electrons. Crystallography and most other physical techniques do not give information about individual molecules.We have obtained striking images of DNA and RNA, using calf thymus DNA and two synthetic polynucleotides, poly(dG-me5dC)·poly(dG-me5dC) and poly(rA)·poly(rU).