Clinical Chemistry 54: 366-370, 2008.
First published December 21, 2007; 10.1373/clinchem.2007.094417
(Clinical Chemistry. 2008;54:366-370.)
© 2008 American Association for Clinical Chemistry, Inc.
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Automation and Analytical Techniques |
Electric Field–Enhanced Sensitivity of Grafted Ligands and Receptors
Ajay Singh Negi and
Ajay Kumar Sooda
Department of Physics, Indian Institute of Science, Bangalore, India.
aAddress correspondence to this author at: Department of Physics, Indian Institute of Science, Bangalore, 560012, India. Fax 91-80-23602602; e-mail asood{at}physics.iisc.ernet.in.
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Abstract
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Background: Particle-based agglutination tests consisting of receptors grafted to colloidal microparticles are useful for detecting small quantities of corresponding ligands of interest in fluid test samples, but detection limits of such tests are limited to a certain concentration and it is most desirable to lower the detection limits and to enhance the rate of recognition of ligands.
Methods: A mixture of receptor-coated colloidal microparticles and corresponding ligand was sandwiched between 2 indium tin oxide–coated glass plates. Electrohydrodynamic drag from an alternating-current electric field applied perpendicular to the plates increased the local concentration of the colloidal particles, improving the chances of ligand-receptor interaction and leading to the aggregation of the colloidal particles.
Results: With this technique the sensitivity of the ligand-receptor recognition was increased by a factor as large as 50.
Conclusions: This method can improve the sensitivity of particle-based agglutination tests used in immunoassays and many other applications such as immunoprecipitation and chemical sniffing.
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Introduction
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Ligands and receptors have a natural affinity toward each other, and in solution they combine to form large insoluble complexes, which then precipitate out. This principle has been used in diagnostic techniques in which antigens and antibodies correspond to ligands and receptors. Grafting of receptors on Brownian microparticles led to the improvement in the detection sensitivity because the larger-sized particles made it possible for even smaller aggregates to be detected by methods such as light scattering (1). These methods are called latex agglutination tests (LATs).1
Since the development of first LAT by Singer in 1956 (2), LATs continue to be widely used for the detection of small quantities of a chemical of interest in fluid test samples. Apart from clinical diagnostics involving detection of antigens and antibodies, commercial LATs are also available for other chemical analytes, e.g., human chorionic gonadotropin, rheumatoid factor (RF), and C-reactive protein. LATs are also used for analyte detection in many other applications, including veterinary medicine, plant health, law enforcement (drugs of abuse in urine), food (antibiotics in milk), and environmental science (3). LATs, being homogeneous assays, do not require washing steps before detection and hence are the simplest available tests today. Some other advantages of these tests are that the procedures are widely applicable and nonhazardous, and test results are obtained in a very short time.
LATs are based on very specific ligand-receptor recognition. These tests mostly use receptor-coated latex microparticles in suspension. On addition of analyte to this particle suspension, agglutination of these particles indicates the presence of corresponding ligand in the analyte. This agglutination happens because the ligand, which has multiple binding sites for the receptor, forms a linkage between 2 or more microparticles. The formation of aggregates is detected visually or using turbidimetry or nephelometry. The agglutination depends on the successful recognition of ligand by the receptor. Apart from other factors, reaction kinetics depend on proximity of the ligand and receptor molecules. At very low concentrations of ligand, agglutination is quite slow, resulting in poor sensitivity of latex agglutination tests. Many techniques have been tried to enhance the rate of agglutination and improve the sensitivity of the tests. The noncavitating standing-wave ultrasound has been used to increase the sensitivity of different latex agglutination tests because of the increased rate of particle collision, because grafted particles are forced into the pressure nodal regions (4). Instead of ordinary latex particles, magnetic colloids have also been used, which upon application of a magnetic field, self-assemble into linear chains, allowing the rapid formation of ligand-receptor links between pairs of neighboring particles (5). Song et al.(6) have used coplanar electric field to form chains of colloidal particles, thereby enhancing the rate of the latex agglutination reaction. In these methods the particles are brought into close contact for quite a long time compared to what would occur through Brownian collisions, thereby greatly enhancing the probability and duration of encounters between the ligand and the receptor. Coplanar electric fields have also been used by Velev et al. (7) to miniaturize the biosensors.
We report a technique that is highly effective in improving the recognition sensitivity between ligands and receptors and hence can also be used as a highly sensitive homogeneous method for biochemical analysis. Our technique involves application of alternating-current fields perpendicular to confining plates that contain latex microparticles coated with an appropriate receptor. Under a perpendicular electric field, most of the particles become confined (thereby increasing their local concentration) to a plane parallel to and situated near to one of the electrodes (gravity favors the bottom) and start coming together to form clusters (8)(9)(10). Because particles in clusters have more neighboring particles than those in chains, this method facilitates the formation of better ligand-receptor links, increasing sensitivity compared to methods in which the particles are aligned in chains. Moreover, the speed of recognition also increases because the particles move toward each other rapidly under the influence of the electric field. We used 2 experiments to test the efficacy of our method, one with colloidal particles coated with streptavidin to detect biotinylated protein–ribonuclease A (RNase A) and another applying the technique to a commercially available reagent set for RF.
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Materials and Methods
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The colloidal suspensions of polystyrene particles (diameter = 0.95 µm) coated with streptavidin with the desired volume fractions (0.2%) were prepared by adding deionized water to the stock suspension (1%) purchased from M/s Bangslab. We used biotinylated RNase A in water as a model ligand. Each molecule of RNase A has on average 5 biotin molecules. This biotinylated RNase A acts as the linkage between 2 or more streptavidin-coated latex particles. We also tested our technique on a commercial latex agglutination test kit for rheumatoid factor (Rhelax RF test, M/s Tulip). This kit comes with a Rhelax RF reagent that is a suspension of polystyrene latex particles (diameter approximately 1 µm) coated with a suitably modified Fc fraction of IgG. The reagent is standardized to detect approximately 10 IU/mL or more of RF. The kit also contains a positive control, which we used as the ligand to be detected, letting X be the concentration of ligand in this pristine control expected to be detected by the test. The Rhelax RF reagent was diluted to 1/10 of initial concentration to facilitate viewing under the microscope. To study the effect of the electric field, 1 volume (3 µL) of streptavidin-coated particles was mixed with 1 volume (3 µL) of different dilutions of biotinylated RNase A, and this mixture was sandwiched between 2 indium-tin oxide–coated glass plates, separated by a spacer of 75 µm thickness. The cell was sealed with wax to prevent evaporation of water. A function generator combined with a potentiometer was used to regulate the strength of the applied electric field. First of all, the threshold electric field (10) for aggregation of particles in the absence of any ligand was determined. An electric field approximately 0.85 times the threshold field was applied for doing the experiments in the presence of ligand. This choice of the applied field minimizes the false-positive (aggregation without ligand) signals while maximizing the sensitivity and rate of recognition. We found that a voltage of 2 V worked well in the case of streptavidin coated particles and a voltage of 3 V was optimal in the Rhelax test. To compare the effects of presence and absence of the electric field, the experiments were also carried out in identical conditions without applying any electric field. A Nikon Optiphot2-Pol polarizing microscope (in transmission mode with 40X objective) combined with a charge-coupled device video camera along with an image-grabbing card (PCI-1411, National Instruments) was used to image the particles. The images thus obtained were processed using the freely available software ImageJ (11). Application of appropriate filters and then adjustment of the threshold converts the images into binary images in which the particles/clusters are black and the background is white. Then, using the built-in tool in ImageJ, we calculated the average area per cluster.
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Results and Discussion
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In the streptavidin-coated particle system, on application of an electric field at a frequency of 1 kHz, the colloidal particles began aggregating into randomly shaped 2-day clusters on the bottom plate. These clusters stay intact even after the field is switched off, thereby confirming that the particles in the clusters are interlinked because of the streptavidin-biotin linkages. The size of clusters formed varied with the concentration of the biotinylated RNase A added. In Fig. 1
, clusters for 4 concentrations of RNase A are shown with the applied field on after 30 min of mixing the streptavidin-coated particles and biotinylated RNase A. At each concentration of RNase A, 5 images of different clusters were taken and average area per cluster was calculated. The variation of average area per cluster with the concentration of RNase A added is shown in Fig. 2
. It is clearly evident from the graph that at a concentration 10 pmol/L, the average area per cluster with field is larger than that without field. We can therefore, safely assert that the detection limit of our technique is 10–100 pmol/L. This detection limit is much better than the approximately 10 nmol/L achievable when no field is applied (see the inset of Fig. 2
).
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Figure 1. Clusters (darker regions) at different concentrations of biotinylated RNase A at an applied field of 2 V.
(A), no RNase A; (B), 10 pmol/L of RNase A; (C), 1 nmol/L of RNase A; (D), 1 µmol/L of RNase A. In the absence of the applied field the clusters are similar to the one shown in (A).
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Figure 2. Mean area per cluster is greater with the applied field (filled circles) than when field is off (open circles) after biotinylated RNase A concentration reaches 10 pmol/L.
The inset shows the mean area per cluster when no field was applied. In the absence of field, the mean area per cluster remains almost constant until the biotinylated RNase A concentration reaches approximately 1 nmol/L, and starts increasing from a biotinylated RNase A concentration of approximately 10 nmol/L. The solid lines are guides for the eye. Wherever the error bars are not visible, they are smaller than the spot size.
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In the Rhelax RF test, the aggregates formed were not confined to the bottom plate (see Fig. 3
). Therefore it was difficult to calculate the average area per cluster, because the image analysis algorithm could not identify the true clusters. Hence, we adopted another way to quantify the aggregation, as demonstrated by Anderson et al. (12) for protein aggregation. When there was no clustering, the particles were distributed uniformly throughout the cell and the transmission of light was spatially uniform, resulting in a uniformly bright image. However, with increased particle clustering, nonhomogeneity in the image brightness increased. The SDs in the pixel intensities of the image were calculated and plotted as the measure of aggregation in the system (see Fig. 4
). The mean of SDs of pixel intensities of 30 images at each concentration with and without field was taken. The images were taken after 10 min of mixing the RF reagent and the positive control in both cases with and without field. As can be seen from the graph (Fig. 4
), the electric field facilitates the aggregation even at a much higher dilution of the positive control. At a dilution of X/50, the difference in the SD between the field on and off is approximately 10%, which is well beyond the uncertainty in the measurement. Taking the starting concentration of the positive control X = 10 IU/mL, we were able to detect concentrations of 0.2 IU/mL (approximately 0.2 µg/L) [using the conversion factor for alpha-fetoprotein of 1 IU/mL = 1 ng/mL (13)]. Therefore, the field-induced aggregation has enhanced the detection sensitivity by 50 times. Because our experiments used 10 times smaller concentrations of antibody-coated latex particles, the sensitivity enhancement may be larger than 50 if the original concentration of the latex particles is used.
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Figure 3. The presence of the electric field aids in agglutination.
(A,C), V = 0 V, after 10 min. Concentration of positive control is X/50. (B,D), V = 3 V, after 10 min. Concentration of positive control X/10.
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Figure 4. The SD of pixel intensity in the presence of the applied field (filled circles) is higher than in the absence of field (empty circles).
The inset shows the ratio between the 2 cases.
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Our experiments show that a perpendicular electric field can be used to enhance the sensitivity of the latex-agglutination tests. In this technique, below the threshold field, the recognition is very specific. Although the simplicity of LATs is somewhat compromised as the electric field is applied, the sensitivity increases significantly. A microscope-based readout adds test complexity and cost compared to a visual readout, which is simpler and less expensive. On a positive note, determining aggregation by the method we describe should improve the reliability of such tests because it eliminates the need for subjective user interpretation. We have demonstrated our technique using polystyrene particles in an alternating-current field, but this technique is not restricted to alternating-current fields. Aggregation of colloidal particles has also been observed in direct-current fields (9)(14). Hence, direct-current fields can also be used to bring the particles closer and improve the sensitivity of the agglutination test. Moreover, instead of polystyrene particles, other particles such as silica can also be used (8). We have used image analysis for detecting agglutination. Other more sensitive methods for detection of agglutination can further improve the sensitivity of this method. The intensity of light scattered by particles dispersed in water depends strongly on the number of particles and the diameter of the particles. With agglutination, the number of particles decreases and their apparent diameters increase. This affects the amount of scattering, and thus changes in scattered light can be a very sensitive indication of agglutination (1). One can use a nephelometer to follow scattered light directly (15) or a spectrophotometer to measure change of absorbance of light (16). Sensitivity 10–15 times better than turbidimetry was reported with the use of particle counters to measure changes in the number of single particles or clumps of particles during agglutination (17). Although we report the application of our technique to a system of relevance to medical diagnostics, it is generic in nature and can be used for other complementary ligand-receptor systems. The receptor/ligand can be a carbohydrate molecule, thus allowing our method to be used for protein-carbohydrate recognition. This method can also be useful in immunoassays and many applications such as immunoprecipitation and chemical sniffing.
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Acknowledgments
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Grant/funding Support: A.S.N. thanks the Council of Scientific and Industrial Research, India, for a Senior Research Fellowship.
Financial Disclosures: None declared.
Acknowledgments: We thank Raghavan Varadharajan and Deepak for biotinylation of RNase A and N. V. Madhusudana for the indium tin oxide plates.
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Footnotes
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1 Nonstandard abbreviations: LAT, latex agglutination test; RF, rheumatoid factor; RNase A, ribonuclease A. 
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References
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Abstract
Introduction
