GET THE APP

Preliminary Development of a Magnetically Assisted Test Strip (MATS) Cartridge and Fluorescent DNA Aptamer-Magnetic Bead Quantum Dot Sandwich Assays for Multiplexed Food Safety Applications
Journal of Biomedical Engineering and Medical Devices

Journal of Biomedical Engineering and Medical Devices
Open Access

ISSN: 2475-7586

Research Article - (2016) Volume 1, Issue 1

Preliminary Development of a Magnetically Assisted Test Strip (MATS) Cartridge and Fluorescent DNA Aptamer-Magnetic Bead Quantum Dot Sandwich Assays for Multiplexed Food Safety Applications

John G Bruno* and Taylor Phillips
Pronucleotein Biotechnologies, Inc., San Antonio, USA
*Corresponding Author: John G Bruno, Pronucleotein Biotechnologies, Inc 4100 NW Loop 410, Suite 230, San Antonio TX 78229, USA Email:

Abstract

Preliminary development of a simple mesofluidic multi-channel plastic cartridge with underlying external magnet to drag DNA aptamer-coated paramagnetic beads through fluids in the channels while developing a sandwich assay with quantum dot-conjugated reporter aptamers is described. This approach is superior to traditional lateral flow test strips in several ways including: 1) the ability to control the speed of lateral flow in the channel versus conventional nitrocellulose analytical membranes with fixed wicking times. 2) The use of aptamers for potentially greater affinity and consistency from batch-to-batch versus comparable antibodies. 3) Superior fluorescence efficiency and intensity provided by quantum dots versus conventional fluorescent dyes and 4) the ability to multiplex based on the various colored emissions of different sized quantum dots when excited with a single ultraviolet source. Development of the system from concept to prototype is described along with illustration of sensitive system performance for several food safety-related targets. The system is also clearly adaptable to rapid multiplex detection and sensitive quantitation of clinical biomarkers, drugs, environmental, veterinary or other target analytes.

Keywords: Aptamer; Fluorescence; Magnetic bead; Mesofluidic; Multiplex; Quantum dot; SELEX.

Introduction

Lateral flow (LF) or immunochromatographic (IC) test strips have long dominated the rapid detection markets in the areas of food safety, clinical (especially pregnancy), drug and other testing for their convenience, speed, portability, minimal training requirements, and low cost which is especially important in resource-limited areas or underdeveloped countries [1]. However, LF or IC assays have several drawbacks including a general lack of sensitivity compared to fluorescence or chemiluminescence methods which stems in part from the use of antibodies and in part from the use of visual assessment of colloidal gold or colored latex particle test lines [1]. While the use of fluorophores or even quantum dots (QDs) with LF or IC test strips has proven to increase sensitivity at least ten-fold [2-6], the use of antibodies still contributes some lack of consistency in performance due to the little acknowledged, but quite real, variability in antibody performance form batch-to-batch [7,8]. DNA aptamers developed by the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process may solve the potential antibody problems because they are proven to have higher affinity than comparable antibodies in some cases [9,10] even leading to low zeptomole level detection [11]. And once an aptamer’s DNA sequence is known it can be reproduced with extremely high fidelity by chemical synthesis from batch-to-batch [9], thus leading to very high assay consistency.

Therefore, our group and many others have been investigating application of aptamers for food safety assays [4,12-29], because food safety testing is an area of “zero tolerance,” i.e., no detectable foodborne pathogens are allowed in tested foods or enrichment broth cultures derived from tested foods. Thus, ultrasensitivity is desired in food safety assays to reduce culture enrichment times enabling faster clearance and sale of refrigerated or frozen food products which saves money for food producers and increases profitability. Our group has demonstrated that aptamers it has developed against Campylobacter jejuni and Listeria monocytogenes can be used in QD or enzymelinked fluorescence aptamer-paramagnetic bead (MB) sandwich assays to achieve detection limits below 10 colony forming units (cfu) or cells per ml [13,15]. Our aptamers and ultralow detection limits even in various foods or culture enrichment broths have been validated by other researchers [11, 16-18] and independent laboratories [13] and led to a recent second place finish among 49 competitors in the U.S. FDA’s inaugural Food Safety Testing Technology Challenge [30-32]. While, our aptamers have proven useful for improving detection limits in traditional LF test strip formats with QDs [4] or tube-based MB fluorescent sandwich assays [13,15], they have thus far only been applied to single target tests. In the present work, we describe our first attempts to develop a multiplexed cartridge in which various aptamer- MB plus aptamer-QD sandwich assays are added to multiple linear mesofluidic channels in a plastic cartridge or tray for simultaneous multiplex testing. These assays are assisted by an external magnet to traverse the channels while the sandwich assays develop and are separated from and washed free of excess aptamer-QD conjugates and any potential food particles or other debris to generate a purified fluorescent assay on MBs for visual or fluorometric detection with a single UV excitation source [33].

More complex external and internal magnetic pumping systems [34-36] including the rotating magnetic wheel pump developed at the Naval Research Laboratories [37] are ingenious and highly useful, but we sought the simplest possible and least expensive system which would still enable the concentrating and purifying advantages of MBs in a small portable package for users in resource-limited environments. Thus, we focused on development of a Magnetically Assisted Test Strip (MATS) cartridge with a simple manually operated or automated external magnet to pull aptamer-coated MBs through fluid or membrane-filled mesofluidic or microfluidic channels as described herein and elsewhere [38]. Only linear movement of the external magnet is required to move the capture aptamer-MB conjugates through the channels to areas of dried and rehydrated reporter aptamer-QD or aptamer-enzyme conjugates and the operator has complete control over the speed of MB movement, thus enabling a major advantage over traditional LF analytical membranes with fixed capillary migration times. Greater reaction times can lead to greater sensitivity by ensuring that binding reactions come to completion or equilibrium. The fluorescence assays can also be washed in the channels with clean buffers following completion of the sandwich assay and prior to analysis in the detection windows shown in the figures.

Materials and Methods

DNA Aptamers, Magnetic Beads, Quantum Dots and Other Reagents or Materials

DNA aptamer development (SELEX) for each of the targets cited in the figures has been described elsewhere in the literature [13, 15, 39]. The actual DNA sequences are not divulged here due to their potential commercial value, but the aptamers used herein are among a subset of the aptamer DNA sequences reported in patents and patent applications [40, 41]. Aptamers biotinylated on their 5’ ends were synthesized and purchased from Integrated DNA Technologies (Coralville, IA). Streptavidin-coated M280 (2.8 μm diameter) Dynal paramagnetic MBs and a sampler kit (catalogue no. Q10151MP) of variously colored streptavidin-coated-QDs were purchased from Life Technologies, Inc. (Carlsbad, CA) and Evident Technologies, Inc. (Troy, NY). Sephadex G25 (PD-10) size-exclusion columns were purchased from GE Helathcare, Inc. Silanizing reagent (~ 5% dimethyldichlorosilane in heptane) was purchased from Sigma- Aldrich, Inc. For some experiments, translucent polypropylene 8- channel low-profile troughs or reservoirs were purchased from Corning Axygen Scientific, Inc. to act as MATS cartridge surrogates for developmental purposes. Campylobacter jejuni (strain 29428) was purchased from American Type Culture Collection (ATCC; Manassas, VA) and staphylococcal enterotoxin B (SEB) was purchased from Sigma-Aldrich, Inc.

Aptamer Conjugation to Magnetic Beads and Quantum Dots

One mg of various 5’ biotinylated capture aptamers specific for each assay target were dissolved in 500 μl of sterile phosphate buffered saline (PBS, pH ~ 7.2) and added to 500 μl of streptavidin-coated M280 MBs (~ 3 x 107 MBs) and gently mixed for 1 h at room temperature (~ 25°C). Aptamer-biotin-streptavidin-MBs were washed three times in 1 ml of sterile PBS after collection of the MBs on a Dynal MPC-S magnetic rack. Five hundred μg of various 5’ biotinylated reporter aptamers specific for each assay target were added to 50 μl of each type of colored streptavidin-QD in 1 ml of sterile PBS and gently mixed at room temperature for 1 h. Aptamer-5’- biotin-streptavidin-QD conjugates were then purified in the void volume (pooled fourth and fifth 1 ml eluted fractions) of a sterile PBSequilibrated PD-10 column.

Magnetically-Assisted Test Strip (MATS) Cartridge, Automated Assay Processor and Assays

A credit card-sized MATS prototype cartridge (Figures 1 and 2) was milled out of polycarbonate plastic slabs by Precision Mold and Tool Group (PMTG), Inc. (San Antonio, TX). The automated magnetic MATS assay processor was designed and built around the use of 8- channel plastic troughs or reservoirs by Taboada Research Instruments, Inc. (TRI; San Antonio, TX). TRI’s unique external magnet design involved putting the North and South poles of a very strong rare Earth magnet in close proximity to one another instead of simply using the edge of a rectangular permanent magnet. The resulting geometry looks something like a vise that is closing when viewed from the side. The magnetic field lines are concentrated at both edges and create a powerful (> 4 Tesla) trap for the magnetic beads, which align themselves in a tight rectangular area that is ideal for fluorescence detection in the final detection window. A screw gear mechanism and servo motor were used to slide the external magnet in a linear fashion underneath the 8-channel trough which emulated the MATS cartridge for initial experiments.

In general, 20 μl of aptamer-QD conjugates were added about 3 cm from the entrance of each channel of the 8-channel troughs or MATS cartridge and allowed to dry overnight. Then 50 μl of aptamer-MB conjugates were added to the entrance of various channels in the 8- channel troughs which were pre-treated with silanizing reagent for 30 min and washed in deionized water prior to experiments to help avoid trailing of the MBs. Varied amounts of the target analytes (Campylobacter jejuni bacteria and staphylococcal enterotoxin B (SEB)) in 50 μl of PBS were added to the channels as shown in the figures and allowed to bind the capture aptamer-MB complexes for 5 min at room temperature. Blanks without the targets were also run to assess background fluorescence levels.

Thereafter, 500 μl of PBS was added to each channel and the underlying external magnet was slowly pulled along the length of the channel either manually or using the automated servo motor mechanism. Reaction times were varied, but 5 min of total time to traverse the channels with the external magnet gave the best results. Once the sandwich assays had formed and been processed at the end of each channel, they were assessed visually, digitally photographed with and without an external UV light source, and siphoned out of the channels (~ 100 μl total volume). Sandwich assays on the MBs were then resuspended in 2 ml of PBS in clear plastic cuvettes and assessed using the UV channel of a handheld Picofluor™ fluorometer (Turner BioSystems, Inc., currently the QuantiFluor™ by Promega Corp., Sunnyvale, CA).

Results

Figure 1A illustrates the basic concept of the MATS cartridge in which a “rolling” fluorescent sandwich assay is developed on the surface of capture aptamer- or antibody-coated MBs added to the entrance of the cartridge, followed by the sample containing the target analyte. After binding to the target analytes, the MBs are pulled to the previously dried, but now rehydrated, reporter aptamer- or antibody- QDs where they are allowed to react for a designated period of time and then pulled to the end of the channel where the sandwich assay on the MBs has left behind much of the matrix and debris or interfering materials. At this point, the stationary MBs which are held in place by the external magnet can be further purified by suctioning out the matrix and washing the MBs several times with clean buffer followed by fluorescence detection resulting from QDs and captured analytes on the MB surfaces. It should be noted here that multiplex detection is possible both by detection of different colored QDs in different channels or different colored QDs in the same channel [33]. Figure 1B shows the results of a very simple proof-of-principle experiment to prove that MBs could be pulled along a buffer-filled mesofluidic (2 mm wide) channel by the edge of a strong permanent magnet.

biomedical-engineering-medical-devices-multichannel-Magnetically

Figure 1A: General concept of the multichannel Magnetically Assisted Test Strip (MATS) cartridge and rolling aptamer-MB or antibody-MB plus aptamer/antibody-QD sandwich assays.

biomedical-engineering-medical-devices-proof-concept

Figure 1B: Simple proof-of-concept experimental results showing that MBs can be pulled along the length of a narrow mesofluidic (2 mm wide) channel cut into polycarbonate by means of the edge of a permanent magnet.

Figure 2A illustrates an exploded view of a credit card-sized prototype MATS cartridge. This design was milled from polycarbonate to produce the prototype cartridge shown in Figure 2B with channels having various diameters from 0.5 to 2 mm. The 1 and 2 mm diameter channels proved to be preferable in experiments because they clogged less frequently at the levels of MBs and target analytes used.

biomedical-engineering-medical-devices-MATS-cartridge

Figure 2A: Exploded view diagram of a proposed MATS cartridge.

biomedical-engineering-medical-devices-polycarbonate-plastic

Figure 2B: Image of an actual MATS prototype milled from polycarbonate plastic.

Figure 3A illustrates tracking of reporter aptamer-QDs in a PD-10 (Sephadex G25) column during the purification process to better enable capture of the fluorescent aptamer-QDs in the column’s void volume. Figure 3B illustrates the ease of multicolored or multiplex detection of various QDs in the prototype MATS cartridge with a single UV excitation source (simple mineral light).

biomedical-engineering-medical-devices-aptamer-biotin

Figure 3A: Purification of aptamer-biotin-streptavidin-QD conjugate in a Sephadex G25/PD-10 column visualized by UV light excitation.

biomedical-engineering-medical-devices-Image-multi-colored

Figure 3B: Image of multi-colored types (various sizes) of QDs in the MATS cartridge prototype showing that only one UV light source is required to induce all three fluorescent color emissions [33]. The far right channel is empty.

Figure 4A is a photo of the special underlying magnet with N and S poles held about 5 mm apart from one another to create a rectangular trap area for the MBs. This special underlying magnet was used in the automated servo motor-driven MATS (8-channel trough) processor shown in Figure 4B. The brown aptamer-MBs are shown at the beginning of each channel in Figure 4C and after stopping to react with the dried reporter aptamer-QDs several cm down the channels in Figure 4D.

biomedical-engineering-medical-devices-Custom-designed

Figure 4A: Custom designed underlying magnet for movement of MBs along the MATS channels having N and S poles adjacent to one another across a small (5 mm) gap to create a rectangular MB trap.

biomedical-engineering-medical-devices-Various-views

Figure 4B-D: Various views of the automated MATS processor using an 8-channel trough and servo motor screw gear mechanism to move the underlying magnet slowly along the MATS channels to develop the fluorescence sandwich assays.

A potential future epifluorescence optical quantitation system for the MATS cartridges in central laboratories is sketched in Figure 5.

biomedical-engineering-medical-devices-Proposed-epifluorescence

Figure 5: Proposed epifluorescence optical design for more sensitive and accurate quantitation of target analytes in the MATS cartridge versus simple visual assessment using a handheld UV light and image analysis.

The sketch illustrates the possible use of objective lenses to magnify fluorescence from the aptamer-QD plus aptamer-MB sandwich assay complexes following excitation through the objective lenses from a UV light source that is reflected down onto the MATS channels by dichroic mirrors. Fluorescent emission light from QDs in the various channels would then be sent through the objective lenses through fluorescence emission filters and to photodiode detectors for sensitive detection and fluorescence quantitation. While this detector design is more sensitive and accurate, it is more complicated and expensive and not required for simple visual assessment in low-resource settings. Indeed, simple manual movement of the underlying magnet and visual assessment of fluorescence from a UV mineral light are the minimal detection requirements.

An example of simple visual assessment of red QD 655 fluorescence following an aptamer-MB Campylobacter jejuni experiment is shown in Figure 6. In the figure, duplicate positive channels containing 100,000 live C. jejuni cells (designated plus (+) in Figure 6) are shown aggregated around the MBs at the end of the channels with strongly visible orange-red fluorescence while the two blank (designated minus (-) in Figure 6) channels demonstrated little or no orange-red fluorescence.

biomedical-engineering-medical-devices-jejuni-aptamer

Figure 6: Visual results of a Campylobacter jejuni aptamer-MB/QD sandwich assay in an 8-channel trough which emulates a MATS cartridge. The two (+) channels received ~ 100,000 live C. jejuni cells per channel while the two (-) channels represented blanks without any bacteria. Problematic trailing MBs is shown in part 3, but this was later solved by first silanizing the channels to maintain a tight band of MBs throughout the assay development process.

Figure 7A illustrates the concept of the aptamer-based MB and aptamer-QD sandwich assay, while Figures 7B and 7C illustrate validation of Campylobacter cell capture from the assay shown in Figure 6. In Figure 7B, the C. jejuni bacteria were stained with Coomassie blue dye and washed in PBS to make the aptamer-MBcaptured cells visible under brightfield microscopy at 1,000X total magnification.

biomedical-engineering-medical-devices-sandwich-assay

Figure 7A: Diagram of the general aptamer-MB plus aptamer-QD sandwich assay.

biomedical-engineering-medical-devices-brightfield-microscopy-image

Figure 7B: 1,000X brightfield microscopy image showing Coomassie blue-stained Campylobacter jejuni cells captured by aptamers on the surface of MBs (arrows).

biomedical-engineering-medical-devices-fluorescence-microscopy

Figure 7C: A fluorescence microscopy image (1,000X) of the same sample as in (B) showing red QD 655 C. jejuni cell detection (arrows).

When this same sample was subjected to UV fluorescence microscopy, red patches or clumps corresponding to the locations of the C. jejuni cells under brightfield microscopy emerged as shown in Figure 7C. Figure 7D illustrates that this assay was sensitive to a level of at least 10 C. jejuni cells per ml as previously published for similar tube-based aptamer-MB plus aptamer-QD assays following assessment of siphoned MB samples resuspended in 2 ml of PBS using a PicofluorTM handheld fluorometer [14,15].

biomedical-engineering-medical-devices-handheld-fluorometer

Figure 7D: Titration curve of the C. jejuni fluorescence aptamer- MB/QD sandwich assay performed in a prototype MATS cartridge demonstrating ultrasensitive detection to a level of 10 or fewer cells per ml as previously reported [15]. Errors bars represent standard deviations of the mean fluorescence values for three independent measurements (N=3). Results were quantified after MB resuspension in 2 ml of PBS using a PicofluorTM handheld fluorometer.

Finally, Figure 8 illustrates that the MATS aptamer-MB/QD assays can be extended to biotoxins such as SEB in PBS buffer with a likely limit of detection (LOD) of 100 ng or less per sample using aptamers previously cited by Bruno and Kiel [39]. Again, SEB aptamer-MB/QD assay samples were siphoned from the end of the MATS channels and fluorescence was quantified using a PicofluorTM handheld fluorometer after resuspension of samples in clear plastic cuvettes with 2 ml of PBS.

biomedical-engineering-medical-devices-handheld-fluorometer

Figure 8: Titration curve for staphylococcal enterotoxin B (SEB) using aptamers developed by Bruno and Kiel [39] used in the fluorescent aptamer-MB/red QD 655 sandwich assay in a prototype MATS cartridge with a 100 ng limit of detection (LOD) in PBS. Results were quantified after resuspension of MBs in 2 ml of PBS using a PicofluorTM handheld fluorometer.

Discussion

The present report documents preliminary development of a simple mesofluidic system designed to be facile and work at low cost in resource-limited environments without sacrificing detection sensitivity, multiplexing ability, or speed. To achieve these goals, our group has32 built upon FDA award winning [30-32] aptamer-MB plus aptamer-QD sandwich assay technology and transferred that technology first to lateral flow test strips without MBs [4] and then to the mesofluidic channels of the prototype MATS cartridge as reported herein.

While the system has been shown to function well and produced sensitive detection of food safety-related analytes (Figures 6 and 8) the MATS design can potentially be improved in several ways. Firstly, the MATS channels might benefit from addition of a paper or other porous solid matrix to better hold back or filter out food or other debris and interferents while still enabling the relatively free movement of the aptamer-MBs and aptamer-QDs in the channels as the sandwich assay rolls along and fully develops at the end. The addition of a paper matrix in the channels could decrease or eliminate washing or back flushing with buffer to purify the MB assay prior to fluorescence detection. One such paper material is Fusion 5 membrane manufactured by Whatman Inc. [42]. Fusion 5 is used a single membrane replacement for traditional multilayered lateral flow test strips in a method known as “boulders in the stream” lateral flow immunoassay [42] and may be suitable for use in MATS channels. Our group is currently investigating this Fusion 5 matrix possibility in MATS channels. In the authors’ long experience with MB-based and lateral flow assays, we have only encountered one other patent application which claimed to move antibody-coated MBs or magnetic particles into and through the analytical membrane of a lateral flow test strip [43], but this patent application has not yet resulted in a commercial product to date.

More sophisticated and sensitive (vs. the human eye) QD fluorescence optical detection and quantitation systems for multiplex (multi-colored) detection in single or multiple channels of a MATS cartridge such as the proposed design shown in Figure 5 are also being investigated. The Figure 5 system will be compared to simple UV light exposure of assays conducted in MATS cartridges followed by color image analysis of digital photos to determine which engineering approach yields the most sensitive and discriminatory fluorescence color quantitation at the best overall manufacturing and operating costs.

In the end, we hope to deliver an ultrasensitive, multiplexed system to the food testing market which combines the concentrating and purifying power of aptamer-MB conjugates with the high affinity and high signal-to-noise ratios of aptamer-QD conjugates in a single easy to use multiplexed cartridge. Of course aptamers are not necessary to the system and aptamers could be replaced by antibodies, if desired, but most aptamers will probably confer greater affinity [9-11], specificity [44,45], and reproducibility [9] to the end user. Finally, it should be clear that if MATS is a success in complex diluted food or enrichment broth samples [13,15], it could also function with body fluids including whole blood, serum and urine, making MATS technology amenable to the much broader clinical biomarker and related diagnostics markets.

Acknowledgements

This work was funded by U.S. Army STTR and SBIR contracts (W911SR-04-P-0085 and W911SR-04-P-0053) as well as an EPA SBIR contract (EP-D-04-027). The authors thank Dr. John Taboada of Taboada Research Instruments, Inc. (TRI; San Antonio, TX) for design and construction of the automated external magnet and automated MATS assay processing system.

References

  1. Posthuma-Trumpie GA, Korf J, van Amerongen A (2009) Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal BioanalChem 393: 569-582.
  2. Berlina AN, Taranova NA, Zherdev AV, Vengerov YY, Dzantiev BB (2013) Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk. Anal BioanalChem 405: 4997-5000.
  3. Berlina AN, Taranova NA, Zherdev AV, Sankov MN, Andreev IV, et al. (2013) Quantum-dot-based immunochromatographic assay for total IgE in human serum. PLoS One 8: e77485.
  4. Bruno JG (2014) Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold. Pathogens 3: 341-355.
  5. Li X, Lu D, Sheng Z, Chen K, Guo X, et al. (2012) A fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip. Talanta 100: 1-6.
  6. Yang H, Li D, He R, Guo Q, Wang K, et al. (2010) A novel quantum dots-based point of care test for syphilis. Nanoscale Res Lett 5: 875-881.
  7. Bordeaux J, Welsh A, Agarwal S, Killiam E, Baquero M, et al. (2010) Antibody validation. Biotechniques 48: 197-209.
  8. Marx V (2013) Finding the right antibody for the job. Nature Methods 10: 703-707.
  9. Jayasena SD (1999) Aptamers: an emerging class of molecules that rival antibodies in diagnostics. ClinChem 45: 1628-1650.
  10. Vivekananda J, Kiel JL (2006) Anti-Francisellatularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-Linked Immobilized Sorbent Assay. Lab Invest 86: 610-618.
  11. Vance SA, Sandros MG (2014) Zeptomole detection of C-Reactive Protein in serum by a nanoparticle amplified surface plasmon resonance imaging aptasensor. Scientific Reports 4: 5129
  12. Davydova A, Vorobjeva M, Pyshnyi D, Altman S, Vlassov V, et al. (2015) Aptamers against pathogenic microorganisms. Crit Rev Microbiol .
  13. Bruno JG, Richarte AM, Phillips T, Savage AA, Sivils JC, et al. (2015) Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for ultrasensitive and rapid Listeria detection. J. Fluorescence. 25: 173-183.
  14. Bruno JG, Carrillo MP, Phillips T, Andrews CJ (2010) A novel screening method for competitive FRET-aptamers applied to E. coli assay development. J Fluoresc 20: 1211-1223.
  15. Bruno JG, Phillips T, Carrillo MP, Crowell R (2009) Plastic-adherent DNA aptamer-magnetic bead and quantum dot sandwich assay for Campylobacter detection. J Fluoresc 19: 427-435.
  16. Queirós RB, Gouveia C, Fernandes JR, Jorge PA (2014) Evanescent wave DNA-aptamer biosensor based on long period gratings for the specific recognition of E. coli outer membrane proteins. BiosensBioelectron 62: 227-233.
  17. Queirós RB, de-los-Santos-Álvarez N, Noronhae JP, Sales MGF (2013) A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins. Sensors Actuators B: Chemical 181: 766-772.
  18. Stratis-Cullum DN, McMasters S, Pellegrino PM (2009) Evaluation of relative aptamer binding to Campylobacter jejuni bacteria using affinity probe capillary electrophoresis. AnalytLett 42: 2389-2402.
  19. Duan N, Wu S, Chen X, Huang Y, Xia Y, et al. (2013) Selection and characterization of aptamers against Salmonella typhimurium using whole-bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX). J Agric Food Chem 61: 3229-3234.
  20. Duan N, Wu S, Zhu C, Ma X, Wang Z, et al. (2012) Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus. Anal ChimActa 723: 1-6.
  21. Dwivedi HP, Smiley RD, Jaykus LA (2010) Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX. ApplMicrobiolBiotechnol 87: 2323-2334.
  22. Hyeon JY, Chon JW, Choi IS, Park C, Kim DE, et al. (2012) Development of RNA aptamers for detection of Salmonella Enteritidis. J Microbiol Methods 89: 79-82.
  23. Lee YJ, Han SR, Maeng JS, Cho YJ, Lee SW (2012) In vitro selection of Escherichia coli O157:H7-specific RNA aptamer. BiochemBiophys Res Commun 417: 414-420.
  24. Ohk SH, Koo OK, Sen T, Yamamoto CM, Bhunia AK (2010) Antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food. J ApplMicrobiol 109: 808-817.
  25. Pan Q, Zhang XL, Wu HY, He PW, Wang F, et al. (2005) Aptamers that preferentially bind type IVB pili and inhibit human monocytic-cell invasion by Salmonella entericaserovartyphi. Antimicrob Agents Chemother 49: 4052-4060.
  26. Singh G, Vajpayee P, Rani N, Jyoti A, Gupta KC, et al. (2012) Bio-capture of S. Typhimurium from surface water by aptamer for culture-free quantification. Ecotoxicol Environ Saf 78: 320-326.
  27. Suh SH, Jaykus LA (2013) Nucleic acid aptamers for capture and detection of Listeria spp. J Biotechnol 167: 454-461.
  28. Suh SH, Dwivedi HP, Choi SJ, Jaykus LA (2014) Selection and characterization of DNA aptamers specific for Listeria species. Anal Biochem 459: 39-45.
  29. Wu WH, Li M, Wang Y, Ouyang HX, Wang L, et al. (2012) Aptasensors for rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium. Nanoscale Res Lett 7: 658.
  30. Anonymous (2015) FDA names first-ever food safety challenge finalists. Food Safety News.
  31. Holliman K (2015) Food safety innovations win awards from FDA. Food Quality & Safety.
  32. Wu J (2015) FDA’s food safety challenge seeks speedier Salmonella detection. US News World Report.
  33. Goldman ER, Clapp AR, Anderson GP, Uyeda HT, Mauro JM, et al. (2004) Multiplexed toxin analysis using four colors of quantum dot fluororeagents. Anal Chem 76: 684-688.
  34. Cao Q, Han X, Li L (2014) Configurations and control of magnetic fields for manipulating magnetic particles in microfluidic applications: magnet systems and manipulation mechanisms. Lab Chip 14: 2762-2777.
  35. Gijs MAM (2004) Magnetic bead handling on-chip: new opportunities for analytical applications. MicrofluidNanofluid 1: 22-40.
  36. Verbarg J, Kamgar-Parsi K, Shields AR, Howell PB Jr, Ligler FS (2012) Spinning magnetic trap for automated microfluidic assay systems. Lab Chip 12: 1793-1799.
  37. Bruno JG (2006) Magnetically assisted test strip cartridge and method for using same. U.S. Patent Application No. 11/433,284.
  38. Bruno JG and Kiel JL (2002) Use of magnetic beads in selection and detection of biotoxinaptamers by electrochemiluminescence and enzymatic methods. Biotechniques 32: 178-180, 182-3.
  39. Bruno JG (2014) Methods of producing homogeneous plastic-adherent aptamer-magnetic bead-fluorophore sandwich assays. European Patent No. EP 2 255 015 B1, WIPO/PCT No. WO2009/104075. Corresponding U.S. Patent Application No. 12/378,515.
  40. Bruno JG (2012) Methods and compositions of nucleic acid ligands for detection of foodborne and waterborne pathogens. U.S. Patent Application No. 13/136,820.
  41. Jones K (2009) FUSION 5: A New Platform For Lateral Flow Immunoassay Tests. In: Lateral Flow Immunoassay, Wong RC, Tse HY eds. Humana Press, New York, pp 115-130.
  42. Kokoris M, Nabavi M, Breidford WL, Gerdes J, Mordue S, et al. (2009) Rapid Magnetic Flow Assays. U.S. Patent Application No. 12/203,779. File September 3, 2008.
  43. Jenison RD, Gill SC, Pardi A, Polisky B (1994) High-resolution molecular discrimination by RNA. Science 263: 1425-1429.
  44. Bruno JG, Carrillo MP, Phillips T, Edge A (2011) Discrimination of recombinant from natural human growth hormone using DNA aptamers. J Biomol Tech 22: 27-36.
Citation: John GB, Taylor P (2016) Preliminary Development of a Magnetically Assisted Test Strip (MATS) Cartridge and Fluorescent DNA Aptamer-Magnetic Bead Quantum Dot Sandwich Assays for Multiplexed Food Safety Applications. J Biomed Eng Med Devic 1:103.

Copyright: © 2016 John G. Bruno, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.