Shape-coded silica nanotubes for multiplexed bioassay: rapid and reliable magnetic decoding protocols

Abstract

Aims: The recent development of 1D barcode arrays has proved their capabilities to be applicable to highly multiplexed bioassays. This article introduces two magnetic decoding protocols for suspension arrays of shape-coded silica nanotubes to process multiplexed assays rapidly and easily, which will benefit the minimization and automation of the arrays.

Methods: In the first protocol, the magnetic nanocrystals are incorporated into the inner voids of barcoded silica nanotubes in order to give the nanotubes magnetic properties. The second protocol is performed by trapping the barcoded silica nanotubes onto streptavidin-modified magnetic beads.

Results: The rapid and easy decoding process was demonstrated by applying the above two protocols to multiplexed assays, resulting in high selectivity. Furthermore, the magnetic bead-trapped barcode nanotubes provided a great opportunity to exclude the use of dye molecules in multiplexed assays by using barcode nanotubes as signals.

Conclusion: The rapid and easy manipulation of encoded carriers using magnetic properties could be used to develop promising suspension arrays for portable bioassays.

Figure 1. Two possible protocols for the magnetic manipulation of barcoded silica nanotubes

(A) After embedding magnetic nanocrystals into the inner voids of SNTs, the template is dissolved to release barcoded magnetic nanotubes followed by modification with probe antibodies. Then, a multiplexed assay is performed using only one dye. Finally, barcoded magnetic nanotubes are isolated by applying a magnetic field and analyzed by optical microscopy. (B) After releasing the SNTs from the template, the SNTs are modified with probe proteins, and then incubated with biotinylated analytes. The specific binding between the probe proteins and the analytes gives biotin-terminated nanotubes that are trapped onto the streptavidin-modified MBs by the biotin–streptavidin interaction. Only the SNTs, which were coupled with MBs, can be isolated by applying magnetic field and then released from the MBs by breaking the bonding of streptavidin and biotin through acid treatment. The shape and amount of the SNTs address the identity and quantity of the analytes, respectively. Therefore, no fluorescence dye is necessary in this protocol. BMNT: Barcoded magnetic nanotube; MB: Magnetic bead; SNT: Silica nanotube.

Figure 2. Barcoded silica nanotubes

Transmission-electron microscopy images of (A) a bare silica nanotube and (B) a barcoded magnetic nanotube. Optical microscope images of (C) silica nanotubes and (D) barcoded magnetic nanotubes. The scale bars are 1 μm in (A & B), and 5 μm in (C & D).

Figure 3. Single- and two-plexed assays using barcoded magnetic nanotubes

(A) Test tubes for single protein detection assays (tube 1) and for multiplexed assays (tube 2). (B) Magnetic field separation of BMNTs on the wall of BioMag^® multi-6 microcentrifuge tube separator. (C) Dark field and (D) fluorescence images of the mixed BMNTs after incubation with one target analyte: red-dye-labeled antirabbit IgG. (E) Dark field and (F) fluorescence images of the mixed BMNTs after incubation with two target analytes: red-dye-labeled antirabbit IgG and red-dye-labeled antimouse IgG. The concentration of each target analyte was 10 μg/ml. The scale bar is 5 μm. BMNT: Barcoded magnetic nanotube.

Figure 4. Cancer marker detection using barcoded magnetic nanotubes

(A & B) Optical (in dark field) and (C) fluorescence microscope images of BMNTs resulted from the sandwich assay for cancer marker detection. BMNT1 and BMNT2 were modified with capture MAbs-AFP and MAbs-CEA, respectively. Only AFP was added as a target analyte in the assay solution. The MAbs of Alexa488-AFP (green) and Alexa488-CEA (green) were used as a fluorescently labeled tag molecule in order to report the interaction between BMNTs and cancer markers. (B) The result of automated identification of BMNTs by NBSee program. The codes for BMNT1 and BMNT2 are decoded as 000111 and 011111, respectively. The scale bar is 5 μm. AFP: α-fetoprotein; BMNT: Barcoded magnetic nanotube; CEA: Carcinoembryonic antigen; MAb: Monocloncal antibody.

Figure 5. Average fluorescence intensity of BMNT1 and -2 in assay 1 (dye-antirabbit IgG was used as an analyte), in assay 2 (both dye-antimouse IgG and dye-antirabbit IgG were analytes) and in cancer marker α-fetoprotein detection (α-fetoprotein was an analyte)

The fluorescence intensity was averaged with ten randomly selected nanotubes of BMNT1 and -2 in each assay. BMNT: Barcoded magnetic nanotube.

Figure 6. Proof-of-concept experiment of protocol 2 using barcoded silica nanotubes coupled with magnetic beads

Optical images of streptavidin-coated magnetic beads (MBs) in phosphate-buffered saline solution (A) before and (B) after incubation with biotin-terminated SNTs. The scale bar is 5 μm. (C) Optical and (D) transmission-electron microscope images of an assembly of MBs at the same area on an index grid where the position of each mesh is labeled with letters and numbers. The scale bar is 2 μm. (E) Optical image of SNTs that were eluted from MBs by acid, and then dried in the air after being collected by filtration. The scale bar is 5 μm. (F) Optical images of the mixture of barcoded SNTs in the bubbles of phosphate-buffered saline before incubation with MBs (top) and after elution of MBs in acid solution (bottom). SNT: Silica nanotube.

Figure 7. Relative number of silica nanotubes versus the concentration of the analyte (biotinylated rabbit IgG)

SNT1, -2 and -3 were modified with biotin, nonspecific antibody and specific antibody, respectively. A small amount of SNT1 (~1/1000 of the number of SNT3 loaded) was used as a control group to monitor biotin–streptavidin binding.