 After neutralizing the pH of vial 1, pH electrode was immediately cleaned and washed in DI water.
Afterward, 441 µl of purified WNA were added at a concentration of 200 µg/ml as the template. The solution was pipette mixed and left overnight to complete the reaction. Non imprinted particles (NIPs) were synthesized under identical conditions for the exception of replacing 441 µl of WNA with HEPES buffer instead. Finally, all particles were left overnight in a rotisserie at room temperature.
After synthesis, MIPs were washed by centrifuging particles at 3200 g for 5 min and discarding the supernatant, followed by addition of 1.2 ml of elution buffer to MIPs. Particles were resuspended in a vortex mixer. If large particles were noted, these were disrupted by sonicating for 1 min in a Branson 2510 sonicator. Afterward, particles were centrifuged, and washing was repeated in triplicate. Any remaining template was removed by suspending the particles in 1.2 ml of a 50% v/v mixture of glacial acetic acid and methanol for 1 h. Particles were then centrifuged, and the supernatant was discarded. Particles were then washed in 1X PBS in triplicate and resuspended in a final volume of 400 μl, no trace of protein could be detected in the supernatant using spectrophotometry with a Micro BCA kit. Particles were then stored as is at room temperature for later use. After particle washing, a total of 7 ± 2.45 mg of microparticles were collected per batch.

Particle characterization
For particle characterization, 10 μl of MIPs were pipetted directly to a 12 mm carbon conductive tab. Particles were then lightly coated with 80:20 gold/palladium target in a sputter coater. Images were obtained using a Hitachi S-4800 SEM. Zeta potential was calculated with a ZS90 zetasizer, where a 50 μl sample was suspended in 1X PBS and diluted as needed by the equipment in a folded capillary cell (DTS1070), determined by total particle count. A total of 6 MIPs, produced in different batches, were compared.

MIP binding characteristics
A LabAlliance Series 3 pump, a Rheodyne injector model 9725i, a GE Tricorn 10/50 column, a LabAlliance UV-VIS model 500 detector with a rise time set to 0.3 s and an SRI model 333 integrator for data collection was used for HPLC studies. MIPs were loaded to empty HPLC column to evaluate their performance. MIPs were compared against nonimprinted particles as controls.
A total of 2 MIP and 2 NIP columns were produced by transferring particles, synthesized as above, to an empty GE Tricorn 10/50 HPLC column. Particles were then immobilized in the column with silica gel by adding 500 µl of TEOS, followed by 100 µl of APS, 25 µl of cAPS, 200 µl of ink and 700 µl of ethanol; template was not used with the silica gel. Afterward, air was intentionally added by rough mixing the solution for a minute with a pipette. In approximately 45 min, solutions gelled in the HPLC columns encapsulating the particles. Finally, column packing was achieved by flowing 2x PBS at a maximum flow rate of 1 ml/min and a maximum column back pressure of 150 psi. If the back pressure was exceeded, the column was repacked. Column flow rates varied from 0.6 ml/min to 1 ml/min. Packed columns were then equilibrated with 2x PBS, as the mobile phase, until the absorbance at 280 nm stabilized. After equilibration, 60 μl of WNA serum was loaded to a 20 μl loop in the sample injector. The sample was then injected at a flow rate of 0.5 ml/min in 2x PBS mobile phase. The first elution peak was recorded and discarded, as it constituted rejected protein from the column. After column equilibration, the mobile phase was changed to elution buffer to release retained antibody from the column. Peaks were then recorded and collected for later analysis with western blot. The collected samples were then desalted with 1x PBS buffer in a Hitrap desalting column, to prevent protein denaturation. Samples were then compared to WNA serum and WN affinity purified antibody.
Western blot was carried with a standard SDS-page acrylamide gel. The following volumes were added per well: 10 μl of molecular weight marker in well one, 5 μl of WNA serum in well two, 20 μl of affinity purified WNA in well three and 40 μl of sample collected from pH control column in well 4. Volumes were estimated based on protein concentration of collected samples. After blotting, the gel was transferred to nitrocellulose paper and developed with fluorescently labeled mice and rabbit anti-antibodies.
We used confocal imaging to determine if MIPs specifically absorbed WNA against controls. MIPs were loaded with fluorescently labeled WNA. As controls, MIPs were loaded with either fluorescently labeled BSA or BGG. As negative controls, NIPs were incubated with fluorescently labeled WNA, BSA, or BGG. All tests were then repeated a total of 4 times. The study was carried with 30 μl of pH matched MIPs or NIPs pipetted to a 0.6 ml centrifuge tube and 20 μl of the appropriate fluorescently labeled protein. Samples were then gently mixed with a pipette and vials were left overnight in a rotisserie to reach equilibrium. Samples were then centrifuged, the supernatant discarded and particles were washed twice with 300 μl of 0.03 M NaCl. Particles were then centrifuged and resuspended in 100 μl of 0.03 M NaCl. Finally, 5 μl of each sample was loaded to a microscope glass slide and viewed under a confocal microscope.
Confocal and standard bright field images were obtained in an inverted Nikon Ti-U microscope, Nikon C1 confocal system, equipped with NIS elements and EZ-C1 software. The images were analyzed using Fiji imaging software [28]. For bright field images, all particles were counted using the particle analysis tool. For confocal images, a histogram was generated and all pixels above the signal noise were counted. The pixels of the confocal images were then multiplied by their signal intensity, added together, and divided by total black pixels obtained from their respective bright field image; the average image fluorescence intensity (AIFI) was thus obtained. After equilibration, particles were then centrifuged at 3200 g for 5 min, the supernatant collected and a micro BCA assay conducted. Particularly, centrifugation could not precipitate all the particles from the supernatant. Thus, the absorbance of the supernatant, containing MIPs without antibody, was obtained after centrifugation; a total of 6 vials were used. Afterward, the baseline was subtracted from the absorbance values of all vials. The free ligand concentration was calculated as the difference between the protein concentration in the supernatant after equilibrium and the loaded protein before equilibrium, normalized by the solution's volume. The bound ligand concentration was then determined by the difference of the total antibody mass added to the particles minus the free ligand mass, normalized by the mass of particles.

Particle immunoassay study
To determine if MIPs could be used as a fluorescent immunoassay, particles were fluorescently labeled and used in a high bind 96 well plate previously loaded with antigen. MIPs were prepared as described, with the addition of 50 µl green UV dye to vial 2. A high protein binding 96 well plate was loaded with 10 µg of purified WNA in 100 µl of HEPES buffer in 9 wells. Afterward, 10 µg of BSA was added to 100 µl of HEPES buffer in 9 wells. The process was repeated with 10 µg of BGG in 9 wells. The well plate was then left overnight in an incubator at 37°C.
BSA in HEPES buffer. The solutions were then loaded with 200 µl of MIPs each. Solutions were left incubating for 1 hour in a rotisserie. Meanwhile, the 96 well plate was washed with 200 µl of 0.01 % (w/v) Tween 20 in HEPES buffer a total of 3 times, per well.
After particle incubation, MIPs in standard buffer solution were added to three wells containing BSA, three wells containing BGG, and three wells containing purified WNA. The process was repeated for MIPs in BSA and Tween solutions. The well plate was then incubated again for two hours and washed five times with 0.01% (w/v) Tween 20 in HEPES. Finally, the well plate was viewed in an Olympus IX71 inverted microscope with a 4X objective, a UV exciter filter, and a green barrier filter block. A total of 27 images were obtained, particles were counted using color threshold and analyze particle filters.

Results and Discussion
Because of the unideal synthesis conditions, we obtained silica particles with a wide size distribution (Figure 1). Where the small particles tend to form bigger aggregates and the collection of aggregates formed µm-sized particles, which are visible to the naked eye. The broad size distribution prevented the use of light scattering techniques for determining average particles size. Nonetheless, zeta potentials could be obtained, where MIPs exhibited broad potential peaks with an average negative potential of −19.5 mV with an SD of 28.7. Due to the large zeta potential of the particles, these will aggregate easily. Thus, the majority of particles will precipitate from the solution after a given amount time.

MIP binding characteristics
The HPLC offset comparison (Figure 2), shows traces obtained from a MIP and a NIP column. The initial peak, rejected protein, was present in both columns, while the MIP column contained a small peak at an elution volume of 3.00 ml. The elution contained no measurable protein by