Research Tools : Detection


Efficient In Vitro Assay for Antigen-Specific Tolerance

Building on their work, UW–Madison researchers have now developed a T cell-bound cytokine (T-CBC) assay for detecting and quantifying regulatory T cells specific to self-antigens or donor alloantigens. The new method comprises (a) culturing the subject’s T cells for 24 hours in the presence of one or more target antigens and (b) analyzing the cultured T cells for expression of a marker (EBi3; TGFβ/LAP) indicative of antigen-specific immune suppression.

Zip-Lignin™ Assay: An Analysis and Validation Tool

The researchers have now developed the most sensitive assay to date for detecting and quantifying Zip-lignin monomers in plants. They modified an existing lignin assay known as DFRC (Derivatization Followed by Reductive Cleavage) that has been in use for almost a decade. They incorporated several new features to improve the sensitivity of the assay, including extended incubation periods and an additional purification step.

The modified DFRC assay is currently the only known technique capable of determining levels of monolignol ester conjugates in plant lignin.

New Mass Spectrometry Detector Uses Optically Active Membrane

UW–Madison researchers and collaborators have developed a mass spectrometry detector that is more sensitive to large molecule impacts and may provide better spatial sensitivity. The detector incorporates a thin membrane made of semiconducting materials. The membrane is optically active, converting the kinetic energy of the molecules that strike its front surface into light photons. The photons are detected and converted to an electrical signal by a photosensor.

Multiplexed Mass Spectrometry Quantification with Neutron-Encoded Mass Tags

UW–Madison researchers have developed customized tagging reagents for MS proteome quantification. Called neutron encoded mass tagging, or ‘NeuCode,’ the method exploits small mass differences, on the order of millidaltons (mDa), between heavy isotopes, such as nitrogen or carbon, which can be coupled with amino acids used during cell culture similarly to SILAC. Through instrument resolution settings, NeuCode labeling allows the user to control when multiplexed information is masked, limit sample complexity, and when it is revealed, produce quantitative data. Furthermore, these mDa mass differences can be incorporated into novel chemical tags that couple to peptides and enable analysis of biological samples from any source. Unlike isobaric tags, however, specialized reporter groups, linkers and charge sites are not required.

Low-Waste and Contamination Device for Isolating Cells from a Biological Sample

UW–Madison researchers have developed an improved microfluidic device for isolating a desired fraction without the sample loss or contamination associated with prior designs.

The device features multiple wells through which a magnet (positioned perpendicularly to gravity) is drawn, attracting the fraction from one well to the next while undesired material settles to the bottom. For this to occur the biological sample first is combined with a magnetically attractive reagent that binds to the targeted fraction and forms a solid-phase substrate.

The sample is deposited in the first well and then magnetically drawn through a second well containing an isolation buffer like oil or wax. The increasingly purified cells are drawn into a final well for extraction or further treatment. Surface tension prevents fluid from spilling between wells.

Devices and Methods for Immobilizing Liquid Crystal Droplets onto a Chemically Functionalized Substrate Surface

UW–Madison researchers have developed devices and methods for immobilizing micrometer-sized liquid domains such as liquid crystal or isotropic oil droplets on a variety of chemically-functionalized surfaces. A multifunctional polymer, which may be a polyamine, is adsorbed at the surface interface of the liquid crystal droplets. Then the droplets are immobilized by covalent bonding, electrostatic interactions or other interactions between the adsorbed polymer and the functionalized substrate surface. The immobilized droplets can be used, for example, in liquid crystal droplet-based sensing devices or devices engineered to possess optical band gaps.

Parallel Measurements by Fluorescence and Mass Spectrometry for Absolute Quantification of Proteins

UW–Madison researchers have developed an improved method for MS-based quantification using an integrated electrospray emitter and fluorescence detector. Solution-phase measurements are employed to overcome the limitations in standard quantification techniques using measured intrinsic fluorescence to quantify selected amino acids. The inventors have developed a label-free technique that exploits the property of intrinsic fluorescence exhibited by tryptophan-containing peptides and proteins as a means to provide both relative and absolute quantification of proteins and peptides in complex mixtures identified through tandem mass spectrometry.

Integration of the emitter and detector permits quantification on a single stream of analyte while placement of the fluorescence detector immediately before the electrospray emitter provides improved correlation between the measurements and reduction of chromatographic dead volume. A continuous capillary may be used to provide not only the electrospray emitter, but also the liquid chromatography column. This combination allows effective analysis of extremely small amounts of material at nanoliter flow rates and a significant reduction in dead volume, which may cause loss of chromatographic resolution and sensitivity.

Microwave Cavity Detector for Highly Sensitive Mass Spectrometry Detection of Large Molecules

UW-Madison researchers have developed a detector for mass spectrometry that employs a tuned microwave cavity to increase the sensitivity of time measurements. The detector can be used in mass spectrometers that provide a source of ionized molecules, which are accelerated in an electric field before reaching the detector. The design includes a cavity of conductive material providing an electromagnetically tuned cavity, which includes an opening positioned to receive the accelerated molecules. An antenna communicates with the cavity to receive an electrical signal caused by electromagnetic resonance of the cavity. Detection electronics receive this electrical signal, which marks the arrival of the ionized molecule in the cavity and allows for time of flight measurement. With proper configuration of the frequency of resonance in the cavity, its modes and its quality factor, time resolutions on the order of one nanosecond are possible. 

Microtube Scaffold for Sensing and Stimulating Nerve Cell Connections

UW-Madison researchers have developed a method to produce a scaffold system for neurons that permits guided growth or interconnection of neurons and sensing or stimulation of neural activity. The method involves growing nerve cells through doped semiconductor microtubes that act as tunable electrodes for sensing and stimulating nerve cell connections. The tubes allow the growth and interconnection of the neurons to be controlled, and sensors and/or stimulating probes incorporated along the length of the tubes can be used to provide precisely located but spatially separated measurements and stimulation.

The tubes are made of semiconducting thin-film nanomembranes, may vary in length and have diameters ranging from one to 100 microns. Cells are placed near the opening of the tube and preferentially grow through the tube. The microtubes form a coaxial probe around the nerve cell growth, effectively coupling an electrode to the neurons. The tube also acts to protect the neuron from a culture solution that may produce ion leakage, affecting signal propagation and introducing signal noise.

Blue-Green Phytochrome-Based Fluorophores with Strong Fluorescence

UW–Madison researchers have created unique blue-green fluorophores with increased fluorescence.  These fluorescent molecules were created by targeted mutation of particular amino acid residues in the phytochrome domain from wild type cyanobacteria such as Thermosynechococcus elongatus.  They have several advantages over currently used reporters such as GFP or luciferase, including their thermostability and small size.  Additionally, different fluorophores can be used to “fine tune” the excitation/emission to a particular wavelength to meet the needs of a specific system or experiment.

Microfluidic Device for Capturing and Analyzing Rare Cells, Including Circulating Tumor Cells

UW-Madison researchers have developed a microfluidic device for concentrating rare cells.  The velocity of flow through the device is manipulated such that particles in suspension, such as cells, are carried to and deposited in a particular location within the device.  The flow in the region where the cells are deposited is slow, so the cells remain in the collection area without any modifications to the surfaces of the device or cells while the fluid that carried them is routed out of the device.  Because particles in suspension enter the collection region but do not leave, many particles may be captured from a relatively dilute suspension.  The cells then can be cultured, stained and imaged for analysis without being removed from the collection area, creating a gentle and efficient way to implement extensive washing and treatment protocols.

Multiphoton Flow Cytometer for High Throughput Analysis of Multicellular Aggregates Like Pancreatic Islets

UW-Madison researchers have developed a system that combines the high throughput characteristics of flow cytometry with the capabilities of MPLSM. This multiphoton flow cytometry system (MPFC) enables deep, high resolution images of large diameter cells and aggregates. 

The multiphoton laser can excite intrinsic cellular fluorophores such as NADH, FAD and collagen, allowing both spectral and lifetime data to be acquired. This information then can be used to reveal information on cellular processes like metabolism, viability and the functional potential of cells, pancreatic islets, embryoid bodies and other entities.

Robust, Moldable Colloidal Liquid Crystal Gels Provide User-Friendly, Portable Sensors

UW-Madison researchers have developed self-supporting and mechanically robust gels that can be molded, easily handled and processed yet retain their responsiveness to chemical and biological species.  These colloidal liquid crystal gels can be used to produce improved liquid crystal-based devices, including wearable, personalized sensors.

The gels are produced by dispersing liquid crystals into colloids, which spontaneously form a network.  The combination of colloids and liquid crystals yields a visco-elastic gel that has high mechanical strength because of the network of colloids.  The high strength allows the gels to be molded into any desired shape, making the detection of chemicals and biochemicals much easier than previous methods using liquid crystals.

Improved Molecule Mass Detection Using Electron Field Emission from Kinetically Impacted Membranes

A UW-Madison researcher has developed an active detector and method for sensing molecules based on the generation of electrons through field emission (FE) and/or secondary electron emission (SEE).  This device is capable of detecting large molecules at higher temperatures than previous devices.

The detector is made of a semiconductor membrane, such as silicon or silicon nitride, with an “external” and “internal” surface.  The external surface contacts the desired molecules, and the internal surface is made of a thin electron emitting layer.  Kinetic energy from the molecules is absorbed through the external surface to the internal surface via vibrational quanta, which cause electrons to be emitted from the internal surface.  The emitted electrons then can be detected by an electron detector. 

Any material that emits electrons via FE or SEE can be used on the internal surface, including highly doped semiconductors and doped diamond materials.  The electron emitting layer can be electrically biased to enhance FE or SEE.

Simpler Culture Screening for Mycobacterium Avium

UW–Madison researchers have developed novel antigen and antibody preparations and kits that can be used in assays to detect MAC bacteria in liquid cultures.

The system uses capture and detection antibodies obtained from animal subjects immunized with Mycobacterium-secreted antigens. During incubation with the sample, capture antibodies will bind to MAC antigens, allowing others to be washed away. Detection antibodies then are added. They may be directly labeled or used with a conjugate enzyme to trigger a detection signal such as a color change.

Automated System Measures Changes in Liquid Crystal Orientation to Detect and Quantify Biomolecules

UW-Madison researchers now have developed an automated system for precisely determining the in-plane orientation of liquid crystals with high spatial resolution to sensitively and quantitatively detect biomolecules.  This system, which allows variations in the anchoring strength of liquid crystals to be measured easily, consists of a modified liquid crystal cell and an automated image recording and analysis system. 

The liquid crystals can be oriented by a variety of mechanisms.  In one embodiment, a liquid crystal cell is composed of two functionalized thin films of gold, which face each other and are separated by a mylar spacer.  One surface orients the liquid crystal with an in-plane anchoring, which is not parallel to the in-plane anchoring of the opposing surface.

Then a series of images are acquired of the liquid crystal film that contacts the surface to be analyzed.  The images are analyzed to yield maps of the twist angle and thus anchoring energy of the liquid crystal across the surface.  This technique effectively condenses a large data set of images into a compact map to reveal features on the analytic surface that were not apparent in the individual images.

High Throughput Assay for Sugar-Mediated Drug Transport

UW-Madison researchers have developed a systematic platform for rapidly assessing the ability of a diverse range of sugars to enhance the uptake and selectivity of sugar conjugates. This invention may lead to the discovery of sugar molecules that improve the delivery of cancer therapeutics.

First, glycorandomization (see WARF reference numbers P04020US and P04455US) is used to generate a library of molecules that differ only by the sugars attached. Then each of these glycosylated molecules is contacted with cells, and their uptake into the cells is assessed relative to that of a corresponding molecule without the attached sugars. To determine selectivity, these sugar conjugates can be contacted with cells from different cell lines and their uptake compared to see if it is elevated in cells from a particular line.

Using this strategy, the researchers found that slight changes in sugar structure can lead to drastic changes in in vitro cellular uptake. They identified sugars that impart up to an eight-fold increase in selective uptake by tumor, rather than normal, cell lines. They also identified sugars that provide greater than a 10-fold increase in uptake as compared to the conventional sugars glucose, 2-deoxyglucose or FDG.

Varied Monodisperse Oil or Liquid Crystal Emulsion Droplets for Improved Nanoviewing, Sensing and Biosensors

UW-Madison researchers have developed a versatile, scalable and highly parallel method of producing monodisperse emulsion droplets in a range of predetermined sizes.  The oil emulsion droplets, in which the oils can be liquid crystal molecules, can be prepared with or without polymeric shells or capsules.

This method is based on templating polyelectrolyte multilayer (PEM) capsules formed by the layer-by-layer adsorption of polyelectrolytes on sacrificial particles.  A polymeric shell is formed around a sacrificial particle, such as silica.  Then the silica is etched away and the shell is infiltrated with an oil.  The shell then can be removed to reveal monodisperse oil or liquid crystal emulsion droplets of a uniform, predetermined size.  These droplets could be used as biosensors to detect enzymatic activity or target analytes, such as bacteria or viruses, in a sample.

Phytochrome-Based Fluorophores with Strong Fluorescence in the Red/Far-Red Region

UW-Madison researchers have developed phytochrome-based fluorophores with strong fluorescence in the red/far-red region.  These fluorophores provide new molecules that can be used to localize and monitor proteins of interest or detect gene expression in cells.  In addition, because they absorb in the red/far-red region, these proteins can be used as reporters in thick samples, including whole mice and plants.  The fluorophores also can be used in combination with molecules that fluoresce at lower wavelengths to detect multiple proteins at the same time.

Robust Substrates Expand the Utility of Surface Plasmon Resonance Imaging for Analysis of Biomolecular Interactions

UW–Madison researchers have developed robust, SPR-compatible substrates. The key to these substrates is a rugged, chemically versatile carbon thin film overlayer placed on an SPR-active metal thin film.

Specifically, the substrates include a support surface capable of transmitting light, a metallic layer adhered to the support surface and a carbonaceous layer deposited on the metallic layer. The substrates also may include biomolecules attached to the carbonaceous, or carbon-rich, layer. These biomolecules may include oligonucleotides, DNA, RNA, proteins, amino acids, peptides or other small biomolecules that can be configured in one or more arrays.

The new substrates are more robust than conventional gold substrates, allowing assays to be performed under higher temperatures and harsher chemical conditions than currently is possible. Additionally, the carbon thin film overlayer is not susceptible to damage from UV irradiation.

Multidimensional Spectrometer

UW-Madison researchers have developed a spectrometer capable of measuring multidimensional spectra in a straightforward manner. The spectrometer utilizes a pulse shaper and a simple collinear geometry so that alignment is easy and data collection can be automated. Researchers can simply insert their samples and select one of several options for collecting 2D IR spectra. With this invention, along with some engineering and computer programming, the powerful new optical techniques are ready to be commercialized in a user-friendly apparatus that will have broad appeal among researchers.

Using Stromal Collagen to Help Diagnose and Characterize Breast Cancer

UW-Madison researchers have developed an imaging method that may assist in diagnosing cancerous and precancerous conditions in breast tissue. Because breast cancer is frequently associated with the increased deposition of proteins, particularly collagen, in the extracellular matrix, the inventors developed three tumor-associated collagen signatures, or TACS, which provide novel markers for localizing and characterizing breast tumors.

To identify breast carcinomas, nonlinear optical microscopy is used to generate high resolution, 3-D images of a test tissue. The images are then analyzed to detect and characterize any TACS that may exist in the tissue. The degree to which the TACS are present correlates with the onset and progression of cancer, thus providing diagnostic information complementary to conventional diagnostic methods.

Methods of Manufacturing and Using Beta-Peptide Lyotropic Liquid Crystals

UW-Madison researchers have developed liquid crystals based on relatively short beta-peptide scaffolds that can be tailored to have unique properties for use in biological assays. Beta-peptides differ from conventional peptides in that certain beta-peptides form stable helices at short, oligomeric lengths, giving rise to robust, asymmetric structures. These helical beta-peptides can self-assemble to form lyotropic liquid crystals in aqueous environments.

To give the liquid crystals more desirable properties, a variety of functional groups can be added to the beta-peptides. The liquid crystals can then be used, for example, to detect an analyte, such as a protein, in a biological sample.

Nanoscale and Microscale Wireless Stimulating Probes Precisely Deliver Electrical Current to Cells

UW-Madison researchers have developed freely dispersable microscale and nanoscale probes that can be activated without a direct wired connection.  Instead, these probes can be triggered remotely by electromagnetic radiation from a laser or other source.

The probes consist of small tubes of strained semiconductor material that overlaps to form a heterojunction semiconductor device.  The overlap region may comprise n and p type doped regions to form a versatile p-n junction, such as a photodiode, which is capable of absorbing electromagnetic radiation. 

The probes receive electromagnetic radiation, such as light, which they then convert to a local electrical potential and subsequently to an electrical current flow.  The current flow could be used to directly stimulate neural cells.  Alternatively, it could be used to trigger the chemical or mechanical release of compounds held by the probes, or to activate a biological system, such as an ion channel.

Smart Leaf Technology - Floating Semiconductor Membranes for Wireless Sensing

UW-Madison researchers have developed wireless sensors made from nanoscale membranes for use in detecting the presence or absence of analytes, systems incorporating the sensors and methods for using the sensors. The “smart leaf” sensors are made of two thin films with opposing front and back surfaces. The surfaces are coated with molecules that react with the target analyte. Upon exposure to the analyte, the molecules alter the geometry of the leaf and change its dielectric response in a manner that depends on the concentration of the target chemical. An electromagnetic source continually exposes an array of sensors to an electromagnetic signal, while a detector regularly scans the sensor array to observe any change in the reflected and/or transmitted radiation. In this way, the presence or even the concentration of a particular analyte may be easily detected without requiring the sensors to be directly wired to a power supply.

Simple Biological Method and Device for Detecting a Toxin or Other Chemical

UW-Madison researchers have developed a simple biological sensing method that can be used to detect toxins or other chemical compounds. The technology uses the elastic instability of swelling hydrogels to act as a trigger when contacted by a designated stimulus, such as a particular chemical.

Two different types of hydrogels are bonded together by a sensitive material, such as a degradable adhesive material specific to a certain enzyme or chemical. The hydrogels swell at different rates, causing them to bend. Because the adhesive restricts the motion of the hydrogels, an elastic force is generated. When the adhesive material contacts the target chemical, the adhesive degrades, releasing the elastic instability and causing the two hydrogels to separate with an explosive motion that is detectable by the naked eye.

Ionizable Isotopic Labeling Reagents for Relative Quantification by Mass Spectrometry

UW-Madison researchers have developed small, inexpensive ionizable tags for non-targeted relative quantification of a variety of biological metabolites, peptides and proteins by liquid chromatography-mass spectrometry (LC-MS). The tags differ in their isotopic composition and react quantitatively with amines or carboxylic acid groups, offering a powerful approach to relative quantification of multiple analytes between two samples by electrospray ionization-mass spectrometry (ESI-MS).

One sample is labeled with an isotopically-light reagent and the other sample is labeled with an isotopically-heavy reagent, which yields a characteristic mass shift in the spectra. The relative intensity for each peak pair allows for the precise determination of the relative concentration of multiple analytes between samples. In addition, these reagents enhance the ionizability of the analytes in positive-ion mode MS, resulting in lower detection limits.

Liquid Crystalline Substrates for Culturing Cells

UW-Madison researchers have developed a method of culturing cells, including hES cells, on liquid crystalline substrates to provide a new means of visualizing developmental changes that occur in the cells. A thin protein layer, such as Matrigel, is deposited on the surface of a thermotropic liquid crystal. Then cells are seeded onto the Matrigel.

As the cells grow and differentiate, they impart subtle structural changes to the protein gel layer. Because the liquid crystal layer is in direct contact with the protein layer, these changes are amplified and transduced by the liquid crystals, where they may be observed easily.

To induce changes in the cells, a stimulus may be introduced to alter the orientation of the liquid crystals. This imparts changes to the structure of the Matrigel, which in turn alters the cellular environment.

Quantitative Comet Assay for Measuring Viral Growth and Resistance to Anti-Viral Compounds

UW-Madison researchers have developed a sensitive means of measuring viral infectivity and replication activity by monitoring flow-induced viral comet formation. A layer of host cells is contacted with a viral sample and cultured in a thin layer of liquid culture medium. Preferably, the host cells are cultured with the virus particles in a microfluidic channel. The liquid medium flows controllably through the channel to enhance the spread of the viral progeny to uninfected host cells. Infected host cells develop an observable indication of viral gene expression, like cell death. The resulting comet-like infections can be digitally imaged and computer-processed for automated quantification of the spread of viral infection.

Cell-Permeable Green Fluorescent Protein

UW-Madison researchers have developed a GFP variant that does not require an internalization tag to enter a living cell. Negatively charged residues on the surface of the protein are replaced with positively charged amino acids, such as arginine. This endows the GFP with a cationic “patch” that attracts it to negatively charged glycosaminoglycans on the cell surface. After it binds to the cell surface, the engineered GFP can easily permeate the cell membrane.

Devices and Methods for Analyte Detection Using Distorted Liquid Crystals

UW-Madison researchers have developed an alternative means of detecting liquid crystal signals in response to biomolecular interactions. This technique involves determining the anchoring strength of a constrained liquid crystal as a function of the molecules bound to the substrate surface. In the absence of any external force, liquid crystals align along the surfaces of a sample with their optical axis along one direction, defined as the “easy axis.” Anchoring strength can be defined as the amount of force required to cause the liquid crystal to not lie along the easy axis. When a liquid crystal is constrained between two surfaces that have their easy axes in two different directions, the liquid crystal is distorted and strained. In that case, the orientation of the liquid crystal is a compromise between two opposing forces: the anchoring strength and the elastic torque resulting from the strain.

To evaluate the anchoring strength of liquid crystals, the researchers developed a slightly modified version of the optical cells typically used to detect biomolecular interaction using liquid crystals. Previous optical cells included two glass substrates separated by thin spacers on each end. One of the surfaces is a reference surface that the liquid crystal is strongly anchored to. The analyte is placed in or on the other substrate surface. In the modified version, a “wedge” optical cell is created by including a spacer on only one end. The easy axis of the substrate surface and the easy axis of the reference surface are rotated from one another by a known angle. If the analyte is present, the orientation of the liquid crystal deviates from the easy axis of the substrate surface in an analyte-dependent manner. The wedge configuration allows the angle of deviation to be measured, enabling the detection and quantification of the analyte.

Improved Sensor Enables Sensitive Detection of Large Molecules in Mass Spectrometry

UW-Madison researchers have developed a new type of sensor for use in a mass spectrometer. Because this sensor does not require the molecule of interest to generate a secondary electron for detection, it avoids the size limit and is capable of detecting larger molecules with more sensitivity than currently available technology.

This device consists of a thin membrane with nano-electromechanical systems (NEMS) pillar resonators distributed in an array fashion on its inner surface. The membrane is set in motion to cause the pillar resonators to vibrate. The vibrational state of these pillars controls the spatial distribution of the electrons that are emitted. Then the molecule of interest interacts with the receiving surface of the membrane, changing the resonance frequency of at least one pillar and altering the electron emission. To sense the molecule, this change can be detected using a fluorescent film or other method.

Polyelectrolyte Multilayer Films at Liquid-Liquid Interfaces

UW-Madison researchers have developed a method for providing a PEM film at a liquid-liquid interface, such as the interface between a liquid crystal (LC) and an aqueous solution. A PEM can be incorporated between an LC and an aqueous solution by sequentially depositing layers of anionic and cationic polyelectrolytes at the interface. This modifies the interface and effectively functionalizes the LC layer. If the appropriate adducts and excipients are in the PEM, the PEM can transduce signals from the aqueous solution to the LC. For example, receptors incorporated in the PEM can recognize and bind ligands in the aqueous solution, resulting in changes that alter the orientation of the LC. This alteration can then be easily observed to determine if the ligand is present in the solution.

Electrospray Ionization Ion Source with Tunable Charge Reduction

The researchers have improved the physical layout of that device by moving the corona discharge and its associated electromagnetic fields outside the charge reduction chamber, making it easier to collect the ions in the mass spectrometer for analysis. This device configuration also provides independent control over the conditions and processes involved in analyte and reagent ion formation, avoids perturbations in the trajectories of analyte ions and charged droplets caused by operation of the reagent ion source, and allows use of a wide range of reagent ion sources.

Versatile Substrate for SPR Detection

UW-Madison researchers have developed a superior SPR chip surface structure that enhances the imaging signal. The new chip consists of a glass/dielectric material/gold-layered structure. The glass support surface and the dielectric layer are transparent to analyzing light. The gold layer consists of metallic islands on top of the hydrophobic dielectric layer. Probe molecules may be attached to the metallic islands. The dielectric layer surrounding the islands promotes “long-range” SPR and increases the sensitivity of imaging.

Using Liquid Crystals to Detect Post-Translationally Modified Peptides

UW-Madison researchers have developed devices and methods that use liquid crystals to distinguish between post-translationally modified peptides and unmodified peptides. A sample containing a post-translationally modified peptide, an unmodified peptide, or a mixture of both is bound to a substrate surface. The surface then is contacted with a recognition agent, such as an antibody, that specifically binds to or forms a complex with the post-translationally modified protein in the sample. A liquid crystal is contacted with the surface, and its orientation is observed. Disruptions in the uniform anchoring of the liquid crystal indicate the presence of post-translationally modified protein.

Device That Uses Dielectrophoresis to Transport and Position Individual Particles/Cells for Analysis

UW-Madison researchers have developed a novel device that uses dielectrophoresis to gently transport individual particles, such as bacteria or other cells, to a fixed point for electrical analysis. Particles are attracted to an electrode edge through dielectrophoresis. Next, a controllable fluid stream precisely transports individual particles along the electrode edge until they reach the desired position for electrical or other analysis. After analysis, the particles can be released at will.

By moving and focusing individual particles, this technology enables electronic “fingerprinting” of distinct cells, bacteria, nanoparticles or other particles.  Every particle within a mixture can be analyzed and specific particles can be detected rapidly, making this inexpensive device potentially useful for quality control in nanomanufacturing, bioterrorism monitoring or monitoring of air and water pollution.

Protein-Acrylamide Copolymer Hydrogels for Measuring Protein Concentration and Activity

UW-Madison researchers have developed a method of specifically attaching proteins to glass surfaces via copolymerization with a polyacrylamide hydrogel. They also have developed techniques for using the arrays formed using this process to detect proteins and measure their concentrations, binding affinities and kinetics.

Their method uses a surface functionalized with an acrylic acid- or acrylamide-based hydrogel. Proteins are labeled with an acrylic moiety and then attached to the functionalized surface through copolymerization with the hydrogel. The attached protein is then available for use in a variety of assays.

Deposition of Samples and Sample Matrix for Enhanced Sensitivity of MALDI Mass Spectrometry

UW-Madison researchers have developed a method of using an ultrasonically actuated microplotter to deposit both the sample and the overlying matrix, resulting in a MALDI target composed of small, homogeneous sample spots. See WARF reference number P01201US for more information about the microplotter itself. This technique results in smaller sample spots than can be obtained with current methods, leading to better mass spectrometry readings. Researchers in both university and industrial settings could use this method for high throughput, high-sensitivity MALDI analyses.

Method and Compositions for Detecting Botulinum Neurotoxin

UW-Madison researchers have developed a fluorescence resonance energy transfer (FRET) method for the sensitive detection of botulinum neurotoxin. The assay uses two fluorescent proteins, such as cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), which are linked together by a molecule that can be recognized and cleaved by botulinum neurotoxin. The emission spectrum of CFP partially overlaps with that of YFP. As a result, when CFP and YFP are very close together, excitation of CFP results in FRET-YFP emission and partial quenching of CFP emission. When botulinum neurotoxin cleaves the linker molecule separating these two fluorescent proteins, FRET is eliminated, i.e., excitation of CFP no longer results in YFP emission and partial quenching of CFP emission.

To detect botulinum neurotoxin, a sample is exposed to the CFP and YFP construct. The FRET signals are measured and compared before and after exposure, with a decrease in FRET after exposure indicating the toxin’s presence. This method is useful for detecting botulinum toxin both in vitro and in living cells.

Fluorescent Assays with Improved Sensitivity

UW-Madison researchers have developed a method of “locking” the fluorescence of a molecule such as rhodamine by linking its two amino groups to a “trimethyl lock.” The lock can then be released through a user-designated interaction with a trigger.

In the trimethyl lock, strain from the steric interaction of three methyl groups forces the formation of a cyclic ester. The fluorescence of the locked rhodamine amide is negligible. But when this compound encounters a trigger molecule, such as an esterase or other enzyme, the bonds of the lock are cleaved to yield rhodamine, increasing the fluorescence 1000-fold. Optionally, the locked fluorescent compound can be conjugated to one or more additional molecules of interest. This invention allows the user to monitor enzymatic reactions and observe the precise movements of molecules.

Use of Liquid Crystals and Affinity Microcontact Printing to Detect Chemicals and Biomolecules

UW-Madison researchers have developed methods for using affinity microcontact printing and liquid crystals to simply and easily detect a ligand or receptor. Affinity microcontact printing captures a specific ligand from a sample and “stamps” the ligand onto a detection surface so that the ligand’s presence can be visualized with liquid crystals.

First, an “affinity stamp” is created by covalently linking a capture protein, which binds the target ligand, to a polydimethylsiloxane (PDMS) base. The stamp is then placed into contact with a sample, and if the target ligand is present in the sample, the stamp will bind it. Next, the stamp is contacted with a detection substrate to transfer the target ligand, if present, from the stamp to the substrate. Liquid crystals are added to the top of the detection substrate, and a functionalized glass slide is placed on top of them. The ligand is present in the sample if the liquid crystal display appears disordered or disrupted.

Liquid Crystals with Reduced Toxicity Toward Living Cells

UW-Madison researchers have now developed liquid crystal compositions that exhibit little or no toxicity towards cells. The compositions include at least two different liquid crystal compounds with chemical functional groups such as fluorine atoms, fluorophenyl groups or difluorophenyl groups. The liquid crystals may be added directly to cell culture media or to components of culture media to enable their use in counting cells.

Inductive Detection for Mass Spectrometry

UW-Madison researchers have developed an enhanced electrospray ionization, time-of-flight mass spectrometer for the analysis of large or complex biomolecules. This device combines a single droplet ion source with multiple, “on-axis”, non-destructive, inductive ion detectors. The implementation of on-axis ion trajectories throughout the mass analyzer, along with multiple detectors, allows pre-acceleration ion velocities to be determined. A second set of ion detectors detects post-acceleration velocities, providing time-of-flight information while reducing ion detection losses of larger, slower-moving biomolecules. The detectors can be configured to simultaneously analyze charge states and mass-to-charge ratios, enabling the determination of absolute mass.

Label-Free, Radio-Frequency Detection of DNA Hybridization at Microelectrodes

UW-Madison researchers have developed a new device and method that use radio-frequency (RF) excitation to directly detect DNA hybridization at microelectrodes. This invention exploits the electrochemical double-layer that forms at solid-liquid interfaces, and, in particular, this layer’s non-linear dielectric properties, which change in response to DNA hybridization events.

The method employs a novel homodyne reflectometer that includes a counter electrode and a working electrode, with the surface of the working electrode functionalized to carry single-stranded DNA molecules. When a low-frequency RF modulation signal is applied to the counter electrode and a high-frequency carrier signal is applied to the working electrode, these signals are mixed by the non-linear behavior of the double-layer at the interface between the working electrode and the solution of attached DNA. Mixing, in turn, generates a new signal that exhibits significant changes in amplitude when DNA hybridizes at the surface.

The dielectric properties of solid-liquid interfaces are known to change in response to biological binding events. What sets this invention apart is that it measures the non-linear response of the double-layer, allowing isolation of the active sensing region from background changes in dielectric properties unrelated to DNA hybridization.

Microwave Dielectric Spectroscopy Method and Device

UW-Madison researchers have developed a method of using dielectric spectroscopy to detect protein conformational changes. This alternative method relies on the fact that proteins in solution are surrounded by one or more shells of “bound” water. In response to changes in a protein’s conformation, bound water is released or rearranged, causing a change in the solution’s permittivity that can be easily measured by using this invention.

Previous dielectric spectroscopy methods have not been widely implemented because they involve complicated analysis. In this invention, data analysis is as simple as in conventional optical spectroscopy. In addition, no labeling of receptor or ligand is required for detection.

Using Liquid Crystals to Detect Interactions at Biomimetic Interfaces

UW-Madison researchers have developed devices and methods for detecting interactions between proteins at interfaces between liquid crystals and aqueous solutions. They discovered it is possible to form biomimetic membranes from phospholipids that spontaneously assemble at the liquid crystal-aqueous interface. These membranes then can be used to detect protein binding and enzymatic activity at the interface.

An aqueous solution containing a surfactant and a receptor molecule, such as a phospholipid, is contacted with the top surface of a liquid crystal. The liquid crystal is in a well or other holding compartment on a substrate like a glass slide, and the aqueous solution may be passed over the surface of the liquid crystal in a flowing stream. The receptor molecule is adsorbed on the top surface of the liquid crystal, forming an interface between the crystal and the solution. If a target compound is present in the solution, it binds to the liquid crystal or the receptor molecule, causing an easily observable change in the orientation of the liquid crystal. Alternatively, a change in the orientation of the liquid crystal may indicate that a reaction of interest, such as enzymatic cleavage or hydrolysis, has occurred.

Optical Imaging of Nanostructured Substrates

UW-Madison researchers have developed devices and methods for imaging phenomena that occur on a nanostructured substrate surface and detecting the presence of a target analyte. Their invention starts with a substrate, such as a molded polymer, that has nanoscale grooves or other topography on its surface. The polymer substrate may include a support, such as a glass slide or plate, and also may be coated with a thin layer of a metal, such as gold or silver. The substrate may include a blocking layer, such as bovine serum albumin, to prevent non-specific adsorption, and may contain surface-bound receptor molecules for the target species.

To image phenomena that occur on the surface, a fluid is placed on top of the substrate, and the surface is observed with polarizing light. To detect an analyte of interest, the substrate is viewed with polarizing light, contacted with a sample and then imaged again. The target analyte is present if the surface appears different after the sample is added.

Fusion Protein Arrays on Metal Substrates for Surface Plasmon Resonance Imaging

UW-Madison researchers have developed surface plasmon resonance-capable arrays in which molecules, such as proteins and nucleic acids, or whole cells, are adhered to a metal substrate. Proteins are immobilized on a chemically modified gold surface in an array format by exploiting an interaction between the enzyme glutathione S-transferase (GST) and the tri-peptide glutathione (GSH). First, a GSH array is created on a chemically modified gold surface. Next, GSH-GST binding is used to immobilize GST fusion proteins onto the gold surface. The GST portion of the fusion protein acts as an anchor to the surface, while the remainder of the protein is accessible for interaction studies with peptides, nucleotides, small molecules or other proteins in solution.

Microfabricated Microbial Growth Assay Method and Apparatus

UW-Madison researchers have developed a microbial growth assay that can be used to rapidly screen substances for their effect on cell growth. The invention includes microbial growth assay wells containing 30 microliters or less of liquid. Two electrodes are placed in each well to measure the capacitance across the liquid. Capacitance or resistance in each well is used to determine the extent of bacterial growth.

This technology represents a significant improvement over commercially available growth assays, most of which rely on flow-through chambers, large sample sizes and long incubation periods. It should provide an economical way to screen potential antibiotics on a massive scale.

Microfluidic Actuation Method and Device

UW-Madison researchers have developed a device and method to achieve pumping and mixing of fluid in lithographically manufactured microfluidic channels. The device starts with microstructures within microcavities, which consist of cantilever elements coupled to a substrate that receives vibrations. An ultrasonic vibrator causes the substrate to vibrate. The motion of fluid within the microcavities can be controlled by selecting the shape of the cantilever elements, their position in the microcavity, the spacing of the cantilever elements in relation to the cavity walls, and the frequency of the vibrations. The vibrations can provide acoustic streaming to pump fluid through the cavity. Alternatively, the vibrations can induce small vortices that trap the fluid and mix the particles within it. Vibration may also cause the particles to concentrate in the vortices, thereby creating a local area for photo-detection of antigen presence. 

Rubbed Substrates for Liquid Crystals

UW-Madison researchers have developed rubbed substrates for liquid crystals. These substrates are easy to prepare. They uniformly align liquid crystals and resist non-specific adsorption of proteins and other contaminants.

The substrates include a blocking compound, such as bovine serum albumin (BSA), which is immobilized on one side of a support to form a biochemical blocking layer. A bifunctional spacer and a surface modifying compound may be used to bind the blocking compound to the surface of the support. An immunoglobulin, peptide, nucleic acid sequence or other agent that is capable of specifically recognizing the target species is also immobilized on the side of the support with the blocking layer. The surface on that side is then mechanically rubbed, so liquid crystals become uniformly anchored when contacted with the substrate.

To detect the presence of a target species, the substrate is incubated with the sample to be tested. Then the liquid crystal is added. Disruptions in the uniform anchoring of the liquid crystal indicate the presence of the target.

New Spectrometer Tool for Studying Biomolecules and Very Thin Films

UW-Madison researchers have developed an instrument for the non-destructive study of surface and interface phenomena, as well as the properties of very thin layers. The instrument employs a near-infrared Fourier transform infrared spectrometer (FTIR) to collect reflectance spectra from a prism-gold film-thin film assembly at a well-defined angle of incidence. The measured reflectance spectra exhibit a pronounced minimum in the IR range from 700-2000 nm due to the effect of the SPR; the position of the minimum can be varied by changing the angle of incidence and the gold film thickness. A shift in wavelength of this minimum position occurs upon the adsorption of molecules onto the thin film surface, due to the change of index of refraction at the interface. The FTIR spectrometer provides high dynamic range and the wavelength stability and reproducibility required to make sensitive SPR wavelength shift measurements and broadens the spectral range over which the SPR measurements can be performed. The FT-SPR technique can be utilized for measurements ranging from sub-monolayer films to films with a thickness of about 200 nm.

Quantitative Characterization of Obliquely-Deposited Gold Substrates in Liquid Crystal Devices

A UW-Madison researcher has developed quantitative methods for analyzing the anisotropy of obliquely deposited metal films. The methods use scanning probe microscopic techniques, such as scanning tunneling and atomic force microscopy. They can be used to characterize the metal films and predict the orientation of liquid crystals supported on the films. They also can be used to design surfaces that differ systematically in their anisotropy, thereby allowing analytes to be detected at different threshold concentrations.

Influenza Reporter Virus Imaged In Vivo

A UW–Madison researcher has engineered an influenza virus carrying an exceptionally small and bright NanoLuc (NLuc) bioluminescent reporter, allowing it to be imaged during infection. Unlike prior attempts, the new reporter virus replicates almost exactly like the natural virus.

It was created by fusing NLuc to the virus’ PA gene. PA and NLuc are separated by a self-cleaving peptide, ensuring that they don’t interfere with each other and are expressed as separate polypeptides.

New Influenza B Virus Enables Real-Time Tracking of Disease Progression

The UW–Madison researcher extended his earlier technology and developed a novel, fully replication competent influenza B reporter virus. It is useful for fast and quantitative measures of viral replication in culture as well as tracking disease progression in infected animals. Importantly, while the present technology is directed at influenza B, the approach is adaptable to any strain of influenza A or B.

The virus stably maintains the NLuc reporter gene, replicates with near-wild type kinetics in culture and is suitable for in vivo imaging of infected mice. Unlike currently available influenza B models, it is high throughput, does not use laborious culture-based assays to detect viral dissemination and load and does not require sacrificing animals.

Bioluminescent Influenza Virions for High Throughput Screening

A UW–Madison researcher has developed a highly sensitive influenza reporter virus suitable for high throughput assays and in vivo imaging. Moreover, this version of the reporter virus produces bioluminescent virions that can be used to quantitate virion production and virion binding to antibodies, target cells or synthetic receptors. These bioluminescent virions are also ideally suited to rapidly test for the presence of neutralizing antibodies resulting from natural infection or vaccination.

The virus was engineered to express the reporter gene (NanoLuc) and to package copies of the NanoLuc protein into virions, making them bioluminescent. This approach is portable and has been applied to multiple influenza A and B virus strains.