Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays

In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 106 virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings.


Size Distribution of Micro-bubbles
The size distribution of the micro-bubbles was investigated by bright field microscopy. 1 µL of compacted micro-bubbles in PBS was diluted with a 5 µL PBS droplet on a glass slide. Two cover slips (0.2 mm) were placed on opposite sides of the droplet to restrict its movement. Another cover slip was placed on top of the droplet and the cover slips were gently moved to disperse the micro-bubbles evenly, thereby forming a single layer in the flat droplet. Three samples were prepared and 15 images were taken from each sample. The number and sizes of micro-bubbles in each image were counted and measured with NIH ImageJ 1.43U (Dr. Wayne Rashand, National Institutes of Health, USA). [1]
In order to deactivate uncoupled fluor molecules, 10 μL of 1.5 M hydroxylamine in PBS, pH 8.5 was added to the solution for 1 h at room temperature. To remove uncoupled fluor molecules, phage was first PEG precipitated as described above and then passed through a Zeba TM desalting column (7 KDa MW) according to the manufacturer's protocol. Fluorescently-labelled M13 particles were stored in the dark at 4°C until used. The M13 phage used here displays an AviTag peptide (a 15-amino-acid peptide that is a substrate for biotin ligase) on the phage tail protein, p3. The AviTag peptide are partially biotinylated during expression in E. coli, and can be more completely biotinylated in vitro through treatment of purified phage particles with the enzyme biotin ligase as we have previously reported. [3]

Protein Biotinylation and Antibody-HRP Conjugation
The biotinylation of BSA, HEL and antibodies was carried out with the EZ-Link sulfo-NHS-LC Biotin kit (Pierce), following the manufacturer's protocol. Briefly, the protein was mixed with sulfo-NHS-LC-Biotin in PBS pH 7.4 (mole ratios, 1:20 for HEL and BSA, and 1:5 for antibodies), and reacted at room temperature for 30 min. The uncoupled sulfo-NHS-LC-Biotin was removed with Zeba TM desalting columns (7 KDa MW) using the manufacturer's protocol. After the biotinylation, the mole ratio of biotin to proteins was estimated using 4´-hydroxyazobenzene-2-carboxylic acid (HABA) assay. The ratio for bHEL was ~2.3, for bBSA ~4.5 and for Abs ~1.1.
The biotinylated detection Abs were then conjugated to HRP through avidin-biotin linkage, by mixing with streptavidin-HRP at a mole ratio of 1:1 in PBS and reacted overnight at 4 °C.

3D Design and Printing
The FI plate and the FI accessory for iPhone 6 Plus were designed using AutoCAD 2012 (Autodesk, Inc. San Rafael, CA, USA) and 3D printed in monochrome plastic resin, using a Stratasys Dimension 1200 3D printer (Eden Prairie, MN, USA), at the UH NSMIT Technology Center (University of Houston, Houston, TX, USA).
The FI plate was designed in 96-well format with standard microtiter spacings to hold the PCR tubes optically-isolated and upside down. As shown in Scheme 1C, the FI plate consists of two parts, the PCR tube holder with 96 wells to hold the PCR tubes containing the reagents of the FIs, and the base for the adaption to the sample inlet of the plate reader. Each well in the PCR holder consists of two parts with different shapes. The top part is of a cylindrical shape, while the bottom part is in the shape of a standard PCR tube. The two parts are connected with a circular window with 3 mm diameter. A PCR tube is inserted into the well from the bottom part and chucked by it, with only one millimeter of its tip protruding into the top part of the well. The three mm high walls of the top parts ensure the protruding tips of the PCR tubes are optically-isolated from each other.
The FI accessory was designed to attach to an iPhone 6 Plus in alignment with the main camera.
We used the Mpow clip-on detachable 10×lens (Mpow Co. Santa Clara, CA, USA) to achieve optimum focus. As shown in Scheme 1D, the accessory consists of one dark box to eliminate the influence of the environmental light, one PCR tube holder to hold the PCR tubes containing the FI reagents, a set of spacers to adjust the focus and a cap to seal the dark box. The accessory is locked to the Mpow lens, which is further attached to the phone with its clip-on design.
Original files of the 3D drawings for the 3D printed FI plate and smart phone FI accessory are available at the following URL: https://drive.google.com/folderview?id=0B7XQKZ2u-Yg0fmIxcE9wNHZlaDhqQmdvWlR4NUFOUjl OWERHYjJET3MxMC1Uc3BibmNTZjQ&usp=sharing

Norwalk virus-like particles
Norwalk virus-like particles (VLPs) were expressed, assembled and purified as reported previously. [4] Briefly, the major capsid proteins (VP1 and VP2) were expressed from a baculovirus vector in Sf9 insect cells. The VLPs were purified using a cesium chloride gradient, and the structure was confirmed by transmission electron microscopy. The VLPs were stored in PBS at 4 °C.

FI for hCG detection in human serum
1 μL of compact immuno-microbubbles was pre-mixed with 20 μL of 100 pM HRP reporters in an optical PCR tube. 10 μL of sample (hCG in 20% human serum in PBS) was added to the PCR tube and incubated on a rotator 10 rpm (30 min). All other experimental details were as described in the main text. Figure S1. Optical blocking of the luminescent signal by 1 μL microbubble layer in PCR tubes. Data represent the mean values ± the standard deviation obtained using three different PCR tubes.       Figure S8. A) Signal profiles of the FI for human chorionic gonadotropin (hCG) detection in 20% human serum. D) Linear response of the maximum CL signal to the amount of hCG in 20% human serum. Mean ± standard deviation; n = 3.