The Walt lab uses optical microfiber arrays to approach a range of research questions.
Single Molecule Enzyme Detection
Single molecule measurements provide unique information about heterogeneous molecular behaviors that are hidden using bulk methods. We separated and enclosed single β-galactosidase molecules with the fluorogenic substrate resorufin- β-galactopyranoside in an array of 50,000 femtoliter-sized chambers on the distal end of an etched optical fiber bundle. According to Poisson statistics an appropriate low enzyme concentration (1 enzyme molecule in 20 microchambers) ensures that the microchambers contain a maximum of only a single enzyme molecule while the rest are empty (Rissin DM, Walt DR, 2006, Nano Lett. ). Trapping single enzyme molecules resolves any problems inherent to surface attachment of the enzyme. The catalytic activity of each enzyme molecule, which resulted in the production of fluorescent resorufin, was detected individually by epifluorescence microscopy (Fig 1). This new single molecule detection method is now pursued commercially by Quanterix, a company that has licensed the technology from Tufts.
Figure 1. Illustration of single enzyme molecule substrate turnover in a fiber bundle array. A schematic section of a 2 mm diameter glass optical fiber bundle containing 50,000 fibers in total is shown in the upper panel. 46-femtoliter microchambers were etched homogenously into the distal end (d) of the fibers. The fiber core is shown in white and the cladding in gray. The fiber bundle was mounted on a custom-built upright epifluorescence microscope and the reaction progress was monitored through the proximal side (p) of the fiber bundle after the microchambers had been sealed by a silicone gasket (gasket not shown). The single enzyme molecules in the microchambers convert a non-fluorescent substrate to fluorescent resorufin (yellow chambers) (Figure from Gorris HH, Rissin DM, Walt DR 2007, Proc Natl Acad Sci USA ).
Activity Distribution of Single Enzyme Molecules
With our novel detection platform for single enzyme molecules we showed that individual β-galactosidase molecules exhibit a distinct catalytic activity which is broadly distributed (“static heterogeneity”) ( Li Z, Hayman RB, Walt DR. 2008 ). In each experiment hundreds of individual enzyme molecules in the fiber bundle array were observed simultaneously – enough data for a thorough statistical analysis of single enzyme molecule kinetics (Fig 2).
Figure 2. Figure 2: Single molecule substrate turnover distribution histograms at substrate concentrations of 25 μM (blue), 50 μM (red), and 150 μM (green). Left: normalized frequency of substrate turnovers (s-1); right: normalized frequency of normalized substrate turnovers (Figure from Li Z, Hayman RB, Walt DR. 2008 ).
The frequency distributions of the normalized velocity of an individual enzyme molecule (vi/<v>) superimpose at substrate concentrations of 25, 50, and 150 µM and have the same coefficient of variation (30 ± 1 %) (Figure 2, right). Therefore, the velocity distribution can be assumed to be a universal function of [S], and - as vi is directly proportional to kcat but not to KM - the single-molecule Michaelis-Menten equation
indicates that variability in kcat but not KM is the source for the wide distribution of vi.
Single Molecule Enzyme Inhibition
When we added the slow-binding inhibitor D-galactal to the enzyme/substrate solution in the microchamber array (Fig 1), we were able to observe for the first enzyme inhibition kinetics at the single molecule level (Gorris HH, Rissin DM, Walt DR. 2007 ). Inhibited and active states of β-galactosidase could be clearly distinguished and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics (Fig 3).
Figure 3. Inhibitor release from single enzyme molecules in a pre-steady-state experiment. β-galactosidase was first saturated with the inhibitor D-galactal. The inhibitor was then 1000-fold diluted such that the inhibitor concentration was too low to rebind to the catalytic sites of the enzyme in a significant amount. The diluted solution was quickly enclosed in the array and sequential fluorescence images of single enzyme substrate turnovers were taken every 30 s. (A) A sequence of these images recorded after closing the chambers (left panel), 1020 s (middle panel), and 1920 s (right panel) shows a delayed onset of substrate turnover that we attribute to stochastic inhibitor release events. (B) Trajectories of fluorescence increase in the indicated microchambers. An empty chamber shows a constant background (red curve). (C) Distribution of off-times with 2 min binning time. (D) The semi-logarithmic plot illustrates a first-order release of inhibitor (Figure from Gorris HH, Rissin DM, Walt DR 2007, Proc Natl Acad Sci USA ).
Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme’s catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk reaction kinetics.
Single Molecule Kinetics of HRP
In contrast to β-galactosidase, horseradish peroxidase (HRP) exhibits product formation rates that are on average 10 times lower at the single molecule level in the femtoliter array than in bulk solution. The redox-reaction mechanism of HRP involves two separate steps of product formation (Fig 4). HRP first oxidizes Amplex Red to non-fluorescent radical intermediates, which subsequently undergo an enzyme independent dismutation reaction to form fluorescent resorufin. If HRP is confined in a femtoliter chamber the dismutation reaction decreases such that less product is formed. This two-step oxidation mechanism of the widely used Amplex Red and other fluorogenic substrates is often overlooked. It is important for single-molecule studies with HRP as well as bulk reactions at low substrate turnover rates.
Figure 4. Figure 4: HRP (purple) catalyzes a one-electron oxidation (red) of non-fluorescent Amplex Red to the non-fluorescent Amplex Red radical. The formation of fluorescent resorufin (yellow) from two Amplex Red radicals is an enzyme independent dismutation reaction. The overall reaction stoichiometry between Amplex Red and H2O2 (green) is 1 : 1 only if all Amplex Red radicals are converted to resorufin (Gorris HH, Walt DR. 2009. J Am Chem Assoc. ).
Single Molecule DNA Detection
In an alternative approach, the array surface was modified with biotin which can efficiently capture single molecules of streptavidin-labeled β-galactosidase (Rissin, DM, Walt DR. J Am Chem Soc. 2006 ). Experiments are under way to use the streptavidin-labeled β-galactosidase as a reporter for the detection of single DNA molecules (Fig 5). After the feasibility of this novel detection method for single DNA molecules has been clearly shown, a range of potential applications opens up for the future.
Figure 5. Illustration of single DNA molecule detection in the fiber array. Single-stranded DNA molecules were first immobilized on the microchamber surface of an etched glass fiber bundle. Upon hybridization with a biotin-labeled complementary DNA-strand and ensuing binding of streptavidin-labeled β-galactosidase, single molecules of DNA were detectable by the enzyme’s substrate turnover.
Nucleic acid hybridization is a fundamental method of molecular biology and plays a central role in diagnostics. We have developed fiber-optic DNA microarrays using etched optical fiber bundles filled with oligonucleotide-functionalized microspheres. Employing fiber bundles comprised of thousands of individually addressable fibers enables massive parallel detection capabilities. This high-density array was fabricated to measure the hybridization of fluorescently labeled targets. Specific hybridization is detected by fluorescence only at the probe positions complementary to the targets.
Harmful Algal Blooms
Harmful algae blooms (HAB) are a serious threat to coastal resources, causing seafood contamination with potent toxins. HAB are often fatal to fish and marine animals. Pre-existing HAB monitoring systems have focused on plankton analysis, but the diversity of planktonic species poses a problem in discriminating toxic species from non-toxic with conventional detection methods. In collaboration with the Woods Hole Oceanographic Institute (WHOI), we developed a sensitive sandwich hybridization assay that combines simple fiber optic microarrays with specific molecular probes to detect Alexandrium fundyense cells. Microarrays were prepared by loading probe-coupled microspheres onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from lysed A. fundyense to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type format.
Figure 6. Illustration of sandwich assay format used for the detection of harmful algal bloom.
We applied the sandwich hybridization for detection of A. fundyense cells in both cultured and field samples. In both cases, as few as 5 cells could be detected within 45 min without any separate amplification step. Our work with A. fundyense cells suggests that fiber-optic microarrays can provide rapid and sensitive HAB detection. In addition, the simplicity of the microarray format offers an opportunity to apply this technology in direct shipboard detection of HAB. Ongoing efforts with WHOI are focused on expanding the number of species from the Gulf of Maine and around Cape Cod.
Pathogens in Food and Water
Escherichia coli O157:H7 and Salmonella spp. are important food pathogens in public health, causing severe illnesses associated with contamination of meat, poultry or dairy foods. Rapid and sensitive detection of these food pathogens is required for food safety surveillance.
We developed a multiplexed DNA microarray with oligonucleotide probes specific for virulence genes (hly A and eae A for E. coli O157:H7, inv A and agf B for Salmonella spp.), which enables simultaneous detection of E. coli O157:H7 and Salmonella spp. Microarrays were prepared by randomly distributing DNA probe-functionalized microspheres (3.1 um diameter) into wells etched at the distal end of 500 micron diameter imaging fiber bundles. Target DNA was amplified from whole cells via single or multiplexed polymerase chain reaction (PCR), and PCR products were used for hybridization without further purification. Hybridization of PCR products to immobilized probes on the microspheres was visualized using Cy3-labeled reverse primers.
Our results show that this microarray format is more sensitive than gel electrophoresis, and that as few as ca. 1 CFU/mL is detected within 30 min for both E. coli O157:H7 and Salmonella spp., with 35 PCR cycles. The fiber optic DNA microarray provides high specificity and avoids false positive/negative signals that are common in other conventional detection methods. Since fiber optic microarrays can be prepared with different types of probe microspheres, this approach shows promise for the detection of multiple food pathogens.
Our research seeks to provide point-of-care clinical diagnostic systems useable by minimally trained personnel for the diagnosis of disease states using saliva as a sample specimen. The final analytical system will integrate microfluidics technology with multiplexed fluorescent arrays in a portable, handheld device.
Figure 7. A diagram of the salivary glands.
Since September 11 and the subsequent Anthrax attacks in 2001, the threat posed by bioterrorism has become real and continues to grow. The need for accurate identification of threatening BWAs is now the focal point for countering bioterrorism. Timely and effective detection of released BWAs is crucial for an appropriate response to mitigate casualties caused by a bioterrorism attack.
We have developed a multiplexed, high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents (BWAs) including Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and Bacillus thuringiensis kurstaki (a BWA stimulant) in our laboratory. 50-mer single-stranded DNA sequences specific to the target BWAs were attached to 3.1-µm microspheres to make 18 different DNA probes. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. Multiplex PCR was applied in conjunction with array hybridization, and the simultaneous detection of these BWAs in real environmental samples has been demonstrated with high sensitivity and specificity.
Genotyping & Molecular Barcoding
Detection and discrimination of pathogenic microorganisms are important for effective treatment and prevention of widespread infection. Bacterial typing is particularly challenging because it is difficult to discriminate between normal and pathogenic serotypes of the same microorganisms.
Our lab has demonstrated the use of a fiber-optic array with cross reactive multilocus sequence typing response to characterize large numbers of closely related strains of E. coli. We are currently using a similar approach to develop an array that will enable the simultaneous serotyping of numerous pathogens based on the cross reactive array approach. To achieve greater numbers of organisms we are using an Illumina BeadStation, a commercial instrument based on the bead-in-fiber technology developed in our lab. This instrument will enable us to use up to 1500 different bead types and evaluate 96 samples simultaneously.
The goal of this project is to design a reversible, fluorescent DNA probe that can be used to determine the dynamic concentration changes of single stranded DNA in solution. The probe consists of a single stranded oligonucleotide that adopts a stem-loop conformation in its non-hybridized state. The stem length and the length of the loop region that is complementary to the target were chosen to allow for reversible binding. A fluorescent indicator is attached to each end of the single strand probe to be used in a ratiometric measurement. The excitation and the emission wavelengths of the two labels, Cy3 and Cy5, allow for fluorescence resonance energy transfer (FRET) in the closed state. Upon hybridization to its complementary target, the stem-loop structure opens up, resulting in a fluorescence intensity increase of the Cy3. The ratio of the Cy5 to Cy3 fluorescence intensities, which is independent of the amount of probe, is a measure of the free target concentration.
Protein assays provide access to biologically relevant information, including qualitative and quantitative information about specific proteins in a given sample matrix. Highly target specific monoclonal antibodies were immobilized on microspheres to create an individually addressed fiber optic array capable of single microsphere interrogation for the simultaneous measurement of multiple proteins. A microsphere based sandwich immunoassay was developed to simultaneously detect two innate immune system proteins found in human saliva. Preliminary results suggest that the degree of multiplexing on our fiber optic platform can be amplified, as the cross-reactivity between detection antibodies and their non-specific targets is relatively low in comparison to the signal generated by the specific binding of detection antibodies with their target.
Figure 8. Duplexed Europium encoding image (left; 600nm emission) and signal image from tagged detection antibody (right; 570nm). Dimmer encoded beads are probes to the target, while brighter encoded beads are probes to a target not present in the sample.
Figure 9. Detection of various concentrations of lactoferrin target protein using a duplexed array. Specific capture to lactoferrin probes shown in red and non-specific capture to immunoglobulin probes shown in blue.
The goal of this project is to develop techniques to encode and transmit information using chemical reactions. This project is part of the larger field of Infochemistry, where information technology and chemistry combine. We are working in collaboration with George Whitesides’ group at Harvard University on this project, which is funded by DARPA. We have developed a detection system capable of receiving the signal from a chemical source hundreds of meters away. The source is known as the infofuse.1 The infofuse uses heat from combustion to thermally excite atomic emission from metal salts. Messages are encoded by spotting different combinations of metal salts. We are continuing to develop new methods for encoding complex signals and for enhancing the ability to detect the signals over longer distances.
Figure 10. (Left) The infofuse works by thermally exciting atomic emission from patterned metal salts. (Image: Rogers, J. 2009. Proc Natl Acad Sci: 106 9123-9127 ); (Right) Detection system constructed to measure the signal from the infofuse.
The overall objective of this work is to create optical/electrochemical hybrid sensor arrays. This multidisciplinary project involves electrical engineering, optics, electrochemistry, and analytical chemistry. We performed a multiplexed electrogenerated chemiluminescence (ECL) sandwich immunoassay. This ECL assay used electrodes that consist of a common metal electrode patterned with an array of wells that hold sensing beads. In addition to the electrode used for the ECL assay, we also created microelectrode arrays with individually addressable electrodes. Some of these microelectrode arrays have on-chip electronics that simulate analytical equipment such as a potentiostat. These arrays show promise for ultrasensitive electrochemical analysis.