Researchers have created a new framework that allows them to fashion hundreds of different microscopic barcodes that incorporate both fluorescent dyes and quantum dots. The barcodes can be recognized using flow cytometry in which particles stream past a laser to be analyzed. The development could allow scientists to create a greater quantity and variety of microscopic barcodes than previously possible, with a range of potential applications including in medicine.

In an article published in a recent issue of Chemistry of Materials, researchers from Shanghai Jiao Tong University describe how they developed the barcodes, which consist of two different-sized magnetic host particles that serve as the core.

Around these cores, the researchers assemble quantum dots—very small semiconductors—that give off specific wavelengths of light. On top of these, the research team embeds carboxylated nanoparticles stained with different amounts of fluorescent dye, fluorescein isothiocyanate (FITC). The quantity of quantum dots and mixing ratios of different fluorescent particles can be varied to create the desired, and unique, barcode.

As a proof of principle, the research team created a 100-barcode library using varying levels of quantum dots and FITC-doped nanoparticles. They analyzed the barcodes using a three-channel flow cytometry system, which measures size, FITC fluorescence, and quantum dot fluorescence.

The “3D” referred to in the study are not the usual three dimensions, but rather the barcode size, fluorescence wavelength spectrum (color), and intensity of light (brightness)—criteria that can be recognized by a laser in flow cytometry. When the laser light encounters each particle, it is refracted by the quantum dots and FITC fluorescent particles within the sphere/barcode as well as the physical size of barcodes, creating a characteristic pattern of light used to identify each one.

Going forward, the system could be used to create many hundreds of barcodes. “Without any trouble, they could get 300 to 400 tags onto these barcodes,” and these could be used to simultaneously stain for or be used to recognize that many different targets, like antibodies, says Garry Nolan, a professor at Stanford University who was not involved in this study.

The researchers, led by Hong Xu, also demonstrated that the barcode system could be used to recognize five different tumor markers that were mixed together in a single vial.  

To create the tiny barcodes, the research team started with the core. These consist of one of two magnetic spheres, with respective diameters of 2.9 µm and 6.2 µm. The spheres were first “functionalized” with a polymer, polyethylenimine (PEI), which allows other materials to be added on top. Next, these spheres were mixed in a solution containing quantum dots (made of cadmium, selenium, zinc, and sulfur), which attach to the sphere surface. This process was repeated several times. To seal this layer, the researchers coated the exterior with a layer of silica and polyelectrolytes, through a condensation reaction. Next, the spheres were placed in a buffer solution of ethanesulfonic acid hydrate containing the FITC fluorescent nanoparticles, which adhere to the outer layer through chemical conjugation. Multiple rounds of washing and drying are required between most of these steps.

By varying the number and color of the quantum dots and fluorescent nanoparticles, the researchers were able to create many distinctive barcodes.

Shuming Nie, a researcher at the University of Illinois who was not involved in the study, says this layer-by-layer fabrication approach allows engineers to spatially separate quantum dots and fluorophores, thus minimizing interaction and interference. However, he says that “the overall technology involves many wet-chemistry steps, each of which looks pretty ‘messy,’” requiring a high degree of precision and effort. For that reason, he says it would be difficult to automate and he does not “anticipate this work would lead to practical applications anytime soon.”  

A good barcoding technology needs to remain stable over time. The researchers showed that after production and 110 days of storage, the fluorescent intensity of the barcodes remained unchanged through the evaluation of flow cytometry. But Nie suggests stability could still be a problem. “Its complicated surface is probably sticky and prone to fouling [via] nonspecific adsorption,” he says.

Xu says the team has been developing the system in cooperation with a Chinese biotechnology company to which it has been transferred, Xu says. It is “now at the stage of applying for clinic approval in China, [and] I believe that this technology is promising to appear on the market soon,” Xu says.

Read the abstract in Chemistry of Materials.