Researchers at Osaka Metropolitan University along with their international collaborators report that they have developed a nanofluidic device capable of capturing single proteins stochastically and detecting them digitally at their naturally high concentrations. This advance could potentially lay the foundation for the future of personalized disease prevention and treatment.

The scientists published their study “Nanofluidic Aptamer Nanoarray to Enable Stochastic Capture of Single Proteins at Normal Concentrations” in Small.

“Single-molecule experiments allow understanding of the diversity, stochasticity, and heterogeneity of molecular behaviors and properties hidden by conventional ensemble-averaged measurements. They hence have great importance and significant impacts in a wide range of fields. Despite significant advances in single-molecule experiments at ultralow concentrations, the capture of single molecules in solution at normal concentrations within natural biomolecular processes remains a formidable challenge,” write the investigators. “

“Here, a high-density, well-defined nanofluidic aptamer nanoarray (NANa) formed via site-specific self-assembly of well-designed aptamer molecules in nanochannels with nano-in-nano gold nanopatterns is presented. The nanofluidic aptamer nanoarray exhibits a high capability to specifically capture target proteins (e.g., platelet-derived growth factor BB; PDGF-BB) to form uniform protein nanoarrays under optimized nanofluidic conditions.

Stochastic capture of single PDGF-BB molecules

“Owing to these fundamental features, the nanofluidic aptamer nanoarray enables the stochastic capture of single PDGF-BB molecules at a normal concentration from a sample with a ultrasmall volume equivalent to a single cell by following Poisson statistics, forming a readily addressable single-protein nanoarray. This approach offers a methodology and device to surpass both the concentration and volume limits of single-protein capture in most conventional methodologies of single-molecule experiments, thus opening an avenue to explore the behavior of individual biomolecules in a manner close to their natural forms, which remains largely unexplored to date.”

Integral to precision medicine is the accurate measurement of biomolecules, such as genes and proteins, within single cells. However, until now, there have been no tools capable of simultaneously handling the minuscule volume of a single cell’s content—typically on the order of picoliters (10−12 L)—and quantifying biomolecules in high-concentration cellular environments, according to the researchers.

aptamer schematic
Schematic illustration of the NANa principle for stochastically capturing and digitally detecting single proteins. [Yan Xu, Osaka Metropolitan University]

The device, named the Nanofluidic Aptamer Nanoarray (or NANa for short), is a nanochannel-based chip designed for the digital assay of individual molecules in a sample with an ultrasmall volume equivalent to that of a single cell. Using synthetic antibodies known as aptamers, NANa can stochastically capture and digitally detect single molecules of target proteins even within high-concentration samples. These aptamers, which bind to specific molecules, are densely arrayed within the nanochannels of the device.

Looking forward, the scientists plan to conduct practical demonstrations with actual cell samples, digitize the measurement data obtained, and explore the potential of integrating AI-based image recognition technology and biological big data.

“Humans are complex organisms consisting of a vast number of cells,” says Yan Xu, PhD, associate professor in the graduate school of engineering at Osaka Metropolitan University. “We hope NANa, which digitizes information on the number of biomolecules in individual cells, will serve as a bridge between life science and information science, paving the way for precision medicine in the future.”

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