Isoskine “pulling full” infrared “skill”

Infrared spectroscopy is an analytical method that identifies compounds by detecting the transition frequency of vibration/rotational energy levels inside molecules to determine the molecular structure of a substance. Its “fast” and “lossless” characteristics are very beneficial to the study of the chemical bonds and functional groups of biomolecules, so it has received widespread attention in the fields of biology and chemistry. However, the weak interaction between micron-level infrared light wavelengths and nano-level biomolecules has become a long-term difficult limit for infrared spectroscopy technology to break through. More importantly, the water environment of in situ detection of biomolecules is the biggest “taboo” of infrared spectroscopy.

To this end, the research team from the National Nanoscience Center (hereinafter referred to as the Nano center) Nanophoton Materials and Devices Laboratory (hereinafter referred to as the photonic room) has independently developed a graphene enhanced liquid phase infrared sensor, “pulling” the “skill” of the infrared spectrum. This sensor not only realizes the vibration fingerprint of in situ identification of nanoscale proteins in the physiological environment, but also innovatively adopts the method of electrical regulation to effectively eliminate water signal interference in the liquid phase environment.

On May 30, the results of the study were published online in Advanced Materials.

Schematic diagram of graphene enhanced liquid phase infrared technology and related experimental data (Courtesy of the research team)

The infrared spectrum puzzle to be solved

In biological research, proteins are complex nanoscale molecular machines, and their nanoprotein crown interfaces, the binding interface between viral protein domains and receptors, and the nanopharmaceutical targeting sites are also at the nanoscale. Yang Xiaoxia, one of the corresponding authors of the paper and a researcher at the National Nanoscience Center, told China Science Daily: “It is important to develop in situ and non-invasive detection techniques with nanoscale resolution to understand biological interfaces and processes in the physiological environment. ”

In the minds of many researchers, infrared spectroscopy, which has been widely used in the identification of substances, is highly anticipated. A beam of infrared light passes through a certain substance, when the vibration / rotation frequency of the group in the molecule of the substance is the same as the specific frequency in the infrared spectrum, the molecule will absorb the energy of the infrared light to complete the “transition”, and the wavelength of the light at that place is absorbed by the material, forming a “vibration fingerprint” with different characteristics. This is the basic principle used by infrared spectroscopy to identify compounds.

However, infrared wavelengths are generally at the micron scale, and there is a size mismatch of more than 3 orders of magnitude with nanoscale biomolecules, resulting in a very weak interaction between light and matter. At the same time, water as a polar molecule, strong infrared absorption always masks the vibrational fingerprint of the key frequency band of biomolecules.

Therefore, how to overcome the two “short boards” of weak signal and water interference has become a big challenge in the field of infrared spectroscopic detection research.

Graphene + plasma excitons

Over the years, scholars have tried everything to achieve the goal of in situ detection of biomolecules using “enhanced” infrared spectroscopy.

As a unique physical phenomenon on conductive materials, the application of “plasmons” is seen as one of the new ways to enhance infrared spectroscopy. In the plasmon phenomenon, the free charge in the incident light-driven material produces a collective oscillation of the optical frequencies, forming an electromagnetic pattern that “focuses” and “amplifies” the signal of the incident light.

At the same time, around 2010, graphene, as a new type of low-dimensional nanomaterial, gradually entered the field of vision of scientific researchers. Graphene has the advantages of monoatomic layer thickness, high carrier mobility, Dirac electron properties, and electrically tunable, making it an ideal medium for enhanced infrared spectroscopy.

Graphene + plasma radicals, what sparks will burst out? In the past, studies have proved that graphene plasmons perform well in the infrared band, which can “delineate” 90% of the electromagnetic field energy in the surface range of 10 nanometers, forming a “hot spot”, and the infrared signal of the molecule to be measured in the hot spot area is effectively amplified.

However, in practice, researchers encounter new difficulties. “While the special structure of graphene brings performance breakthroughs, it also makes its plasmon effect susceptible to strong interference from the surrounding dielectric environment.” Wu Chenchen, a doctoral student at the National Center for Nanoscience, told China Science News.

In order to solve the problem of the susceptibility of graphene plasmons to interference, since 2015, the research team of the Nanocentric Photonics Chamber has broken through the interference of the substrate dielectric environment through the study of graphene nanostructure design and plasma exciton regulation law, and has realized the high sensitivity detection of trace solid phase organic molecule films and harmful gas molecules, and the relevant research results have been published in journals such as Nature Communications and Advanced Materials.

Ideas become reality

After conquering the detection of solid and gas phase molecules, the scientific research team developed a “new skill” for graphene plasma excitons, that is, liquid phase molecular detection.

Wu Chenchen introduced that eliminating water interference is the biggest challenge encountered by molecular detection in the physiological environment. On the one hand, the electrical regulation of graphene through the electric layer can deduct the background signal outside the plasmon hot spot in situ; on the other hand, the hydrophobic surface of graphene can effectively adsorb the protein molecules in the solution to its hot spot area, and exclude the water molecules from the hot spot area, which synergizes and can effectively amplify the infrared signal of the protein molecule.

The idea seems easy, but it’s not that simple to actually make a physical object. The research team first designed an ultra-thin transmitted infrared liquid flow cell to ensure the stable optical path and high transmittance of infrared light in the liquid environment. Then, graphene nanostructures that are electrically regulated effectively in the physiological environment are constructed. After 3 years of unremitting efforts, a tunable graphene plasma excitation element enhanced liquid phase infrared sensor is finally released.

From the moment he entered the team, Wu Chenchen soaked in this experiment almost every day from morning to night, and went to the micro-nano processing laboratory and the infrared spectroscopy laboratory. “The circuit design from the infrared liquid flow cell to the sensor theoretically seems to be able to go through, but after preparing the sensor in the micro-nano processing laboratory and testing the infrared spectrum, it was found that there was no expected result.” She said, “It’s a lot of failure, repeatedly consulting the literature, talking to teachers, summarizing the reasons, redesigning and re-preparing the sensor.” Of course, she also got an unexpected harvest, micro-nano processing technology has been greatly improved.

Experiments have proved that this liquid phase infrared sensor effectively stimulates the tunable graphene plasmon response in the physiological environment, which not only successfully suppresses the signal interference in the water environment, but also improves the sensitivity of spectral detection to the level of 2 nanometers. On this basis, further experiments identified the vibration fingerprints of the nanoscale proteins “amide I band” and “amide II band” in situ, and successfully monitored the hydrogen-deuterium proton exchange process of the nano protein.

“Almost starting from scratch, looking at this step-by-step research of the real technology, there is a strong sense of achievement.” Wu Chenchen admitted.

Even more exciting is the self-designed tunable graphene plasmon-enhanced liquid-phase infrared sensor as a removable accessory that is compatible with the measurement modes of commercial microinverter spectrometers.

Wu Chenchen, a doctoral student at the National Nanoscience Center, is the first author of this paper, and Researcher Dai Qing and Researcher Yang Xiaoxia are co-corresponding authors. (Source: China Science Daily Gan Xiao)

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