Enhancement of Heterogeneous Assays Using Fluorescent Magnetic

Jun 18, 2014 - Katie A. Edwards and Antje J. Baeumner*. Cornell University, Department of Biological and Environmental Engineering, 140 Riley-Robb Hal...
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Enhancement of Heterogeneous Assays Using Fluorescent Magnetic Liposomes Katie A. Edwards and Antje J. Baeumner* Cornell University, Department of Biological and Environmental Engineering, 140 Riley-Robb Hall, Ithaca, New York 14853, United States S Supporting Information *

ABSTRACT: Interactions between solution phase analytes and surface immobilized biorecognition elements in heterogeneous binding assay formats, such as enzyme-linked immunosorbent assays (ELISAs), are often hindered by mass transfer limitations. In order to improve detection limits and decrease assay times, an applied magnetic field can be used to promote target binding events if the species used for signal generation is rendered magnetic. Here, a ferromagnetic metal oxide-oleic acid complex was incorporated into the lipid bilayer of fluorescent dye-encapsulating liposomes, allowing for their influence under a magnetic field while maintaining their high interior encapsulation volume for signaling molecules. In a high-throughput sandwich-hybridization assay, these DNA-tagged liposomes yielded enhanced sensitivity, in addition to reduced assay times and reagent concentrations, when used with an underlying magnet. These magnetic signaling reagents offer superior performance and adaptability to standard assay formats.

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loaded with the drug have been utilized either by placing the affected site within an external magnetic field12 or through implanting a magnet within a tumor. 13 For imaging applications, liposomes loaded with paramagnetic gadolinium species offer improved MRI contrast.14 Combining paramagnetic properties for MRI response with fluorescent labels has extended the utility to provide dual purpose liposomes capable of providing in vivo fluorescence cellular imaging as an orthogonal measurement.15 However, to our knowledge, the great potential for magnetic liposomes as analytical reagents for in vitro assays remains unstudied. Nonmagnetic liposomes offer well-established advantages as signaling reagents which include ease of functionalization of the lipid bilayer with hydrophilic or hydrophobic biorecognition elements; assay sensitivity stemming from the encapsulation of hundreds of thousands of signaling molecules, such as fluorescent dyes or electrochemical markers, within their aqueous cores; instantaneous signal provided through surfactant-induced release of encapsulated contents; and the protective nature of the interior toward encapsulants conferring their increased long-term stability.16−18 They can provide signal as intact species through measurement of changes in optical density, refractive index, or mass. They have also been widely employed in microfluidic, microtiter plate, and flow-injection analysis platforms using fluorescence or electrochemical detection, among others, to yield signal

ssay formats relying on interactions between solution phase analytes and immobilized supports, such as in enzyme-linked immunosorbent assays (ELISAs), are widely used in food, environmental, and clinical analyses. Interactions in such heterogeneous assay formats are dependent on many factors including the surface concentration of binding sites, concentration of solution phase molecules, affinity of binding partners, reaction rate constants, and diffusion constants of the solution phase species.1−5 In such formats, depletion of solution phase analytes or labeled biorecognition elements (BREs) near the solid phase upon binding and the subsequent need for transport to the surface from the bulk solution can result in suboptimal detection limits and lengthy incubation times in order to attain the desired level of sensitivity.6−8 This is especially the case for larger, nanoparticle-based labels which are of increasing interest as signaling reagents. To mitigate these limitations, approaches to increase interactions between surface bound and solution phase species, such as applying perpendicular flow to force analyte streams closer to binding surfaces in microfluidic channels,9 are often sought. The approach described within is to instead modify the labeled species such that it is capable of being drawn to the underlying surface under the influence of a magnetic field. Such an approach offers the potential for increasing binding events with surface-captured target molecules, while allowing application in standard assay platforms with the only modification being the incorporation of a magnet. Liposomes incorporating magnetic nanoparticles have been studied for use as tissue specific drug delivery vehicles for chemotherapy and as magnetic resonance imaging (MRI) reagents.10,11 In drug delivery applications, magnetic liposomes © XXXX American Chemical Society

Received: April 3, 2014 Accepted: June 10, 2014

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Figure 1. (Top) Interactions of bulky (∼350 nm), DNA reporter probe labeled fluorescent dye-encapsulating liposomes with target sequences hybridized to immobilized capture probes are subject to mass transfer limitations to the analyte captured on the immobilization surface. (Bottom) An underlying magnetic field is used to draw these liposomes incorporating an iron oxide-oleic acid complex toward the binding surface in this heterogeneous sandwich hybridization assay format. The application of the magnetic field helps to overcome mass transfer limitations yielding increased binding interactions of liposomes with surface captured DNA or RNA target molecules, affording increased assay sensitivity, reduced reagent concentrations, and reduced assay times.

resulting from measurement of their large payload of released materials when lysed.16−18 Described here are BRE-tagged, fluorescent dye-encapsulating liposomes that have also been functionalized with an iron oxide-oleic acid complex to provide for their influence under a magnetic field. Imparting magnetic properties to liposomes was envisioned to maximize their signaling potential in heterogeneous formats through overcoming diffusion constraints yielding reduced analysis time and increased binding events, which when coupled with their large marker payload, would be expected to enhance sensitivity (Figure 1). Here, it is demonstrated that the magnetic field indeed helps to overcome mass transport limitations of these bulky ∼300 nm species to the binding surface. The resulting close proximity promotes

interactions between the liposomes whose bilayers are functionalized with DNA reporter probes and DNA or RNA target molecules that are hybridized with DNA capture probes immobilized in 96-well microtiter plates. The goal is to lower the limit of detection while improving assay efficiency through reducing the signaling reagent concentrations and assay time required. Other encapsulants, BREs, and assay platforms could be utilized with this approach, with great potential for application in microfluidics allowing also for directional control.

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MATERIALS AND METHODS 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], sodium salt (DPPG), and the extrusion membranes were purchased from B

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probe modified liposomes17 incorporating iron oxide-oleic acid rather than a biotinylated reporter probe and streptavidin conjugated liposomes.23 Briefly, 5′-amine labeled capture probe was covalently immobilized in a black amine-binding microtiter plate; then, the plate was blocked with 0.1% (w/v) casein, sodium salt in TBS for 1 h. Synthetic target DNA or NASBAamplified heat shock RNA extracted from 5 C. parvum oocysts (lysis, extraction, and amplification carried out as previously described26) was added at various dilutions in hybridization buffer (600 mM sodium chloride, 60 mM sodium citrate, pH 7.0, 20% (v/v) formamide, 0.1% (w/v) Ficoll) in triplicate and incubated for 1 h at low speed on a vortex with plate adapter. Liposomes encapsulating sulforhodamine B dye and tagged with both a reporter probe and the iron oxide conjugate were diluted to 25 μM phospholipid in hybridization buffer and incubated with the captured target for 1 h, during which time, the plates were removed from the magnet and placed on the vortex for 5 s twice. The plates were washed with 3 × 200 μL of hybridization buffer; then, those liposomes remaining bound were lysed with 50 μL of 30 mM OG. The fluorescence intensity from the released dye was measured at λex = 540/35 nm and λem = 590/20 nm. Wash steps were carried out in between all additions as described.23 In each experiment, two identical plates were run side by side per assay: one with a magnetic plate (consisting of 96 tube magnets embedded within a plastic housing (Figure 2a)) placed underneath and the other without the magnetic plate.

Avanti Polar Lipids (Alabaster, AL). The iron oxide-oleic acid conjugate was purchased from NN-Labs, LLC (Fayetteville, AR). Block magnets were purchased from K&J Magnetics (Jamison, PA). Sulforhodamine B (SRB) was purchased from Invitrogen (Frederick, MD). Clear and black Immobilizer Amino microtiter plates were purchased from Nunc (Roskilde, Denmark.) The Costar clear medium binding 96-well microtiter plates (for Bartlett assay) were purchased from Corning (Corning, NY). All other reagents used in these experiments were purchased from VWR (Bridgeport, NJ.) All buffers were prepared with HPLC grade water, manufactured by JT Baker (Phillipsburg, NJ). The liposome size distribution was determined by dynamic light scattering with a DynaPro LSR (Proterion Corporation, Piscataway, NJ) using the Dynamics (version 6.3.01) software program and the Cumulants method of analysis. Fluorescence and visible measurements were made using FLX800 and PowerWave XS microtiter plate readers, respectively (Bio-Tek Instruments, Winooski, VT). Liposome Preparation. SRB-encapsulating liposomes were prepared using the reverse phase evaporation method19,20 as described previously using a 3′-cholesterol modified reporter probe at 0.013 mol % of total lipid21 but additionally with 5−15 nm iron oxide-oleic acid at 0.5−8 mol % of total lipid. The phospholipid content for each liposome batch was determined using the Bartlett assay,22 with the procedure carried out as previously described.23 Dynamic light scattering measurements and the encapsulation efficiency assay were carried out as detailed previously.24 To assess the influence of a magnetic field on various preparations, liposomes were diluted to 100 μM in HEPESsaline sucrose buffer (HSS: 10 mM HEPES, 200 mM sucrose, 200 mM sodium chloride). 200 μL was pipetted onto a glass coverslip situated over a sheet of 20 lb white paper to aid in visualization and a block magnet (grade 40, 0.5 in. × 0.5 in. × 0.125 in. NdFeB magnet, 6 lbs pull force) to qualitatively assess the influence of the magnetic field by the observed shape formation. The buffer used in these experiments was that which is often employed to maintain high osmolarity conditions required for successful liposome encapsulant retention.21 The high sucrose content renders this buffer relatively viscous (1.234 cP); hence, in conjunction with the use of a relatively weak magnet, these screenings were carried out under stringent conditions, compared to those subsequently evaluated below. Microfluidic Retention of Magnetic Liposomes via Permanent Magnets. 50 μL of the above liposomes at 200 μM phospholipid with 6 mol % of the 15 nm iron oxide-oleic acid complex were introduced at 5 μL/min, into a microfluidic device. The device was produced as described and was blocked with 0.01% (w/v) bovine serum albumin in PBS and washed with HSS prior to the introduction of liposomes.25 The device was situated onto a 4 in. silicon wafer to provide a flat, dark background for fluorescence imaging in either the absence or presence of an underlying 1 in. × 1 in. × 0.1875 in. NdFeB magnet. Unretained liposomes within the channels were removed via flow of HSS (100 at 10 μL/min); then, 30 mM n-octy-β-D-glucopyranoside (OG) was introduced to lyse liposomes remaining in the channels prior to imaging the device using a fluorescence microscope. Microtiter Plate Sandwich-Hybridization Analyses and Optimizations. The microtiter plate-based assay was carried out as detailed previously with modifications. The plate preparation, target hybridizations, and data analysis were carried out as described23 but utilized 3′-cholesteryl reporter

Figure 2. Directional control of magnetic liposomes via permanent magnets. A 96-well magnetic plate (a) was placed under a glass coverslip onto which dye-encapsulating liposomes without (b) and with (c) an iron oxide complex were pipetted. A sheet of 20 lb white paper was inserted between the plate and the coverslip to aid in visualization. Liposomes prepared with the iron oxide-oleic acid complex were drawn to the underlying magnet whereas those without were not.

Optimizations to obtain the aforementioned conditions included liposome concentrations ranging from 10 to 75 μM phospholipid; liposome incubation times ranging from 15 min to 2 h; and using liposomes prepared with 5, 10, or 15 nm of iron oxide-oleic acid at 0.5 to 8 mol %. The assay was also run where liposomes and target DNA were simultaneously incubated for 1 h in the presence of the immobilized capture probe to assess the ability of the liposomes to draw the target to the binding surface and further reduce assay time. Data processing for all experiments was carried out as described previously.23 Influence of Magnetic Field Strength. To investigate the influence of magnetic field strength, neodymium iron boron (NdFeB) block magnets with 1 in. × 1 in. and thickness ranging from 0.0625 in. to 1 in. were used in lieu of the commercially available 96-well magnetic plate. These magnets C

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(experimentally similar to that in Figure 2). Liposomes with 8 mol % 5 nm complexes could be readily prepared but were not observed to be influenced by a magnetic field, while those attempted with lipid mixtures containing 8 mol % 15 nm complexes precipitated during purification and could not be recovered. Those prepared with 4 mol % or 6 mol % of the 15 nm complex were successfully influenced by a magnetic field, whereas those with lesser concentrations or particle diameters were not when used under relatively high viscosity buffer conditions with a relatively weak magnet (6 lbs pull force). Future studies will determine if lowering the buffer viscosity and increasing the magnetic force will positively affect the magnetic influence of liposomes with lower iron oxide concentrations and smaller particle diameters. In terms of dye encapsulation, incorporation of the 15 nm iron oxide-oleic acid complexes at 4 or 6 mol % yielded 1.34 and 1.27 mol of sulforhodamine B (SRB) dye per mol of phospholipid, respectively. These values indicate a positive impact of the iron oxide-oleic acid complexes on SRB encapsulation versus encapsulation values of 0.57 mol of SRB per mol of phospholipid for similar probe-labeled liposomes lacking the complexes. Ultimately, the liposome preparation with 6 mol % 15 nm iron oxide-oleic acid complex in a lipid mixture composed of cholesterol, DPPC, DPPG, and a cholesteryl modified DNA probe was found to be optimal due to relative ease of preparation, high encapsulation efficiency, and maximal magnetic influence. Using dynamic light scattering, hydrodynamic diameters of 357 ± 28 nm were determined, yielding an approximation of 1.1 million molecules of SRB dye per liposome and thus the potential for a substantial signal enhancement per binding event. Directional Control and Retention. To demonstrate directional control, a 96-well plate with tube magnets (Figure 2a) was placed under a glass coverslip onto which a solution of liposomes was dispensed. These magnets drew the liposomes with iron oxide functionalization toward the bottom of the solution in circular patterns, consistent with the geometry of the magnets (Figure 2c), whereas liposomes without the iron oxide functionalization remained unaffected by the magnet (Figure 2b). Similarly, such liposomes could be captured while flowing and retained within microfluidic channels by an underlying permanent magnet (Figure S-1, Supporting Information). These straightforward experiments successfully demonstrated the ability to direct liposomes toward the binding surface under static or dynamic conditions which was the first step in utilizing them to overcome diffusion limitations in heterogeneous formats. Application to Heterogeneous Assays. Once it was confirmed that the liposomes could be directed using permanent magnets, the magnetic plate shown in Figure 2 was situated under commercially available microtiter plates to evaluate the impact of drawing the liposomes toward the binding surface in a standard assay format. Capture probe DNA-functionalized microtiter plates23 were used with reporter probe-tagged, dye-encapsulating21 magnetic liposomes in sandwich hybridization assays for synthetic target DNA (Figure 1) and heat shock mRNA extracted from Cryptosporidium parvum, that was amplified using nucleic acid sequence-based amplification (NASBA).26 The liposome incubation step was carried out side by side on two identical plates with and without the magnetic plate to identify the effects of the magnetic influence. The resulting fluorescence intensity in the presence of the magnet was significantly increased over the entire

were either grade N42 or grade N52 and were magnetized through thickness, yielding pull strengths ranging from 7.01 to 94.6 lbs. These magnets were situated under 9 wells in the top corner of the magnetic plate, and the plates were balanced using polypropylene inserts on the opposite corner of the plate to maintain a level surface and to provide a nonmagnetized control on the same plate. The above sandwich hybridization procedure was carried out except using clear microtiter plates to aid in visualization, and situation of the underlying magnet and mixing during the liposome incubations did not take place to ensure that plate alignment over the magnets was maintained. Concentrations of synthetic target employed were 0, 2, 20, and 200 nm. The thickness of the microtiter plate was 0.8 mm, as reported by the plate manufacturer (https://static.thermoscientific.com/ images/D03032~.pdf). This value was used as the distance from the magnet surface in field strength calculations using the magnet manufacturer’s magnet calculator (http://www. kjmagnetics.com/calculator.asp). At this distance from the various block magnets, liposomes could be subjected to magnetic fields ranging from 727 to 6079 G at the base of the wells, decaying exponentially with further distance from the base of the wells and reaching approximately 669−5107 G at the surface of the 100 μL solution. Caution: Strong magnets can cause physical injuries due to pinching of body parts between other magnets or metal objects, interference with pacemakers, and damage to sensitive electronic equipment. They must be handled with appropriate care and signage.

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RESULTS AND DISCUSSION The intended design for these liposomes was to allow control by magnetic fields while retaining high levels of dye encapsulation and functional surface BREs. Such liposomes were envisioned to retain well-established binding and signaling abilities, while adding control of their motion and reducing diffusion limitations. Properties. Initial studies focused on the synthesis of such liposomes, utilizing a commercially available monodisperse complex (15 nm ± 1 nm) between iron oxide and oleic acid to afford magnetic properties. The concentration and particle size of the iron oxide-oleic acid complex within the overall lipid composition was varied and the effect on the magnetization of liposomes studied qualitatively. Specifically, the mol % of this lipid added ranged from 0.5% to 8.0% of total lipid with diameters of the iron oxide complex ranging from 5 to 15 nm. The oleic acid imparts control over size uniformity and prevents aggregation of such nanoparticles, while yielding improved dispersion in organic solvents.27,28 Oleic acid is a fatty acid with a long monounsaturated hydrocarbon tail and polar carboxylic acid headgroup. The carboxylic acid groups form coordination complexes with the iron oxide nanoparticles while the hydrocarbon chains extend into the surrounding organic media often used for dissolution. It is assumed that the hydrocarbon chain incorporates into the liposomal lipid bilayer while the polar complexes face the inner and outer aqueous solutions. The optimal formulation was a balance between successful preparation, overcoming liposome buoyancy and solvent viscosity to ensure maximal influence by a magnetic field, and retention of liposomal dye-encapsulation and binding abilities. These initial screenings for influence under a magnetic field were qualitative, observing formation of liposome patterns using a glass coverslip placed over a block magnet D

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concentration range tested (10 pM to 100 nM synthetic target) versus that obtained in its absence (Figure 3). This was also

Figure 3. Calibration curve for synthetic target DNA in the presence and absence of a magnetic field. Reporter probe-tagged magnetic liposomes diluted to a phospholipid concentration of 25 μM were used for the sandwich hybridization-based detection of synthetic target DNA ranging from 10 pM to 100 nM. Liposomes remaining bound were lysed with 30 mM OG to release their encapsulated SRB dye, and fluorescence intensity was measured at λ540/590 nm. Incubation of the liposomes with the hybridized complex between target DNA and immobilized capture probe took place in the presence (■) or absence (▲) of a 96-well magnetic plate. Each point is the average of triplicate determinations, and error bars represent one standard deviation.

Figure 4. Influence of magnetic field on incubation time (top) and phospholipid concentration (bottom) on signal intensity in a sandwich hybridization assay format. Synthetic C. parvum target DNA was introduced at 50 nM to an amine modified capture probe immobilized in amine-binding microtiter plates. Reporter probe-tagged liposomes diluted to a phospholipid concentration of 25 μM were incubated with these hybridized complexes in the presence (red ■) or absence (green ▲) of a 96-well magnetic plate. Each point is the average of triplicate determinations, and error bars represent one standard deviation.

observed for dilutions of NASBA amplified RNA extracted from 5 C. parvum oocysts ranging from 1:1 280 000 to 1:1250. Meanwhile, nonspecific binding with the plate surface was only minimally increased (1.3 times) over that of the same liposomes without an applied magnetic field leading to significantly improved signal-to-noise ratios over the entire concentration range tested (Figure S-2, Supporting Information). For synthetic DNA, the limit of quantitation (LOQ), using the criteria of the background signal plus ten times its standard deviation, decreased approximately 15-fold using the underlying magnetic plate, yielding a LOQ of 35 pM versus 535 pM in the absence of the magnet. Similarly, RNA dilutions of 1:20 000 could be quantified in the presence of the magnet, whereas in its absence, this increased to only dilutions of 1:2500. The liposomes could also be simultaneously incubated with the target, thus utilizing the magnet to draw the target to the binding surface as well, allowing for the omission of a separate incubation and washing step (Figure S-3, Supporting Information). Influence of Magnetic Field on Incubation Time and Liposome Concentration. Advantages in assay efficiency in terms of reduced signaling reagent incubation times and concentrations were also realized when using the magnetic field to increase interactions of signaling species with surface-bound molecules. The effect of liposome incubation time and concentration was evaluated by comparing the signal intensity for sandwich hybridization-based detection of synthetic DNA both with and without the underlying magnetic plate for 15 to 120 min incubations and using phospholipid concentrations from 10 to 75 μm, respectively (Figure 4). The signal obtained after 15 min in the presence of the magnet exceeded that at 120 min in its absence, indicating that the assay time could be reduced at least 8-fold when using the magnet. This is beneficial for microtiter plate-based assays to improve sample throughput. However, perhaps equally significant, the ability to reduce

interaction times using magnetic fields is an important finding for improving assay performance in flow-based devices, such as in microfluidics, where the luxury of extended incubation times is not available. Similarly, the signal obtained using 10 μM phospholipid concentration in the presence of the magnet was approximately the same as that obtained using 50 μM in its absence, indicating that the signaling reagent concentration could be reduced at least 5-fold when using the magnet. This translates to lower reagent costs and lower background signals. The potential for reduced assay times and lower reagent concentrations while still obtaining the same performance offers significant advantages in terms of throughput, cost, and reduced background, respectively. However, if improved assay performance was required, it could be achieved through extended incubation times or higher liposome concentrations in this magnetic enhancement strategy since the signal intensity increased linearly with both parameters to a greater degree in the presence of the magnetic plate. Influence of Magnet Strength. The above benefits were demonstrated using tubular 96-well plate magnets which are best suited for immunomagnetic separations in round-bottom microtiter plates. With the flat-bottom plates currently optimized for the sandwich hybridization assay employed here, only a small portion of the functionalized well surface was in direct contact with the underlying magnets which drew the liposomes to the binding surface at the outer edge of the bottom surface of the wells (Figure 2). As a 100 μL volume of the well was functionalized with the amine modified capture probe in these plates, roughly only 7.4% of the functionalized E

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well surface area was in contact with the magnets. The benefit of the magnetic liposomes would be expected to be enhanced by an appropriately designed magnet encompassing a greater area of the plate, for example, through employing magnets in between as well as at the base of the wells. To determine if a more suitable magnet design could indeed improve performance, flat 1 in. × 1 in. block magnets of varying heights and magnet grades were utilized to cover the complete bottom surfaces of 9 wells. Target DNA concentrations of 0, 0.2, and 2 nM were analyzed in triplicate for each magnet pull strength, which ranged from 7.0 to 94.6 lbs, corresponding to 727−6079 G at the distance of the solution at the internal base of the well, and the results were compared to those obtained in the absence of such magnets. Here, while still not directly addressing the sides of the wells, these magnets were in direct contact with the entire well base, rather that just a narrow circular pattern. (By contrast to the protocol using the 96-well plate magnet, the assay plate was neither removed from the underlying magnet nor mixed during the liposome incubation period to ensure that proper alignment with the 1 in. × 1 in. magnets was retained for the duration of the experiment. With an improved magnet configuration, mixing at this step would likely improve assay reproducibility.) An average signal-to-noise ratio of 10 to 1 was obtained for a 2 nM target concentration in the absence of the magnet. Using block magnets of any strength, the response improved for both the 0.2 and 2 nM concentrations of target over that obtained in the absence of magnets (Figure 5). The response generally increased with increasing magnet strength, reaching an impressive signal-to-noise ratio of 55 to 1 at a target concentration of 2 nM for the magnet with 43.2 lbs pull force. These results compare favorably to the signal-to-noise ratio of 36.5 to 1 obtained at the higher 2.5 nM target concentration using the 96-well magnetic plate, indicating that the flat magnets indeed offered an improvement to assay response. At the same time, even magnets with pull forces as low as 18 lbs could be used while still significantly increasing the assay performance, yielding a signal-to-noise ratio of 49:1. The signalto-noise ratio declined slightly at magnet strengths higher than 43.2 lbs pull force due to increased nonspecific interactions of the liposomes with the binding surface. Additionally, while not studied here, it is known that liposomes without imparted magnetic properties undergo deformation and leakage of contents in the presence of strong applied magnetic fields.29 Thus, from both an assay performance and a convenient, safe handling perspective, magnets with intermediate strengths were preferred. The ability to utilize moderate strength magnets will facilitate incorporation of these liposomes into microfluidic devices as well as their integration into existing high-throughput sample handling platforms.

Figure 5. Influence of block magnet strength on target recognition. Synthetic C. parvum target DNA was introduced at 0, 0.2, and 2.0 nM (legend) to an amine modified capture probe immobilized in aminebinding microtiter plates. Reporter probe-tagged liposomes diluted to a phospholipid concentration of 25 μM were incubated with these hybridized complexes in the presence or absence of 1 in. × 1 in. flat magnets with varied thickness (0.0625−1 in.) and pull forces (7.0− 94.6 lbs, x-axis). (A) Fluorescence intensity values (log scale) versus target concentrations are plotted. Each point is the average of triplicate determinations, and error bars represent one standard deviation. (B) Results from (A) plotted in terms of signal-to-noise ratios (signal at 0.2 or 2.0 nM target DNA divided by that obtained in its absence).

influence under a magnetic field even under stringent conditions. Our previous work has shown that similar liposomes lacking the oleic acid conjugate are stable for at least a year when stored at 4 or 21 °C.30 Consistent with this longevity, the magnetic liposomes described within retained their ability to be influenced by a magnetic field, signal enhancement capabilities, and hybridization functions over at least a 3 year period when stored at 4 °C. A full stability study on the impact of the oleic acid conjugate on the liposomes is the subject of future work. The magnetic liposomes resulting from these studies were shown to be able to be directed toward surfaces; retained in flow-based microfluidic devices; had a positive impact on sulforhodamine B dye encapsulation; and, in the presence of a permanent magnetic field, ultimately improved the sensitivity, reduced assay times, and reagent consumption in heterogeneous sandwich assays through overcoming diffusion limitations. A substantial 15-fold improvement in the limit of quantitation for nucleic acid sequences was obtained using a 96-well magnetic plate to draw these liposomes in a circular pattern toward the analytes captured on the binding surface of standard microtiter plates. Subsequent use of magnets which covered the complete base of the wells indicated that this result could be improved further by a magnet design encompassing more of the well area. This microtiter plate format successfully demonstrated proof of principle for

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CONCLUSIONS Described within is the development and utility of markerencapsulating, BRE-tagged magnetic liposomes for signal enhancement in analytical assays. Studies to develop a magnetically influenced liposome formulation yielded an optimal lipid composition including a 6 mol % 15 nm oleic acid-iron oxide complex in a lipid mixture composed of cholesterol, DPPC, DPPG, and a cholesteryl modified DNA probe to afford recognition capabilities. The encapsulant was 150 mM sulforhodamine B dye, which has been used extensively to provide signal in fluorescence-based quantification methods. This formulation of liposomes was selected due to ease of preparation, high encapsulation efficiency, and F

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this technology in high-throughput analytical applications which may be readily implemented with standard, commercially available equipment. Such liposomes will likely find utility in other platforms, for example, used in a similar manner to immunomagnetic separation but both preconcentrating and simultaneously labeling analytes from complex matrices and drawing them to binding surfaces. In an analogous manner to prior work using magnetic nanoparticles to direct analyte complexes toward binding surfaces,31 these liposomes add sensitive fluorescent signaling capabilities and could also be used to provide an orthogonal measurement approach for immunoassays currently relying on magnetic species alone as markers.32 However, the greatest versatility of these liposomes may be realized in microfluidic platforms, where there can readily be envisioned microfluidic devices taking advantage of region-specific accumulation and release; integration of electromagnets providing controlled motion and mixing options; and devices utilizing separation via gradient magnetic fields. Developments from the drug delivery arena for magnetic liposomes for triggered release via thermosensitive lipid compositions33 and release through localized heating via alternating magnetic fields34,35 could readily be coupled with the enhanced detection strategy described here. The demonstrated and universal advantages of these magnetic liposomes for signaling purposes offers many exciting detection opportunities and greatly expands the toolbox for method development.

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ASSOCIATED CONTENT

* Supporting Information S

Images from preliminary microfluidic studies, a signal-to-noise plot for the results in Figure 3, and results from combined incubation of magnetic liposomes with target DNA. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 607-255-5433. Fax: 607255-4449. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Michael Cate of NN-Labs, LLC for review of technical content as it pertains to their products and Barbara Leonard of Cornell University for providing the RNA samples used in this study. Funding for this work was provided by Cornell University.

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REFERENCES

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dx.doi.org/10.1021/ac501219u | Anal. Chem. XXXX, XXX, XXX−XXX