11409 VALLEY VIEW RD
EDEN PRAIRIE,MN 55344
Non Technical Summary
This Phase II Small Business Innovation Research proposal responds to food safety market needs by developing a high-throughput pathogen detector that will reduce the potential for disease outbreaks and costly food recalls. The device developed in this program addresses a broad sector of food producing markets. This project will focus on detecting live Salmonella organisms in industry-relevant large sample volumes faster, and with significantly higher sensitivity than state-of-the-art methods. The detection method uses DNA and RNA aptamers as biochemical "hooks" between Salmonella and magnetic nanoparticles. The magnetic nanoparticles are then detected by a novel tunneling magnetoresistance (TMR) lab-on-a-chip sensor. The key innovation is a unique microfluidics architecture that localizes Salmonella and bound magnetic nanoparticles to the location on the TMR sensor with the highest sensitivity while dramatically increasing throughput. NVE Corporation has assembled a team of experts from academia and industry for microfluidics design and fabrication, aptamer and magnetic nanoparticle creation, and magnetic TMR sensor production, yielding a highly specific and integrated detection solution. A feasibility prototype was successfully tested in the Phase I program. The goal of the Phase II is the construction of a high-throughput bench-top system with faster, higher-sensitivity detection of Salmonella than otherwise possible.This file MUST be converted to PDF prior to attachment in the electronic application package
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Goals / Objectives
The primary goal of the Phase II program is to develop a prototype benchtop sensor system that accepts a 100ml sample, preconcentrates it, and provides a yes-or-no indication of Salmonella enterica in less than 20minutes. We will accomplish this goal by completing the following technical objectives:Optimize sensor and concentrator surface treatments to minimize chemical adherence, reduce reactions from other matrix elements, and maximize magnetic particle adherence. In phase I, channel clogging problems were anticipated but not fully resolved.Develop a magnetophoretic device that successfully separates unbound magnetic nanoparticles from bound Salmonella-MNP conjugates with at least 99% efficiency. Removing unbound MNP is important to minimize false positives. Other chip-scale separation technologies such as dielectric electrophoresis will be considered if necessary.Develop scalable multichannel electronic signal processing that supports two 16×16 sensor arrays. Phase I prototype measurements were made with laboratory electronics on individual sensors. The Phase II design will be built from discrete components on printed circuit boards to create a standalone test system.Develop a pre-measurement sample preparation protocol for filtration and magnetic concentration using commercially available tools. This protocol will ensure that the sample fluids are sufficiently mixed with functionalized MNPs for effective Salmonella-MNP tagging. Filtration must remove particulates large enough to plug sensor holes without removing Salmonella. The ratio of sample volume and MNPs will also be optimized.MicroPlumbers Microsciences (MPM) and Diagnostic Biosensors (DiagBio). Develop a microfluidic manifold for the fluidic samples with electrical connections to two sensor chips, the magnetophoretic concentrator, and electronics.University of Florida (UF). Scale MNP coating processes for commercial manufacturing. Functionalized MNPs have been produced in small quantities suitable for prototyping. A large-scale process is needed for commercialization.University of Minnesota (UMN). Complete additional SELEX processes to refine the Salmonella enterica aptamer. Additional characterization including imaging number of MNP per bacteria will further enhance sensor efficacy.Demonstrate a compact benchtop detection system with a sample preparation protocol, magnetophoretic separator, integrated next-generation sensor chip, test electronics and software.Evaluate the prototype system for accuracy and lowest detectable limit. The goal of one CFU will be statistically compared to qPCR for several Salmonella enterica serovars.The sensor system will be documented and a datasheet and other collaterals generated for sales and marketing.
METHODS and EFFORTSNVE's approach combines existing magnetic nanoparticle (MNP) and aptamer technologies with a novel magnetophoretic concentrator and spintronic tunneling magnetoresistance (TMR) sensor chips.A sample of poultry or fresh vegetable wash water with live Salmonella enters from the top-left and is mixed with a suspension of functionalized MNPs coated with Salmonella-targeting aptamers. This mixture is then magnetically concentrated using commercially available methods. Waste is drained and the remaining sample fluid is transferred into a magnetophoretic concentrator where large magnetic field gradients further separate Salmonella-MNP conjugates from waste materials including unbound MNPs and nonmagnetic plant and dirt detritus. The remaining sample then flows by TMR sensors on a second chip. A change in the output signal from the TMR sensors indicates live Salmonella.With this approach, a large sample volume can be quickly concentrated and measured by TMR sensors embedded in microfluidic channels. A liter-sized sample of fresh vegetable wash water could be tested for one Salmonella CFU in minutes with an optimized, scaled-up system. We will detail the methods in the individual componentsMagnetic NanoparticlesSeveral varieties of iron oxide MNPs are in use today . Prof. Rinaldi's group at UF used thermal decomposition of metal organic precursors in a high boiling point solvent developed previously to synthesize the particles for the program. This method yields very uniform-sized nanoparticles. Oleic acid-coated nanoparticles were first exchanged with 2000 Da. polyethylene glycol (PEG) silane. The PEG silane coated iron oxide MNPs were functionalized; first with dicarboxylic acid PEG, and then aptamer-designed molecules. The dicarboxylic acid PEG was used as a binding molecule between the particles and the aptamer using the EDC and sulfo-NHS chemistry to covalent bond carboxylic acids with amines groups.AptamersStandard cell-SELEX (systematic evolution of ligands by exponential enrichment) was used by Prof. Sreevatsan's laboratory [24,31] to clone Salmonella aptamers in the Phase I program. Successful aptamer synthesis was verified by gel-PCR with live Salmonella.Aptamers present several advantages over antibodies, four of which are important for our intended applications: (1) Aptamers can readily be synthesized on a large scale using standard automated nucleic acid chemistry, which reduces system cost. (2) Aptamers are stable at room temperature and easily stored. (3) Aptamers can readily be modified to yield selective luminescent probes or beacons. (4) Aptamers detect only live bacteria, an advantage over antibodies since dead bacteria are not food safety threats.Magnetophoretic ConcentratingMagnetophoresis is a method to separate magnetic objects in the laminar flow of an aqueous medium. Our sensor will use magnetophoresis to separate Salmonella-MNP conjugates from unbound MNP and other nonmagnetic waste materials such as excess water, plant and other process materials. A spherical MNP with attached Salmonella will follow a trajectory dictated by the magnetic force and the Stokes drag.Simple equations are used to determine the trajectories of MNP-Salmonella conjugates and unbound MNPs in laminar flow. The relatively large effective radius of Salmonella compared to the free MNPs results in the bound Salmonella drifting slower along the field gradient compared to unbound MNP. Concentration using this approach has been demonstrated by others and NVE has built, tested, and patented similar concentrators in the past. We will design a magnetophoretic separator in the Phase II.Tunneling Magnetoresistance SensorsSpintronic TMR sensors produce a linear response voltage proportional to applied magnetic fields from the MNPs. A TMR sensor is typically composed of multiple sensor elements comprising a Wheatstone bridge. In this configuration, two tunneling magnetoresistors are active and two serve as reference resistors and are inactive. The active sensor elements are located adjacent the through-hole, while the reference elements are placed away from the hole. This allows the sensor to produce temperature compensated, self-referenced differential signal that can be easily amplified for maximum sensitivity.A basic TMR device consists of two ferromagnetic layers separated by an ultrathin insulating spacer material. The resistance of the material stack depends on the quantum mechanical spin tunneling of electrons between the two ferromagnetic layers. More electron tunneling states are available if the ferromagnetic layers are aligned parallel, producing a lower resistance compared to the antiparallel condition. The sensor chip uses an array of TMR sensors to monitor the change in magnetic field created by the flow of Salmonella and magnetic particles.Maximizing sensor sensitivity requires minimizing the distance between the TMR elements and the MNPs. NVE has previously developed a wet potassium hydroxide backside wafer etch to create the microfluidic channel under the sensor with only a 200nm silicon nitride film between the sensor and channel. This geometry, however, did not confine the Salmonella to the vicinity of the SiN film, and the average distance between sensor and Salmonella was greater than 100µm. Despite this limitation, concentrations as low as 106 Salmonella per milliliter, equating to approximately 30 Salmonella + MNP under the sensor detection, area have been detected in previous programs. The design drawback of large sensor-to-MNP distance in previous designs is remedied in this program with a unique through-hole design that passes the Salmonella less than 6µm from the sensor.EVALUATIONIN this program, the evaluation will come though the accomplisment of the milestones of the project.The completion of design activities of an integrated sensor system designates MILESTONE 1.Completion of assembly and test of prototype components occurs at the end of the first year of the program and is the project's MILESTONE 2.Testing the fully assembled Prototype system will be MILESTONE 3.MILESTONE 4 will occur after both unbound magnetic particles and live pathogen testing, when NVE has established sensor sensitivity limits.