Metasurfaces‐Driven Hyperspectral Imaging via Multiplexed Plasmonic Resonance Energy Transfer

Obtaining single–molecular–level fingerprints of biomolecules and electron–transfer dynamic imaging in living cells are critically demanded in postgenomic life sciences and medicine. However, the possible solution called plasmonic resonance energy transfer (PRET) spectroscopy remains challenging due to the fixed scattering spectrum of a plasmonic nanoparticle and limited multiplexing. Here, multiplexed metasurfaces‐driven PRET hyperspectral imaging, to probe biological light–matter interactions, is reported. Pixelated metasurfaces with engineered scattering spectra are first designed over the entire visible range by the precision nanoengineering of gap plasmon and grating effects of metasurface clusters. Pixelated metasurfaces are created and their full dark‐field coloration is optically characterized with visible color palettes and high‐resolution color printings of the art pieces. Furthermore, three different biomolecules (i.e., chlorophyll a, chlorophyll b, and cytochrome c) are applied on metasurfaces for color palettes to obtain selective molecular fingerprint imaging due to the unique biological light–matter interactions with application‐specific biomedical metasurfaces. This metasurface‐driven PRET hyperspectral imaging will open up a new path for multiplexed real‐time molecular sensing and imaging methods.


Introduction
Single-molecular-level spectroscopic imaging and real-time electron-transfer dynamic imaging in living cells are critically demanded in postgenomic life sciences and medicine. Therefore, real-time monitoring of such activities inside the cell has been developed using fluorescence and chemical luminescence. [1][2][3] However, these methods require complex pretreatment and have low durability due to photobleaching, limiting the long-term monitoring of cells. To overcome these limitations and obtain real-time electronic state and electron-transfer imaging, plasmonic resonance energy transfer (PRET)-based molecular imaging using plasmonic nanoparticles as a probe has been proposed as a non-destructive and label-free method for real-time monitoring in living cells. [4] PRET-based molecular imaging enables not only quantitative biomolecule detection, but also observes dynamic biological activities in cells. [5][6][7] PRET has been demonstrated based on the direct energy transfer between the plasmonic nanoparticles and adsorbed biomolecules, which are superior for cellular compatibility. When the scattering spectrum of the plasmonic nanoparticle and the absorption spectrum of the biomolecule overlap, the scattering spectrum of the conjugated particle results in quantized plasmon quenching dips observed using a hyperspectral system. Thus, PRET can detect selective molecular fingerprints or electronic states of the corresponding molecules. The electronic energy state measurement at visible wavelengths is favorable for real-time intracellular monitoring of cellular activity, including cell apoptosis, necrosis, and electron transfer. [7,8] For instance, the quenching dips in the scattering spectrum of cytochrome c (Cyt c) change depending on the reduction and oxidation of the molecules. [7,8] Thus, PRET-based living cell imaging has shown massive potential for application in cellular imaging, systems biology, and molecular diagnostics. However, the current plasmonic nanoparticle-based PRET is limited in monitoring the large-scale spectral range of detection. The limited controls of multiplexed plasmonic nanoparticles in precision printed matter cause restricted monitoring of the entire spatial region, limiting the continuous tracking of chemicals or profiling secreted molecules of live cells. [8] Moreover, because individual plasmonic nanoparticles have specific and narrow spectral widths, only molecules with absorption peaks near the plasmon resonance of the nanoparticles can be detected, which may undermine a multiplexed PRET method for monitoring various types of molecules. Recently, a graphene-covered plasmonic nanoparticle interface was proposed for fine-tuning the scattering spectrum to increase the PRET signal. However, the spectral shift was under 50 nm, which is not applicable for multiplexed PRET. [9] Metasurfaces composed of materials with subwavelength scale structures have emerged as a breakthrough platform exhibiting various exciting optical responses that are not found in nature. [10][11][12] Thanks to strong light and metasurface interactions, the engineering of the properties of light, such as amplitude, phase, and polarization, is possible by controlling the geometry, periodicity, and material of the structures, known as meta-atoms. Therefore, many advantages, like ultracompact features, substantial field enhancement of the subwavelength resonators, and the ability to effectively manipulate light, make metasurfaces a prime candidate for future nanophotonic biosensors. [13] Exploiting the practical light engineering of metasurfaces, nanobiosensors with high-quality factors (Q factors) detecting molecular absorption fingerprint with a pixelated metasurface, and detecting biomarker-induced resonance shift by single-wavelength detection have recently been demonstrated. [14][15][16][17] These biosensing methods are highly sensitive due to the high Q factor of the nanobiosensor; however, additional antibody treatments and data post-processing steps are required. In addition, resonance shift-based detection methods lack selectivity for simultaneously detecting different biomolecules. Therefore, a new molecular absorption-based spectroscopy method with high selectivity and void of the complex additional post processes is required.
Here, we propose a metasurface-driven multiplexed nanospectroscopy platform based on PRET. The metasurface is designed for engineering the scattering spectrum using gap plasmon and grating effects. The metasurface array selectively deflects obliquely incident light toward the collection region of the objective resulting in an engineered scattering peak. The grating vector efficiently manipulates the diffracted light by controlling the periodicity of the meta-atoms in the metasurface. For simultaneous PRET detection of multiple biomolecules, we designed a pixelated metasurface consisting of 10 μm × 10 μm sized metapixels arrays. Each metapixels are composed of clustered aluminum (Al) disks with different periods that vary the resonance frequency of each metapixel. Thus, the scattering peak of the metapixels can cover the entire visible regime and effectively widen the spectral range for PRET detection (Figure 1a). When the metapixels scattering peak matches the absorption frequency of the particular conjugated biomolecule, strong PRET occurs between the metasurface and biomolecules resulting in deep quenching dips in the scattering spectrum. The extensive monitoring regime and high spatial resolution of our metasurfacebased molecular display, reaching ≈1.5 μm, advance molecular sensing by making continuous monitoring in the entire cell region possible. Moreover, PRET of multiple types of biomolecules with different absorption frequencies in the visible regime can be independently detected by the metapixels with the optimized scattering spectrum covering the visible regime.

Results and Discussions
In contrast to bright-field microscopy, dark-field microscopy blocks the central region of the incident light to make the light obliquely incident on the sample. [18] Here, the dark-field spectrum is formed by collecting the scattered light that reaches the collection region of the objective, which is determined by the numerical aperture (NA) of the objective expressed as NA = sin c (1) where c is the angle of the collection regime of the objective. Since our dark-field microscopy uses an objective with NA = 0.45 (MPlanFL N, 20×), i ranges from 40°to 55°and c ranges from 0°to 27°. To achieve a spectral peak at the desired frequency in the darkfield spectrum, we design a metasurface using Al nanodisks in a metal-insulator-metal (MIM) multilayer configuration, which produces strong gap plasmon resonances. By utilizing the gap plasmon resonance of the MIM metasurface and the diffraction generated by the periodic meta-atoms, the desired wavelengths of obliquely incident light with incident angle i ranging from 40°to 55°are selectively deflected to the collection region of the objective, resulting in engineered dark-field scattering in the visible with full dark-field coloration for each metapixels (Figure 1b).
The molecular absorption spectrum can be obtained by conventional absorption spectroscopic method; however, it does not Figure 1. Schematic illustration of the metasurfaces for multiplexed PRET molecular imaging. a) Schematic of scattering engineered metapixels in the dark-field for multiplexed nanospectroscopy based on PRET. Strong PRET occurs when the metapixels scattering peak matches the distinctive molecular absorption peaks. b) Scattering spectra of each metapixels. Blue, green, and red lines: scattering spectrum of the left, middle, and right metapixel of (a), respectively. c) Molecular absorption spectrum of different biomolecules using a conventional absorption spectroscopic method with a cuvette, which does not allow capturing electron -transfer signals of secreted biomolecules from cells. d) Scattering spectrum of the biomolecule-conjugated metapixels resulting in a quenching dip at the molecule absorption peak, which allows capturing electron-transfer signals from secreted biomolecules from cells. Blue, green, and red lines: scattering spectrum of the biomolecule-conjugated left, middle, and right metapixel of (a), respectively. allow capture of electron-transfer signals in a single molecular level ( Figure 1c). When the absorption peak of the conjugated biomolecules spectrally overlaps with the scattering peak of each metapixels, strong PRET occurs at the molecular absorption peak reducing the scattering intensity. Therefore, plasmon quenching dips in the dark-field scattering spectrum can be obtained at the molecular absorption peak near the vicinity of the metasurfaces, where electron transfer occurs [8] (Figure 1d).
To selectively diffract the target wavelength range of the obliquely incident white light to the collective objective, the metasurface consists of a MIM multilayer configuration for the formation of strong gap plasmon resonance (Figure 2a[i]). It consists of Al disk on top of an aluminum oxide (Al 2 O 3 ) spacer, and an Al film. The height of the Al disks (H), and thickness of the Al 2 O 3 layer (T) are chosen to be 44 and 5 nm, respectively, and the Al disks form a 2 × 2 clustered array to achieve an enhanced scattering induced by gap plasmon resonance ( Figure 2a[ii]). The diameter of the Al disk (D), small gap (G) between clustered Al disks, and large gap (L) between each cluster are adjusted to manipulate the wavelength of scattering peak in the dark-field scattering spectrum. The period (P) of the clustered Al disk is therefore defined as 2D + L + G. Figure 2a[iii] shows a dark-field image of the designed gap plasmon-based metasurface that exhibits a saturated green color by scattering 550 nm light to produce a peak in the dark-field spectrum. The geometric parameters are D = 100 nm, L = 240, and G = 60 nm. The bright dots in the green dark-field image are attributed to fabrication defects and specks of dust that scatter the incident white light.
Since the input light is obliquely incident, the metasurface is designed to control the −1st order diffracted light to fall within the range of the collection regime. Here, the diffraction efficiency strongly depends on the periodicity of the clustered Al disks, gap plasmon resonance, and the polarization mode of the incident light. First, the −1st order diffraction is strongly enhanced only if the incident polarization state of the oblique incident beam is TM mode ( Figure S1, Supporting Information). The 0th order reflection is similar for both TM and TE polarized incidence; however, the −1st order diffraction strongly occurs under TM polarization due to strong coupling between the incident electromagnetic waves and gap plasmons. To effectively control the wavelength selectivity of the −1st order diffraction efficiency, the effect of the Al 2 O 3 thickness T is investigated ( Figure S2, Supporting Information). When T increases, only the −1st order diffraction efficiency in the unwanted lower wavelength region increases, hindering the wavelength selective scattering peak in the visible regime. Thus, T = 5 nm is fixed to achieve a strong gap plasmon driven diffraction engineered metasurface that effectively shifts the −1st order diffraction peak wavelength when the period is modulated (Figure 2b). The higher order diffraction efficiencies are not shown since they are negligibly weak compared to −1st order diffraction.
At the −1st order diffraction peak, the 2nd order gap plasmon resonance is formed (Figure 2c). Two magnetic field maxima are formed and the electric field is highest at each side and the center of the gap, resulting in two anti-parallel current loops ( Figure S3, Supporting Information). Therefore, at the gap plasmon resonance, the incident light couples to the gap plasmon and the −1st order diffraction enhances while the 0th order reduces. In contrast to the diffraction peak, at the diffraction dip, magnetic fields form a maximum lobe at the side of the gaps extending to the air, reducing the −1st order diffraction. Thus, the metasurface allows a straightforward gap plasmonic resonance engineering that enhances −1st order diffraction. Principle of the spectrally engineered dark-field scattering spectrum for metasurface-driven PRET imaging. a) i) Schematic of dark-field microscopy. The i is 40-55°, and c is 0-27°; ii) geometric parameters of the clustered Al disks are diameter D, height H, the length between clusters L, cluster period P, Al 2 O 3 thickness T, and the gap between disks G; iii) example of the dark-field image of the metasurface selectively deflecting green wavelength regime to the collection regime. b) −1st order diffraction efficiency of the clustered disk arrayed metasurface for the i = 55°with D = 160 nm, G = 60 nm, and P ranging from 580 to 620 nm. c) Electric and magnetic fields at = 524 and = 536 nm. d) i) Wavevector diagram of the single disk arrayed metasurface showing the dark black image in the dark-field; ii) wavevector diagram of the clustered disk arrayed metasurface showing the saturated blue image in the dark-field. e) Normalized measured scattering spectrum of the metasurface with D = 120 nm, G = 20 nm, and P ranging from 440 to 540 nm. The dashed lines indicate the cut for i = 40°and 55°, respectively. f) Calculated (solid line) and measured (square) cut of the metasurface.
Due to the periodically arranged Al disks on the substrate, the diffracted light is deflected by the grating vector with the output angles calculated using the grating equation where k out , k inc , k grating , and m are the output wave vector, input wave vector, grating vector of the metasurface, and the order of diffraction, respectively. [19] The grating vector can be expressed as 2 /P and k out and k inc are denoted as 2 / . Therefore, the output angle of the −1st order diffracted light of the metasurface depends on P and . Note that diffraction only occurs when the period of the array is larger than /(1 + sin i ) which is the condition that matches the grating equation (Note S1, Supporting Information). The simulated diffraction efficiency using the finite element method also supports the diffraction condition ( Figure S4, Supporting Information). When P matches the grating equation, diffraction occurs and the output angle of −1st order diffraction increases as the wavelength increases reaching the boundary of the collection regime of the objective. This specific incident wavelength is known as cutoff wavelength ( cut ) where the −1st order-diffracted light outcouples the collection regime and is calculated using the grating equation as where c is the angle of collection regime of the objective. [18] The input wavelength exceeding the cut does not reach the collection objective. Because the i exists as a range from i,1 to i,2 , the cut also varies depending on the i . The single disk arrayed metasurface shows an example of the cut (Figure 2d[i]). At the cut , for the i,2 angle of incidence, the output angle of the −1st order diffracted light reaches the boundary of the collection regime. However, the single disk arrayed metasurface has a periodicity of P, while the clustered disk arrayed metasurface doubles the periodicity to 2P, therefore, the grating vector of the clustered disk arrayed metasurface is half of the single disk arrayed metasurface. Under the same i , the clustered disk arrayed metasurface has a larger cut than the single disk arrayed metasurface according to Equation (3). The cut of the single disk arrayed metasurface is lower than the visible regime, resulting in dark black color in the dark-field image. In contrast, the clustered disk arrayed metasurface has the cut near the blue wavelength regime, thus, deflecting the blue light to the collection regime, achieving saturated blue dark-field color (Figure 2d[ii]). Figure 2e shows the dark-field spectrum of the clustered disk arrayed metasurface. Here, P of the clusters is varied from 470 to 570 nm, and D and G of the Al clusters are fixed at 120 and 20 nm, respectively, for precise observation of the cut . The darkfield spectrum peak shifted from 500 to 600 nm, and the cut suppresses the unwanted higher wavelengths. For P = 440 nm and i = 40°, the cut is calculated as 483 nm, and for i = 55°, the cut is calculated as 560 nm using Equation (3). This agrees with the measured dark-field spectrum of the clustered metasurface with varying P. The continuous increase of the cut for the i in the range of 40-55°results in continuous decreases of the scattering intensity between the two cut . The calculated cut for i = 50°and 55°depending on the period is compared with the measured cut (Figure 2f). The cut generally agrees well, indicating the robustness of the grating equation for the diffraction of the clustered disk arrayed metasurface.
To fully utilize the −1st order diffraction and the cut of the clustered disk arrayed metasurface, a pixelated metasurface consisting of an array of 16 × 7 metapixels formed by the clustered Al disk was fabricated. The scattering peak of each metapixel is modulated over the full visible regime in the dark-field by controlling D, G, and L. The geometry-engineerable spectral modulation in the visible regime is achieved resulting in full darkfield color palettes by varying L from 180 to 330 nm, D from 100 to 180 nm, and G from 20 to 80 nm (Figure 3a). The scattering spectrum of each metapixel is measured and plotted on the CIE 1931 Chromaticity Diagram with the sRGB gamut shown as a reference (Figure 3b). The wide color gamut indicates the strong spectral engineering of our designed metasurface. In addition, we have chosen metapixels that show the scattering peak that continuously increases the wavelength from 475 to 625 nm with equal intervals. Figure 3c shows the simulated and measured scattering spectra of the chosen metapixels. For the calculation of the scattering spectra of the metasurfaces, the diffraction was averaged for i ranging from 40°to 55°, and the source spectrum was multiplied to the diffraction. Some slight wavelength shifts of the scattering spectra are observed, which can be attributed to fabrication errors in the dimensions of the Al disks slightly varying from the designed geometric parameters.
In addition, to investigate the minimum size of the metasurface arrays that can effectively manipulate diffraction, which determines the resolution of the molecular display, the minimum pixel size that can possess the designed dark-field colors is investigated ( Figure S5, Supporting Information). Three repeated clustered Al disks successfully formed the grating vector achieving the designed dark-field color. As a proof of concept of the high resolution and independent functionality of each pixel, we used the clustered Al disks to print a high-resolution art piece with saturated dark-field colors and demonstrated the famous art pieces "The Starry Night" and "Café Terrace at Night" by Vincent van Gogh (Figure 3d,e). The colors of the art pieces were matched with the experimentally obtained color of the metasurface pixel by pixel by minimizing the mean square of the difference between the RGB values. The pixel size was determined by considering the most significant period structures to repeat at least three times to produce the desired dark-field colors. Therefore, 2.5 μm × 2.5 μm sized pixels span the 500 μm × 500 μm metasurface (Figure 3f). Note that in contrast to bright-field colors, dark black colors in the dark-field can be obtained by placing blank spaces in the pixel where diffraction does not occur. The entire light at the specific pixel reflects in the 0th order direction, which outcouples to the collection objective ( Figure S6, Supporting Information).
To demonstrate the advantages of our spectrally engineerable metasurfaces for multiplexed PRET molecular imaging, multiple types of biomolecules are placed on an array of metapixels. We prepared chlorophyll a (Chl a), chlorophyll b (Chl b), and reduced Cyt c for metasurface-driven PRET measurements. The molecular absorption peak was measured for each molecule where Chl b exists at 460 and 650 nm (Figure 4a). The reduced Cyt c absorption peaks were observed at 520 and 550 nm, and 655 nm for Chl a (Figure 4b,c). Three metapixels with the scattering spectral peaks at 625, 550, and 475 nm shown in Figure 3c were chosen to overlap the absorption peak of each molecule spectrally. The biomolecules were placed on the array of metapixels in solution, and the PRET signal was measured at the specific metapixels using the dark-field hyperspectral system shown in Figure 3a. As a result, the quenching dip resulting from PRET between the metapixels and the biomolecules appeared at the molecular absorption peaks. Moreover, the quenching dip was maximized when the metapixels scattering spectrum perfectly overlapped the biomolecule absorption spectrum (Figure 4d-f). Because the metapixel with the scattering peak at 625 nm has a broad scattering spectrum, fingerprint quenching dip appeared at Chl a, Chl b, and Cyt c absorption peaks. The dark-field spectrum varied due to the different spectral positions of quenching dips. Thus, the dark-field color of the metapixel with 625 nm spectral peak significantly changed from red to yellow, saturated red, and lime for the interaction with Chl a, Chl b, and Cyt c, respectively ( Figure S7, Supporting Information). In addition, the dark-field color of the metapixel with 550 and 475 nm spectral peaks also changed depending on the type of conjugated biomolecules ( Figures S8 and S9, Supporting Information). This dark-field color change shows the potential of the metasurface as a future colorimetric detection and monitoring platform of biomolecules and understanding of biomolecular interactions.
Photobleaching of biomolecules arising due to light exposure has been characterized by monitoring scattering spectrum evolution as a function of source exposure time ( Figure S10, Supporting Information). Based on the exponential decay of the Chl a fluorescence intensity peak existing at 665 nm, we determine the photobleaching rate of 8 × 10 −5 s −1 . This photobleaching rate is low compared to those frequently observed in single-molecule studies and implies that less than 0.5% of the initial biomolecule number photobleached within one hyperspectral image acquisition. [21] In order to compare the strength of the quenching dip depending on the spectral overlap, d PRET , the scattering intensity difference of metapixels with and without the molecular interaction at the molecular absorption frequency is calculated. For a clear comparison, d PRET for metapixels that vary the spectral peak with the constant 25 nm intervals shown in Figure 3c was calculated (Figure 4g). The d PRET continuously increases as the spectral peak of the metapixels approaches the absorption frequency for each molecule and reaches the highest when the spectral peak matches the molecular absorption peak, resulting in the enhancement of the PRET sensitivity. The minus sign of d PRET for the Chl a molecule at 475-550 nm metapixels is due to the fluorescence signal of the Chl a existing at 665 nm. In addition, to investigate the sensitivity limit of our device, we conducted experiments using two different metapixels with scattering peaks at 625 and 550 nm, respectively. Minimum concentration of 4 μg mL −1 of Chl a on the metapixel resulted in a quenching dip at the absorption peak of Chl a with positive d PRET , which indicates the sensitivity of our platform of minimum measurable concentration as 4.48 μM. In contrast, no quenching dip occurred on the metapixel with a scattering peak at 550 nm. This further supports the importance of spectral matching between the metapixel and biomolecule for PRET ( Figure S11, Supporting Information). The measured results indicate that the wide engineerable range of the scattering peak of the metasurface advances PRET sensing in terms of sensitivity and multiplexity.
Moreover, to further investigate the multiplexity and the independent PRET quenching dips of different biomolecules, PRET signals were measured for mixed solutions of Chl a, Chl b, and Cyt c. The quenching dips at the molecular absorption peaks strongly appear for the mixed biomolecules solutions (Figure 4h). The d PRET for the mixed solution was calculated, showing a similar tendency to independent molecule-conjugated PRET measurement, which verifies the sensitivity increases in favor of the spectral overlap between the metasurface and molecular peak (Figure 4i). The minus sign of the Cyt c and Chl a is due to the shift of the 475 nm metapixel due to the substantial dip at the Chl b absorption peak and the fluorescence peak of the Chl a, respectively. Hence, the results indicate that the designed spectrum from metapixels can be optimized to independently measure the mixed biomolecule signals and increase the sensitivity enabling multiplexed PRET.
We further monitored the reactive oxygen species (ROS) of living cells in real-time using Cyt c, which is suitable for monitoring ROS secreted from living cells because of its absorption spectrum change depending on the redox energy state. We cultured cells on  [20] spectral-matched green-colored metasurface to monitor ROS released under different conditions via Cyt c-mediated metasurfacedriven PRET (Figure 5a). Cells were cultured on the metasurface (Figure 5b) under the following conditions: HDF as normal cells, A375P as tumor cells, and doxorubicin (DOX)-exposed A375P as drug-treated tumor cells. Previous studies have shown that tumor cells release more ROS than normal cells, and cells can release more ROS under stressed conditions. [22] In particular, DOX is well known to induce ROS production by one-electron redox cycling of anthracyclines within cells. [23] Since fully reduced Cyt c can be gradually oxidized by ROS secreted from living cells cultured on metasurface, this can be observed through changes in the quenching dip by Cyt c (Figure 5c) for DOX-exposed A375P. Zooming in on the quenching dip regime, and plotting the d PRET by the elapsed time, the changes were clearly observed as shown in Figures 5d and 5e, respectively. In contrast, the quenching dip of the HDF and A375P are barely visible as shown in Figure 5f,g.
To quantitatively compare the change of ROS secreted from cells, we plot the Δd PRET for three different conditions (Figure 5h), where Δd PRET = d PRET (t = 0) − d PRET (t) and t indicates time. The results show a clear division between three different cell conditions, thereby indicating that quantitative monitoring of ROS secreted from cells can be observed by our metasurface, signifying the availability of the metasurface as a real-time electronic energy state measurement platform.

Conclusion
We have demonstrated a label-free, non-destructive metasurfacedriven spectroscopic imaging approach using PRET that provides strong dark-field spectral engineering using a combination of gap plasmons and grating effects. Using simple dark-field microscopy, the pixelated metasurfaces can provide a spectrally engineered scattering peak covering the entire visible regime Figure 5. Metasurface-driven real-time PRET imaging to detect reactive oxygen species (ROS) secreted from cells. a) Schematic illustration of ROS monitoring of living cells on the metasurface. b) Bright-field and dark-field images of the DOX-exposed A375P cells cultured on the metasurface. c) Time-dependent spectral change of quenching dip of the DOX-exposed A375P (scale bar: 50 μm.) d) Magnified time-dependent spectral change of quenching dip of the dotted line in (c). e) Time-dependent d PRET of the DOX-exposed A375P. f,g) Time-dependent spectral change of the HDF and A375P, respectively. h) Average values of time-dependent Δd PRET . Data are presented as mean ± standard error of the mean. For reproducibility, independent experiments were repeated three times.
obtaining multiple scattering spectrums depending on the spatial positions of the metapixels to overlap the molecular absorption peak spectrally. Therefore, the sensitivity of the signals significantly increased thanks to the substantial quenching dip of PRET. Moreover, the color change due to the quenching dip of the different biomolecules exhibits the possibility of applying a colorimetric monitoring device that indicates the dynamical molecular state or types of molecules by dark-field color.
Compared to the previous individual plasmonic nanoparticlebased PRET, the versatility and robustness due to the natural formation of a 2-3 nm oxide layer of our Al-disk arrayed metasurface will provide the convenient, selective, precision detection method via multiplexed PRET. In addition, due to our chipbased PRET with periodic array of nanostructures, quantitative real-time monitoring within identical spatial regions and tracking various secreted molecular signals from live cells can be possible. Our platform can thus detect electron transfer between molecules, ROS generation of cells, and secretomes outside of the cell for cell-to-cell communication monitoring. Also, due to the chip-based PRET sensing, our platform can advance the compatibility of sample preparation and processing techniques like microfluidics and optical trapping. By broadening the scattering spectrum of the metasurface from the visible to the infrared regime, other biomolecules, including proteins, and antibodies, can also be detected by metasurface-driven PRET.
The metasurface is highly advantageous for manipulating light at the nanoscale, promising for future biosensors and imaging devices. However, low-cost and wafer-scale fabrication issues are unsolved yet. To overcome these constraints, many efforts have been proceeded by fabricating metasurfaces using nano-imprint and deep-ultraviolet lithography. [24][25][26][27] Therefore, by adopting the fabrication methods for mass production, our probing method of biological light-matter interactions in metasurfaces via multiplexed PRET nanospectroscopy will enable real-time