Metal-Amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA)

This paper reports the development of Metal-amplified Density Assays, or MADAs — a method of conducting quantitative or multiplexed assays, including immunoassays, by measuring metal-amplified changes in the density of biomolecule-adsorbed beads using Magnetic Levitation (MagLev). The binding of target analytes (i.e. proteins, antibodies, antigens, complementary strands of DNA) to their specific ligands immobilized on the surface of the beads, followed by a chemical amplification of the change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules and electroless deposition of metals such as gold and silver), translates analyte binding events into measurable changes in the levitation height. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the scaling dynamics of the assay. We term a MADA— when performed with antigens and antibodies— a Density-Linked ImmunoSorbent Assay, or DeLISA. As proof of principle, we conducted a competitive immunoassay to quantify the concentration of the aminoglycoside antibiotic neomycin in whole milk, and a multiplexed indirect immunoassay with colored beads to detect antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in samples of serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no power or electricity to obtain a quantitative readout of analyte concentration. Thus, the method, with further development, has the potential to be useful in resource-limited or point-of-care settings. step


Introduction
Immunoassays that do not require electricity or specialized equipment, that are capable of providing quantitative readouts, and that can detect multiple analytes in a single sample (i.e. multiplexed assays), are objectives of the rapidly developing field of point-of-care (POC) diagnostics. Lowered costs and simple procedures are especially important for assays intended for resource-limited settings 1, 2 . Polymeric particles or beads, used initially to improve the sensitivity of flocculation assays 3 , have proven to be useful alternatives to macroscopic planar surfaces (e.g. microtiter plates) for immobilizing biomolecules for a number of types of immunoassays 4,5 . The high ratio of surface-to-volume 6 , the improved kinetics of mass transport 7 , the ability to multiplex 5,[8][9][10] , the ability to separate paramagnetic beads with bound biomolecules magnetically [11][12][13] , and the development of mechanisms to automate steps by using microfluidics 13,14 or centrifugation 15 has led to successful commercial adoption [16][17][18] of beadbased assays, and to their deployment in the field 19 .
Bead-based assays are typically read out by fluorimetry 5 , chemiluminescence 20 , or colorimetry 21,22 . These methods rely on measuring the intensity of light -either generated from excitation of fluorophores or chemical reactions, or attenuated through the development of strongly absorbing colored compounds -and require electronics and signal processing to obtain a quantitative readout. Qualitative readouts, while possible with the naked eye for colorimetric assays, may be subject to the variations in human interpretation of color, or may be biased due to variations in the intensity or the quality of the ambient light 20 . Recently, an electrochemical method, which required a powered electrochemical analyzer, was used to read out quantitatively a bead-based assay 19 . A fundamentally new technique of reading bead-based assays -one that provides an alternative to electrochemical methods, and that does not employ measurements of the intensity of light -has the potential to be useful, especially if it offers a means of quantitation that does not require specialized electronics or electricity. This paper reports a bead-based assay -Metal-amplified Density Assays (MADAs)that is based on detecting a change in the density of a bead; it is read by monitoring the levitation height of polystyrene beads presenting biomolecules (e.g. proteins, antigens, antibodies, or nucleic acid) on their surfaces in a Magnetic Levitation (MagLev) device, following a step in which analyte binding-events causes electroless deposition of metal on the bead.
The MagLev device consists of two permanent magnets arranged coaxially with likepoles facing 23 . Due to opposing buoyant and magnetic forces, beads -when suspended in a paramagnetic solution in the MagLev device -levitate at a static 'levitation height' against gravity [23][24][25][26][27][28][29][30][31][32][33] . The binding of target analytes (i.e. proteins, antibodies, antigens, complementary strands of DNA) to their cognate ligands immobilized on the surface of the beads, followed by a chemically amplified change in the density of the bead (achieved by using gold nanoparticlelabeled proteins and electroless deposition of metals such as gold and silver), translates analyte binding events into measurable changes in the levitation height of the bead. This change in levitation height (the signal of the assay) depended on the concentration of analyte that was present in the sample.
To obtain an understanding of the mechanism of MADAs, we implemented a MADA that quantified the concentration of gold-labeled streptavidin using polystyrene beads with biotin on their surfaces. A minimal mathematical model, based on the diffusion-limited growth of independent hemispherical nuclei (i.e. the gold nanoparticles) on a surface, adequately described the time dynamics of the assay. Measurements of the number of adsorbed gold-labeled streptavidin molecules on the surfaces of the beads using inductively-coupled plasma mass spectrometry (ICP-MS) further matched the predictions of the minimal model. The addition of free gold nanoparticles in solution modified the deposition kinetics of gold on the beads, leading, after an initial period of development, to dramatically slower changes in levitation height. The slowing down of the kinetics suggests a means of designing time independent assays.
We term a MADA-when performed with antigens and antibodies-a Density-Linked ImmunoSorbent Assay, or DeLISA. We conducted DeLISAs to detect for the presence of small molecules and proteins (specifically antibodies) in complex biological fluids. We chose two different immunoassay formats -a competitive immunoassay to quantify the concentration of the aminoglycoside antibiotic neomycin in whole milk, and a multiplexed indirect immunoassay (including a positive and negative control) to detect antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in samples of serum. MADAs, including DeLISA, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no power or electricity to obtain a quantitative readout of analyte concentration.

Background
The MagLev device that we used was similar to the ones previously described 23,33,34 . Finiteelement simulations based on the parameters (dimensions, strength of the magnetic field, magnetic susceptibility of the solution) of this device show that, to a good approximation, the gradient of the magnetic field is linear, with a constant slope between the surface of the top magnet to the surface of the bottom magnet, and that the magnetic field is zero at the center of the device 17 . In this configuration, Eq. 1 describes the relation between the density and the levitation height of the center of volume of homogenous spherical objects (i.e. beads) in the MagLev device 23,34 .
In this equation, ρ bead (kg m -3 ) and ρ buffer (kg m -3 ) are the densities of the bead and the buffer, χ bead (χ bead ~ 10 -5 for diamagnetic objects) and χ buffer (χ buffer ~ 1.8 × 10 -4 for 1.00 M MnCl 2 23 ) are the magnetic susceptibilities (dimensionless) of the bead and the buffer, d (m) is the separation distance between the magnets, B o is the magnitude of the magnetic field at the surface of the magnets, µ 0 = 4 π × 10 −2 (N A − 2 ) is the magnetic permeability of free space, and g (m s − 2 ) is the acceleration due to gravity.
We have used MagLev in the past to measure changes in the levitation height of porous gel beads due to the binding and release of bovine carbonic anhydrase (BCA) to (aryl)sulfonamide ligands 24,35 . In one version of these assays, BCA diffuses into the beads and binds to the immobilized ligands. The accumulation of protein in the beads due to this process results in a change in the density and levitation height of the beads. Although the use of gel-immobilized ligands in MagLev provides a label-free method to detect the concentration of protein in solution, the method worked best with small proteins < 60 kDa having high diffusion constants in the pores of the beads, and required relatively long-reaction times (hours to days). These limitations made the method, without modification, unsuitable for most applications of immunoassay. In this paper, we use a chemical reaction on the surface of non-porous beads to measure the concentration of proteins in solution. As we describe in the next section, the general principles of DeLISA are similar to existing immunoassays 20 , and thus allow the adoption of established protocols and reagents. As a result, we could use commercially available primary and secondary antibody pairs that had been optimized for use in conventional ELISAs and western blots (i.e. those with a high binding constant) for DeLISA.

Results and Discussion
Principles of Metal-amplified Density Assays (MADAs) For a DeLISA, this objective is achieved by exposing the beads to secondary antibodies conjugated to gold nanoparticles after the primary incubation step. Note that DeLISA -an immunoassay that detects the binding of antigens and antibodies -is an example of a general class of other potential MADAs. We envision that assays that detect DNA or RNA, or that detect glycans or lectins could be implemented by using gold-nanoparticles modified with nucleic acids 36,37 or sugars 38 .
After the primary and secondary incubation steps, the beads are then loaded into a capillary containing a paramagnetic salt (MnCl 2 ) dissolved in a commercial gold amplification solution (the developing buffer). The gold amplification solution, which contains gold ions, a reducing agent, and various proprietary stabilizers, causes gold ions in solution to reduce to elemental gold on the surface of the gold nanoparticles 39 . Gold and silver amplification solutions are used, conventionally, to increase the size, and hence the scattering cross-section, of gold nanoparticle labels in tissue slices for optical and electron microscopy [39][40][41] , and to conduct assays with optical readouts [42][43][44] . We reasoned that in addition to increasing optical and electron contrast, the deposition of elemental gold onto the surface of an object with density lower than that of gold should lead to an increase in the density of the object.
The rate at which gold is deposited on the bead (and hence the change in density and levitation height of the bead) should, in principle, depend on the number of bound gold nanoparticles on the surface of the bead. This dependence allows a density-based assay for the concentration of a target analyte in the sample.

Principle
To test the concept of a MADA, we designed a model assay that quantified the concentration of gold-labeled streptavidin in a buffer solution using electroless deposition of gold. We used polystyrene beads with biotin covalently immobilized on their surface. We chose this model system because: i) the reagents were commercially available and well-characterized, ii) the binding of gold-labeled streptavidin to antibodies conjugated with biotin is a standard mechanism of detection in many immunoassays 20 , iii) the characteristics of the binding of biotin to streptavidin in solution are known [45][46][47] , and the binding of biotin to streptavidin-modified surfaces has been studied [48][49][50] . The binding constant for biotin and streptavidin in solution is K d ~ 10 -14 M -1 ; this reaction is generally considered a good model for irreversible binding. On a surface, binding is claimed to be less favorable-conjugation of streptavidin with fluorophores, DNA, or solid surfaces decreases the K d by about 4-5 orders of magnitude 48,49 . In contrast, antibodies have K d that range from 10 -6 M -1 to 10 -12 M -1 20 . Thus, the binding constant of the gold-labeled streptavidin is close to that expected for a tight binding antigen-antibody pair.
Antibodies with appropriate K d are selected for heterogeneous immunoassays, as part of the design of the assay, to ensure that the antibodies do not desorb significantly during wash steps.
Generally, antibodies with K d below 10 -8 M -1 are unsuitable for immunoassay 20 , due to the rapid unbinding of the antibodies during wash steps. The change in the color of the beads is indicative of the growth of the nanoparticles on the surfaces of the beads. Scanning electron micrographs of the surface of a bead exposed to 3000 pM of gold-labeled streptavidin at 0, 30 and 60 minutes confirmed that the nanoparticles on the surfaces of the beads, which were not visible prior to amplification, increased in size and eventually coalesced to form a film (Fig. 2C). Both these observations confirm that the amount of gold that was deposited on the surface of the bead increased with time in the developing buffer.

A Model for MADA
The decrease in levitation height of the beads is due to the increase in the density of the beads. In the developing buffer, the 10-nm diameter gold nanoparticles that are adsorbed on the surface of the bead -due to analyte binding events -grow in size with time due to the deposition of metallic gold from solution ( Fig. 4A). As the nanoparticles increase in size, they eventually touch and form a contiguous film on the surface of the bead (Fig. 4A). The electrochemical deposition of metal from solution onto an electrode has been studied extensively [51][52][53][54] . The initial stages of the process of deposition is typically modeled as the nucleation and growth of hemispherical nanoparticles (nuclei) on the electrode surface. The kinetics of deposition and growth in an electroless deposition system, such as in a MADA, is expected to be more complicated since the surfaces of the nanoparticles serve as both anode and cathode and as a catalyst for the deposition 55 . The addition of chemicals such as stabilizers and growth modulators likely complicate further the system 39, 55, 56 .
In this equation, C 1 is a constant that depends on experimental conditions and = .
In this equation,

Changing the Dynamics of an AuMADA by Adding Free Gold Nanoparticles in Solution
A goal of immunoassays is to obtain a time-independent readout after an initial period of development. This feature is particularly valuable in the field or in busy settings where careful control over developing times might be difficult. Current assays often have strict time limits within which the assay must be read 20 , and the MADA, due to the time-dependent deposition of metal, also has similar limitations.
We hypothesized that AuMADA might be made less dependent on time by adding free gold nanoparticles in the developing buffer. These free nanoparticles would compete for gold ions in the solution and serve as sinks. Once all the gold ions were depleted from the developing buffer, the levitation height of the beads should stop changing. Fig. 5A, and B, shows the evolution of the levitation height of beads that had been previously exposed to 3000 pM of goldlabeled streptavidin (similar to bead in Fig. 2A). The developing buffer in Fig. 5A was doped to a final concentration of 1 x 10 10 gold nanoparticles/mL and the buffer in Fig. 5B was doped to 2 x 10 10 20 nm diameter gold nanoparticles/mL. As expected, with larger concentrations of free nanoparticles in solution, the change in levitation height slowed down at an earlier time, leading to a smaller net change in the levitation height (Fig. 5B). The levitation height of the bead, however, does not stop changing completely, at least within the timescale of a 2 hour experiment. We attribute these observations to residual low concentrations of reactants that continues the deposition process on the surfaces of the beads. For example, free gold nanoparticles that increase in size sufficiently so that they sediment to the bottom of the capillary Such a study is beyond the scope of this work.

Density-linked Immunosorbent Assays, DeLISAs
In the next two sections, we demonstrate AuMADAs applied to the binding of antigens and antibodies (immunoassays), i.e. DeLISAs. Fig. 6A and B shows schematically the steps of the two immunoassays that we implemented. Both these assays are heterogeneous immunoassays, and thus have brief wash steps to remove unbound and non-specifically bound antibodies after the incubation steps 20 . Modifications of assay procedures, for example by using a gold-labeled analyte (homogenous immunoassay format) 20 , might eliminate the necessity for washing. Like other heterogeneous immunoassays, improper or excessive washing can decrease the final signal 20 of these assays.
The first assay was a quantitative competitive assay for the concentration of the small molecule antibiotic neomycin (an aminoglycoside) in milk (Fig. 6A). In this assay, the antigen was neomycin, the primary antibody was a monoclonal mouse anti-neomycin immunoglobulin G (IgG), and the secondary antibody was a polyclonal goat anti-mouse-IgG conjugated to 10 nm diameter gold nanoparticles. In a competitive immunoassay for neomycin, a fixed concentration of mouse anti-neomycin IgG was added to the samples of milk before the addition of polystyrene beads with neomycin on their surfaces. Free neomycin, if present in the milk, binds to the antineomycin IgG, thus reducing the amount of anti-neomycin antibody available for binding to the neomycin on the surfaces of the beads. The higher the concentration of free neomycin in the milk sample, the less anti-neomycin IgG is available to bind to the neomycin on the beads. Hence, the strength of a signal that is generated from the bound molecules on the bead will be inversely correlated with the concentration of free neomycin in the milk sample, i.e. a sample with no neomycin will generate the strongest signal, and samples with increasing concentrations of neomycin will generate weaker signals.
The second assay was a multiplexed indirect immunoassay for antibodies against syphilis and Hepatitis C (HepC) in serum (Fig 6B). The antigens were Treponema palladium p47 protein on polystyrene beads, which were colored blue, Hepatitis C virus NS3 protein on polystyrene beads, which were colored pink, bovine serum albumin (BSA) on polystyrene beads, which were colored red and human IgG on polystyrene beads, which were colored yellow. The red and yellow beads served as negative and positive controls respectively. The primary antibodies were what is assayed in the human serum. In a serum sample, they consist of polyclonal antibodies of varying avidity and affinity that arise due immune response to the disease causing agent 20 . The secondary antibody was polyclonal goat anti-human IgG (γ-chain specific) conjugated to 10 nm gold nanoparticles. In a properly implemented assay, the presence of the primary antibody is required to allow binding of the gold-labeled secondary antibody onto the beads. Thus, in this assay, the signal, i.e. the electroless deposition of gold and the change in levitation height, is positively correlated with the concentration of primary antibody in a serum sample.

A Quantitative Competitive DeLISA for Neomycin Residues in Whole Milk
We chose this particular assay since, i) quantitation of antibiotic residues is important in the milk industry, and tables of maximum allowable limits (MRL) of residues in milk are published 59,60 , ii) the assay must work on whole milk, which is a complex suspension of proteins, lipids and sugars, and iii) the antigen-antibody pair had been optimized by the manufacturer for ELISA. The MRL for neomycin in milk is 500 ppb 59, 60 . Indeed, beads that were exposed to the antibiotic-free milk had a levitation height of 27 mm, while the beads exposed to the sample with 500 ppb of neomycin levitated at 35 mm (they did not change levitation height). Fig. 7B shows a plot of the levitation height of beads exposed to a range of neomycin concentrations. At 30 minutes, milk samples with residues above the MRL can be distinguished from sample with residues below the MRL. The milk sample that was at the MRL could not be distinguished from those that were above the MRL. After a further 30 minutes in the levitation buffer (i.e. after a total time of 60 minutes), the beads decreased in levitation height further (Fig. 7C) and the milk sample that was at the MRL can be distinguished from those samples with antibiotic residues above the limit (Fig. 7D).

A Multiplexed Indirect DeLISA for Syphilis and Hepatitis C (HepC) in Blood Serum
Bead-based assays are particularly suited for multiplexing. Immobilizing different antigens onto distinguishable beads allows the simultaneous detection of many different disease targets from a single sample 5, 10 .  beads after 30 minutes in the developing buffer. We observed that for the complement of beads that were exposed to Sample 1, only the yellow bead (Human IgG) decreased in levitation height ( Fig. 8B). For the beads exposed to Sample 2, the pink (HCV NS3) and yellow bead (Human IgG) decreased in levitation height (Fig. 8C). For beads exposed to Sample 3, the pink (HCV NS3), blue (T. Pallidum p47) and yellow (Human IgG) beads changed levitation height (Fig.   8D). As expected, the red (BSA) beads which served as negative controls did not change levitation height for all the assays. Thus, the beads only changed levitation height when their cognate antibody was present in the serum sample. The positive and negative controls assured that the assay conditions were appropriate and served as an additional check that the deposition of gold and the change in levitation height were specific to the presence of the disease antibodies in the samples. nitric acid (70%) were purchased from EMD. We purchased 20 nm gold nanoparticles from BBI International. Square glass capillaries with an inner diameter of 2 mm were purchased from VitroCom, 1 mm inner diameter untreated micro-hematocrit polycarbonate capillaries (SafeCrit)

Conclusions
were purchased from VWR. Certified antibiotic-free whole milk (Shaw Farm Dairy, Dracut, MA) was purchased from a local store.

Procedure for Dyeing the Polystyrene Beads
For the multiplex assay, we dyed the colorless polystyrene beads to allow visual discrimination of the various disease targets. Four milligrams of dye (Sudan Red, Reactive Blue, Alazarin Yellow, or Fat Brown) was dissolved in 1.5 mL of 10:1 toluene:ethanol. After ten minutes, the dye solutions were passed through cotton filters to remove any remaining particulates.
Approximately 300 mg of polystyrene beads were then added to the dye solution and gently rocked for one hour. The beads were then thoroughly rinsed with ethanol and dried in vacuo for at least four hours (typically overnight).

Functionalization and Covalent Grafting of Proteins onto Polystyrene Beads
To functionalize the polystyrene beads with carboxylic groups, we oxidized them with a dilute solution of potassium permanganate and sulfuric acid in water 61 . It is important to stress that mixing concentrated aqueous solutions of potassium permanganate with concentrated aqueous solution of sulfuric acid is highly hazardous. In contrast, handling dilute solutions of those same reagents has not been reported to be a source of hazard. For those reasons, we first prepared a dilute aqueous solution of sulfuric acid (1.2N, 1/30 dilution, 10 mL) to which we slowly added potassium permanganate (5 w/V %) with constant stirring, thus avoiding variations in the concentration of permanganate in the reaction mixture. The polystyrene beads were suspended in this mixture, which was maintained at 60°C for 30 minutes in a water bath. During the course of the reaction, the surface of the polystyrene beads was partially oxidized to yield surface carboxylic groups, while potassium permanganate was reduced to insoluble brown manganese (IV) dioxide which deposited on the surface of the carboxylated beads. We then washed the beads by vortexing in 6 N hydrochloric acid (10 mL) to dissolve this brown manganese oxide layer. We repeated the washing step until the final solution was colorless (typically 5-6 times). We

Procedure for the Quantitative Competitive Immunoassay for Neomycin in Milk
We prepared milk samples spiked with neomycin by making a stock solution of neomycin in 10 mL of whole organic certified antibiotic-free milk at a concentration of 10,000 ppb. We then diluted this stock solution with milk to 1,000 ppb, and serially diluted to obtain a range of neomycin concentrations from 250 ppb to 1,000 ppb. The MRL of neomycin is 500 ppb, samples with concentrations far from this value are not relevant practically. We performed the competitive assay by following the manufacturer's (Silver Lake Research) protocol with minor modifications. Briefly, we diluted the monoclonal murine anti-neomycin antibodies 1:1500 times in PBS from the stock obtained from the manufacturer. We added 25 µL of the milk sample and 25 µL of the diluted antibody into an Eppendorf tube. We added seven beads functionalized with a BSA-neomycin conjugate into the Eppendorf tube and incubated for 30 minutes at room temperature. We removed the milk sample from the Eppendorf, pipetted in 100 µL of PBS and gently washed the beads by pipetting 100 µL of fresh buffer to remove all traces of milk. We then added 50 µL of 10 nm gold-labeled goat anti-mouse-IgG that was diluted 1:100 times in PBS as recommended by the manufacturer. We incubated the mixture for 30 minutes at room temperature. We removed the gold-labeled antibody, pipetted briefly with 100 µL of deionized water to remove any non-specifically bound gold-labeled antibody, and then immediately transferred the beads into an Eppendorf with developing buffer. We loaded the beads into capillaries through capillary action and placed them in the MagLev device.

Procedure for Preparing Simulated Diseased and Normal Serum Samples
Antibody concentrations can often vary widely from patient to patient and also depends strongly on the progression of the disease 20  minutes, the serum samples were pipetted out, and 100 µL of PBS buffer was pipetted in once and discarded. This step removed any remaining sample from the supernatant and removed any non-specifically bound antibodies from the surface of the beads. The beads were then exposed to gold-labeled goat anti-human IgG, and incubated for 15 minutes. The beads were transferred into 100 µL of deionized water and then immediately into the developing buffer and loaded into individual capillaries.

Preparation of the Samples for ICP-MS Analysis
A dilute solution of aqua regia was prepared by mixing trace analysis grade nitric acid, hydrochloric acid and MilliQ water in a 1:3:2 ratio. Seven beads exposed to samples of differing concentrations of gold-labeled streptavidin were placed in 0.87 mL of freshly prepared dilute aqua regia for 20 minutes. To each sample, we added 50 µL of a 1000 ppb solution of bismuth (prepared by a dilution of a 1000 ppm ICP grade standard, which was prepared by dissolving high purity bismuth metal in 5% nitric acid) in MilliQ water to serve as an internal standard. We  of a biotin-labeled polystyrene bead in the developing buffer in the MagLev device. The bead was previously exposed to a sample containing 3000 pM of gold-labeled streptavidin. The bead, which was initially colorless, changed color and decreased in levitation height, reaching the bottom of the capillary at 21 minutes. B) A bead that was previously exposed to 30 pM of goldlabeled streptavidin was still 3 mm above the bottom of the capillary after 1 hour in the developing buffer. The white substance at the bottom of the capillaries was tacky wax that was used to seal the capillaries. C) SEM images of the surface of the bead (exposed to 3000 pM gold-labeled streptavidin) at t= 0, 30, and 60 minutes. The gold nanoparticles, which were initially too small to be observed, grew and formed an almost continuous film of gold at 1 hour.
Scale bar 500 nm.    A DeLISA for neomycin in whole milk. Seven beads were exposed to samples of milk that were spiked with various concentrations of neomycin. Photographs of the levitation height of the beads after 30 minutes in the developing buffer. The left image is of beads exposed to a sample containing 0 ppb and the right image is of beads exposed to a sample containing 500 ppb of neomycin. The black arrows and the dashed rectangular boxes are guides to the eye showing the location of the beads. B) Plot of the levitation height versus the concentration of neomycin in the samples of milk at 30 minutes. The beads exposed to samples with no antibiotic residues and residues below the MRL (concentration marked with a dashed vertical line in the plots) have sunk below the beads exposed to samples with antibiotic residues above the MRL. The dotted horizontal line is a guide that shows the average levitation height of all these beads at the beginning of the development period. C, D) At 60 minutes, the milk sample at the MRL can be distinguished from the samples that exceed the MRL, while beads exposed to the milk samples with a high concentration of neomycin did not change levitation height. the yellow negative control bead decreased in levitation height for the beads exposed to Sample 1 which was negative for both syphilis and HepC, C) the pink and yellow beads decreased in levitation height for the beads exposed to Sample 2 which was positive for HepC, D) the pink, blue and yellow beads decreased in levitation height for the beads exposed to Sample 3 which was positive for both syphilis and HepC. The red beads were negative controls which should not (and did not) change in levitation height for all samples. The dashed black circles are guides to the eye showing the location of the beads. Note that the coloring of the bead disrupts the optical appearance (e.g. the apparent color, by eye) of the gold that is deposited on the surface.