Article

Rapid Whole Blood Bioassays Using Microwave-Accelerated Metal-Enhanced Fluorescence

Kadir Aslan *

Morgan State University, Department of Chemistry, 1700 East Cold Spring Lane Baltimore, MD 21251. 

* Corresponding author. Email: kadir.aslan@morgan.edu  

Citation: K. Aslan, et al., Rapid Whole Blood Bioassays Using Microwave-Accelerated Metal-Enhanced Fluorescence. Nano Biomed. Eng., 2010, 2(1): 1-7.

DOI: 10.5101/nbe.v2i1.p1-7

Abstract

The proof-of-principle demonstration of rapid whole blood bioassays based on microwave-accelerated metal-enhanced fluorescence (MAMEF) method using silver nanoparticle-deposited surfaces is presented. In this regard, spherical silver nanoparticles were deposited onto glass slides (silver nanoparticle films, SNFs) in a highly reproducible manner, which was assessed by optical absorption spectroscopy. Atomic force microscopy was employed to determine the size of the deposited silver nanoparticles. A model bioassay, based on the well-known interactions of biotinylated bovine serum albumin (b-BSA) and streptavidin was constructed on SNFs. The model bioassay was run at room temperature (metal-enhanced fluorescence (MEF)-based bioassay without microwave heating) for 60 minutes and with microwave heating (MAMEF-based bioassay) for 1 minute. In contrast to MEF-based bioassays that only allowed the use of samples in buffer solution, MAMEF-based bioassays afforded the use of whole blood samples. A lower detection limit of 1 nM and 0.01 nM for b-BSA was determined in MEF-based and MAMEF-based bioassays, respectively. 

Keywords: Bioassays; Plasmonics; Surface plasmons; Silver nanoparticles; Gold nanoparticles; Surface plasmon resonance; Plasmon controlled fluorescence; Metal-enhanced fluorescence; Microwave heating 

1. Introduction

Bioassays are commonly used for the detection of biomolecules and analytes present in biologically rele-vant samples [1]. The quantitative detection of biomol-ecules and analytes are typically carried out by labeling the antibody/protein with an enzyme, [1] magnetic par-ticles [2-3]or fluorophores [4-5] followed by the con-version the measured signal to the concentration of the unknown biomolecules or analytes. Fluorescence-based readout for signal transduction is one of the most wide-ly used technique in bioassays. Although well estab-lished, the sensitivity of the fluorescence-based bioas-says is mainly affected by the quantum yield of the fluorophore used to label the detector antibody/protein and the type of optical detectors used in collecting the fluorescence readout [6]. In addition, the total assay time is controlled by the binding kinetics of the pro-teins, which takes up to 20 minutes with automated miniature instruments including sample preparation or several hours in a typical laboratory setting [7]. For whole blood samples, additional time is required to separate whole blood component for the detection of desired biomolecules and analytes. In this regard, to address the two major shortcomings of fluorescence-based bioassays currently in use today; i.e., bioassay sensitivity and rapidity, a new platform technology called microwave-accelerated metal-enhanced fluorescence (MAMEF), was recently intro-duced [8-9]. The MAMEF method couples the benefits of low power microwave heating with metal-enhanced fluorescence (MEF) [10]. In MAMEF, the MEF phe-nomenon increases the sensitivity of the assays, while the use of low power microwave heating kinetically accelerates assays to completion within a few seconds [8]. There are three components of the MAMEF method: (I) plasmon-supporting nanoparticles (i.e., silver and gold), (II) electromagnetic energy (microwave region) and (III) an aqueous bioassay medium. The plasmon-supporting nanoparticles serve as (1) a surface for the attachment of one of the biorecognition partners (2) as an enhancer of the fluorescence emission (MEF effect) [11]and (3) a microwave transparent material for the selective heating of water, which occurs as the aqueous medium is heated by microwaves to a higher tempera-ture than the plasmon-supporting nanoparticles. Subse-quently a temperature gradient between the warmer (than room temperature) aqueous medium and the colder nanoparticles (silver nanoparticles are transpar-ent to microwaves at 2.45 GHz and remain at room temperature) is created. This temperature gradient re-sults in mass-transfer of biomolecules from the warmer medium to the surface of the nanoparticles, where the biorecognition events are rapidly driven to completion. Although the MAMEF method provides the means to increasing the sensitivity of the bioassays while short-ening the total assay time, to date, it was only demon-strated for bioassays that employ samples in buffer so-lutions. Given the increasing demand for bioassays for complex biological samples (such as serum, whole blood and saliva), there is still a need for bioassay techniques those routinely evaluates these complex biological samples. In this study, the proof-of-principle demonstration of whole blood bioassays using MAMEF method is pre-sented. In this regard, first freshly prepared spherical silver nanoparticles were deposited onto glass slides in a highly reproducible manner. Optical absorption spec-troscopy was employed to evaluate the reproducibility of the deposition method. The surface plasmon reso-nance peak at 430 nm for silver nanoparticles deposited onto 10 different glass slides showed a minimal ~2% variation, which provided a direct evidence for the ef-fectiveness of this straightforward deposition method. Atomic force microscopy was employed to determine the size of the silver nanoparticles, where the height of the silver nanoparticles was found to be~100 nm. MEF-based detection of a model protein, b-BSA, in buffer in the concentration range of 0.1-1000 nM was achieved within 60 minutes at room temperature. The incorpora-tion of low power microwave heating into MEF-based bioassays (that is, MAMEF-based bioassay) afforded the detection of < 0.01 nM b-BSA from whole blood samples in 1 minute. In addition to the reduced assay times, the use of microwave heating in MEF-based bi-oassays also afforded for the expansion of the detecta-ble concentration range of biomolecules/analytes in complex biological samples using MEF-based bioas-says without the need for expensive detectors and op-tics. 

2. Materials and Methods

Bovine-biotinamidocaproyl-labeled albumin (b-BSA), Fluorescein isothiocyanate (FITC)-avidin, whole blood, silver nitrate (99.9%), trisodium citrate, press-to-seal silicone isolators (8 well, D × diam. 1.0 mm × 9 mm) and silane-prepTM glass (amino) slides were pur-chased from Sigma-Aldrich. All chemicals were used as received. The synthesis of spherical silver nanoparticles was performed using the following procedure: 2 ml of 11.6 mM trisodium citrate solution was added drop wise to a heated (90˚C) 98 ml aqueous solution of 6.5 mM of silver nitrate while stirring. The mixture was kept heat-ed for 10 minutes and then it was cooled to room tem-perature. All glassware used was treated with “piranha solution” (3:7 v/v; 30% hydrogen perox-ide/concentrated sulfuric acid: CAUTION! piranha solution reacts violently with most organic materials and should be handled with extreme care) and rinsed with deionized water at least three times before use. The coating of the amino slides with spherical silver nanoparticles was accomplished by incubating the ami-no slides in a freshly prepared silver nanoparticle solu-tion for 30 minutes. The amino slides were coated with silver nanoparticles due to the binding of silver to the terminal amine groups of the silane. An 8-well silicon isolator was attached to the SNFs before the construc-tion of the bioassays. Atomic Force Microscopy (AFM) images were col-lected with a Veeco Atomic Force Microscope (TMX 2100 Explorer SPM), which is equipped with a dry scanner. Surfaces were imaged in air, in a tapping mode of operation, using non-contact mode cantilever. The AFM scanner was calibrated using a standard cali-bration grid as well as by using gold nanoparticles, 100 nm in diameter from Ted Pella. Images were analyzed using SPMLab software. Figure 1A show the construction of the model bioas-say used in this paper, which is based on the well-known interactions of biotin and streptavidin. Biotin groups are introduced to the surface of SNFs through incubation of b-BSA at room temperature or using mi-crowave heating. The fluorophore (FITC)-labeled avi-din was allowed to bind b-BSA by incubation at room temperature or using microwave heating. In the MAMEF-based whole blood bioassay, a solution of b-BSA in phosphate buffer (pH=7) is mixed with whole blood (50% v/v mixture, final volume: 50 μl, b-BSA final concentration range: 0.01-5000 nM). These mix-tures or a buffer solution (control sample, no b-BSA) was placed inside the wells of silicon isolators and the SNFs were heated for 30 seconds in a commercially available microwave oven (Emerson, maximum power 700 W microwave oven, Model: MW8784B, power setting 3 was used). The unbound material was re-moved by rinsing with phosphate buffer three times. Then, 50 μl of 10 μM FITC- avidin was subsequently added to the b-BSA coated wells and heated for 30 se-conds in the microwave cavity, followed by rinsing with buffer to remove the unbound material. In the bioassay run at room temperature (MEF-based bioassay, no microwave heating), b-BSA in buffer (concentration range: 0.01-5000 nM) was incubated inside the wells of the silicon isolators for 30 minutes. The unbound material was removed by rinsing with phosphate buffer three times. Subsequently, 50 μl of 10 μM FITC-avidin was incubated on biotinylated surfac-es at room temperature for 30 minutes. The unbound material was again removed by rinsing with phosphate buffer three times. In all the experiments performed with low power microwaves and SNFs with silicon isolators, there was no evidence of drying of the aque-ous media. The absorption spectrum of SNFs was measured us-ing a Varian Cary 100Bio spectrophotometer. Fluores-cence measurements were undertaken using a Jaz™ spectrofluorometer (Ocean Optics, Inc., FL, USA), which allows the collection of fluorescence spectrum of samples using a fiber optic (Figure 1B). A broad spectrum halogen lamp (from Ocean Optics) was used as excitation source. A variable bandwidth filter (Ocean Optics Inc., FL, USA), which has center wavelength of 450 nm and transmission bandwidth at ~25 nm FWHM was placed in front of the excitation source. In addition a 500 nm long pass filter (Ocean Optics Inc., FL, USA) was placed in front of the detec-tor to block the excess excitation light. 

Figure 1. (A) Model bioassay constructed on SNFs. MAMEF-based bioassay was run with 30 seconds of low-power microwave heating in each step of the bioassay (total assay time: 1 minute). MEF-based bioassay was run at room temperature for 30 minutes each step (total assay time: 60 minutes). A control experiment, where b-BSA is omitted from the surface is also run to determine the background emission in the bioassays. (B) Sample geometry for fluorescence emission measurements.

3. Results and Discussion

Since the MAMEF-based bioassays employ silver nanoparticles, it is important to validate the reproduci-bility of the deposition of spherical silver nanoparticles from solution onto glass surfaces. In this regard, 10 different SNFs were prepared and the absorption spec-trum of each slide was measured (Figure 2A). As shown in Figure 2A, surface plasmon resonance (SPR) peak for SNFs occur at 430 nm and has the absorption value of 0.28 ± 0.006. The standard deviation for the SPR peak of 10 different samples was calculated to be ~%2.1, proving the highly reproducible deposition of silver nanoparticles onto amino glass slides. Figure 2B shows a typical AFM image (5x5 micrometer2) of SNFs. The horizontal cross-section in the AFM image reveals that the height of the silver nanoparticles is ~100 nm. Previous studies on MEF have shown that the optimum size for silver nanoparticles is ~100 nm [8, 10]. One of the most commonly used methods for the deposition of silver nanoparticles onto planar substrates in literature is called silver island films (SIFs). SIFs are deposited onto glass [10] (and plastic [12]) surfaces in a hetero-geneous manner  (up to %20 deviation in the wave-length of the SPR peak and broad absorption spectrum at wavelengths >500 nm). SIFs are typically used in the proof-of-principle demonstration of new methodologies, where quantitative measurements are not done [8, 13]. In this regard, the deposition of spherical silver nano-particles onto glass slides as presented in this work yield highly reliable surfaces for quantitative detection of biomolecules and analytes based on MEF. Figure 3 shows the summary of the results of the model bioassay run at room temperature (MEF-based bioassay) and with microwave heating (MAMEF-based bioassay). The total time for the bioassay was 60 minutes and 1 minute for the MEF-based and MAMEF-based bioassays, respectively. Figure 3A-Top shows the fluorescence emission spectrum of FITC measured from SNFs containing various amounts of b-BSA (in buffer concentration range: 0.01 - 5000 nM) in MEF-based bioassay. A control experiment, where b-BSA was omitted from the surface, was also run to de-termine the background emission due to the non-specific binding of FITC-Avidin to the surfaces. Figure 3A-Top shows an increase in emission intensity of FITC-avidin as the concentration of b-BSA is increased in buffer. Figure 3A-Bottom shows the emission inten-sity of FITC at 520 nm levels off when <0.1 nM and >1000 nM of b-BSA was used, indicating the con-centration range for b-BSA that can be measured by this method. The background emission intensity was significantly lower than the emission intensities meas-ured for all samples. That is, the extent of non-specific binding of FITC-avidin was minimal. Figure 3B-Top shows the fluorescence emission spec-trum of FITC measured from SNFs containing various amounts of b-BSA (in whole blood, concentration range: 0.01 - 5000 nM) in MAMEF-based bioassay. A control experiment, where b-BSA was omitted from the surface (whole blood sample was mixed with buffer without b-BSA), was also run to determine the back-ground emission due to the non-specific binding of FITC-Avidin to the surfaces. Figure 3B-Bottom shows the emission intensity of FITC at 520 nm increases as the concentration of b-BSA is increased in whole blood. The background emission intensity is ~2-fold less than the intensity measured for the lowest b-BSA concentra-tion, which implies that the lower detection limit is expected to be < 0.01 nM. In addition, since the emis-sion intensity value does not level off at 5000 nM, the upper detection limit in the MAMEF-based bioassay is predicted to be >5000 nM. It is important to note that MAMEF-based method is designed for bioassays that employ surfaces plasmon-supporting metal nanoparti-cles. It was previously shown that the sensitivity of the bioassays carried out using glass surfaces without the metal nanoparticles were significantly less than those surfaces with metal nanoparticles. [8] This is attributed to the fact that the use of plasmon-supporting metal nanoparticles results in the increase of the fluorescence emission and the temperature gradient between the aqueous medium and metal surface, as described in the Introduction section. In this regard, the use of low power microwave heating in bioassays on glass surfac-es without metal nanoparticles was not attempted in this study. In order to visually compare the results for the MEF-based and MAMEF-based bioassays, the normalized fluorescence emission intensity at 520 nm measured in MEF-based and MAMEF-based bioassays were plotted and shown in Figure 4. Figure 4 reveals that the emis-sion intensity values for the MAMEF-based bioassay (Mw 1 min, SNFs, whole blood) do not level off within the range of concentration of b-BSA studied here. On the other hand, the emission intensity values for the MEF-based bioassay (RT, SNFs, buffer) levels off at <0.1 nM and >1000 nM of b-BSA. That is, the incorpo-ration of microwave heating in MEF-based bioassays afforded for the expansion of the detectable concentra-tion range of biomolecules using MEF-based bioassays without the need for expensive more sensitive detectors and optics. One reason for the ob-served expansion of the detectable concentration range using microwave heating in MEF-based bioassays is thought to be the significant decrease in the extent of non-specific binding of biomolecules to the surface of the SNFs (and glass) [8]. It is important to note that whole blood assays were also carried out at room temperature (data not shown). In these experiments, no detectable fluorescence emis-sion was measured from the samples (similar to back-ground emission). This is attributed to the fact that whole blood completely coagulates within ~3 minutes on the surface of SNFs at room temperature. The co-agulation of whole blood results in the confinement of the b-BSA molecules in whole blood. That is, the bind-ing of b-BSA to the surface of SNFs was prevented. On the other hand, the MAMEF-based bioassays using whole blood samples were successfully completed due to the reduced assay time (30 seconds). It was previ-ously shown that 30 seconds of microwave heating of the samples in buffer provided sufficient time to allow the binding of b-BSA to the surface of SNFs [8, 14]. It is interesting to note that planar metal thin films were also used in conjunction with microwave heating in bioassays [15]. Subsequently, the data presented in the present work proves that the binding of proteins to the surface of silver nanoparticles using microwave heat-ing is possible even when mixed with whole blood, which was never shown before.

Figure 2. (A) Absorption spectrum for SNFs (the average for 10 different SNFs is plotted). (B) Atomic force mi-croscope image of typical SNFs.

Figure 3. Fluorescence emission spectrum of FITC measured from SNFs containing various amounts of b-BSA (A) in MEF-based bioassay (room temperature; no microwave heating; b-BSA in buffer, total assay time = 60 minutes), (B) in MAMEF-based bioassay (microwave heating; b-BSA in whole blood; total assay time = 1 minute)

4. Conclusions

The combined use of low power microwave heating and spherical silver nanoparticles deposited to glass slides in MEF-based bioassays for whole bloodsamples is presented. In this regard, the reproducible deposition of silver nanoparticles onto glass slides were achieved by immersing the amino-coated slides in freshly pre-pared solution of silver nanoparticles. Optical absorp-tion spectroscopy studies revealed that the surface plasmon resonance peak at 430 nm for silver nanopar-ticles deposited onto 10 different glass slides showed a minimal ~2% variation, proving the effectiveness of this straightforward deposition method. Atomic force microscopy studies showed the height of the silver nanoparticles deposited onto glass slides were ~100 nm. MEF-based detection of a model protein (b-BSA) in buffer in the concentration range of 0.1-1000 nM was achieved within 60 minutes at room temperature. On the other hand, MEF-based detection of b-BSA in whole blood samples was not successful due to the coagulation of whole blood at room temperature. The incorporation of low power microwave heating into MEF-based detection scheme (MAMEF-based bioas-say) afforded the detection of b-BSA from whole blood samples as low as 0.01 nM in 1 minute. In the MAMEF-based bioassay, the use of microwave heat-ing also affords for the expansion of the detectable concentration range of biomolecules/analytes in com-plex biological samples using MEF-based bioassays without the need for expensive detectors and optics.

Figure 4. Comparison of the normalized fluorescence emission intensity of FITC-streptavidin versus concentration of b-BSA measured in MAMEF-based (Mw, 1min) and MEF-based (RT: room temperature) bioassays. The control experiment, where b-BSA is omitted from the surface, is also shown to indicate the extent of background emission in the bioassays.

Acknowledgements

The project described was supported by Award Number 7-K25EB007565-03 from the National Institute of Biomedical Imaging and Bioengineering. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Biomedical Imaging and Bioengineering or the National Institutes of Health.

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Received 18 January, 2010; accepted 18 February, 2010; published online 5 March, 2010. 

Copyright: (c) 2010 K. Aslan. This is an open-access article distributed under the terms of the Creative Commons Attribu-tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.