Top‐Down Fabrication of Ordered Nanophotonic Structures for Biomedical Applications

The size, distribution, and specific shape of ordered nanophotonic structures are crucial for their biomedical applications. Bottom‐up approaches such as self‐assembly, emulsification, and precipitation are commonly fabricated nanophotonic structures, which often lack control of nanophotonic structures morphologies and monodispersed sizes. On the contrary, top‐down nanofabrication techniques offer the advantages of high fidelity and high controllability and are employed in the fabrication of nanophotonic structures. This review focuses on top‐down nanofabrication techniques to fabricate ordered nanophotonic structures and their biomedical applications. Several top‐down approaches used in the semiconductor industry and other fields requiring micro‐ and nanopatterns are used, including electron beam lithography/ion beam lithography, photolithography, interference lithography, nanoimprint lithography, nanosphere lithography, nanotransfer lithography, and nano‐electrodeposition. Various current and emerging biomedical applications of the ordered nanophotonic structures are also covered: i) surface‐enhanced Raman scattering, ii) plasmonics, including surface plasmon resonance and localized surface plasmon resonance, and iii) fluorescence enhancement. Finally, a future perspective of nanophotonic structures fabricated by top‐down techniques in biomedical applications is also summarized.


Introduction
Nanophotonic structures are structures with a nanophotonic effect, and these structures can control the behavior of light on the nanometer scale and the interaction of nanometerscale objects with light.Nanophotonic structures have great potential in biomedical applications, such as drug delivery, [1] biosensing, [2] and diagnostic imaging. [3]To date, bottom-up approaches are still the main approaches to form nanostructures DOI: 10.1002/admi.202300856 for these applications. [4]These bottomup approaches include emulsification, [5] precipitation, [6] and self-assembly [7] of constitutional units such as block copolymers, nucleation or growth of crystals with the advantages of being readily accessible, low-cost, and productive but lacking precise control of structure shape, size, and uniformity [8] which are crucial for their employment in biomedical applications. [9]Compared with bottom-up methods, top-down approaches have the advantages of high consistency, high controllability of size, and uniformity of the pattern.However, they also have several drawbacks, such as the need for an extra step of surface functionalization, higher equipment costs, limited access to fabrication facilities, [10] and relatively lower fabrication throughput, which limit their further use in bio-applications.
Top-down lithographic methods can fabricate size-and shape-controlled nanostructures alone or in combination with other additive or subtractive fabrication techniques, such as reactive ion etching (RIE), [11] thermal/e-beam evaporation, [12] atomic layer deposition, [13] and physical vapor deposition, [14] E-beam lithography [15] and ion beam lithography can directly pattern ultrasmall structural units and fabricate masks or molds for other lithography approaches (e.g., photolithography, nanoimprint lithography, and nano-electrodeposition).However, the low throughput and high cost hinder these techniques in actual applications.Photolithography is the most reliable, productive, and convenient technique and has been well-developed and widely used by the conventional semiconductor industry and other fields requiring micro-and nanopatterns. [16]Interference lithography, as a special photolithography technique, has also been widely used for the large-area fabrication of periodic nanostructures. [17]oth the commonly used positive and negative tone photoresists can be employed for interference lithography, for example, SU-8 negative photoresists and AZ series positive photoresists.However, these photoresists are often incompatible with biomedical applications.The development process in photolithography may also cause undesirable effects on particle fabrication.Nanosphere lithography fabricates well-ordered 2D nanoparticle arrays by employing a layer of colloidal particles as an etching or a material deposition mask with the advantages of being inexpensive, simple to implement, and having highthroughput. [18]However, there is still a challenge to fabricating large-area long-range flawless nanoparticle arrays. [19]The above issues of top-down approaches can be solved by nanoimprint lithography, [20] which replicates nanopatterns from a nanostructured master mold in a simple, parallel, and cost-effective way.Recently, nanotransfer lithography and nano-electrodeposition have been developed with the advantages of cost-effectiveness, large area, but the ultrasmall patterns still depend on template fabricated by electron beam lithography or other high-resolution lithographies.Nano-electrodeposition has successfully extended solution processing to fabricate nanophotonic structures. [21]12d,22] In recent years, nanoimprint lithographies and their derivatives have been developed to realize nanopatterning on various wafer-scale substrates accurately. [23]With the employment of flexible and soft templates, the imprint process conditions are mild and compatible with various biomaterials, making it one of the most promising technologies for fabricating nanoparticles in biomedical applications with the advantages of scalability and size-and shape-controlled.This review briefly introduces several commonly used top-down lithographic techniques and compares their differences, advantages, and disadvantages.We have listed several examples of nanoparticles fabricated by top-down approaches in a variety of current and emerging biomedical applications: i) surface-enhanced Raman scattering (SERS), ii) plasmonics, including surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), and iii) fluorescence enhancement.

Overview
Ordered nanophotonic structures have attracted growing interest in many biomedical applications, such as drug delivery, [24] photothermal therapy, [25] and biosensors. [26]Each application has diverse nanostructures requirements, which may require different fabrication methods.To meet the increasing demand for fabricating ordered nanophotonic structures in various shapes, sizes, and materials, many semiconductor manufacturing nanofabrication processes [27] have been adopted to fabricate monodispersed particles for biomedical applications.This section briefly introduces several top-down fabrication methods that can be used to fabricate ordered nanophotonic structures for biomedical applications.

Charged Particle Lithography (Electron Beam and Ion Beam)
Charged particle lithography mainly contain electron beam lithography and ion beam lithography. [28]E-beam lithography (EBL) is a parallel maskless and precise lithography technique with a high-resolution below sub 10 nm for fabricating arbitrary nanometer-scale patterns and a capable method to fabricate 3D micro-and nanostructures. [29]As shown in Figure 1a-i, it works by focusing and accelerating an extremely narrow scanning ebeam toward a thin layer of exposure-sensitive resist material coated on the substrate.The electron-electron collision in the interaction volume of the incident electrons induced the modification of the resist layer's chemical properties (e.g., solubility).After development, the scanning e-beam pattern is obtained on a negative resist, and the opposite pattern can be obtained on a positive resist.However, EBL is a time-consuming and expensive lithography technique.Figure 1a-ii shows a scanning electron microscope (SEM) image of straight ridges created by EBL followed by alumina evaporation and liftoff, and Figure 1a-ii-iv shows SEM images of the EBL fabricated SU-8 lines and tubes.
Apart from e-beams, other charged particle beams could also be used as tools for lithography.For example, helium ions are implemented in the newly introduced helium ion microscope for high-resolution imaging and patterning. [30]It has been reported that helium ion beam lithography (HIBL) can attain sub-4 nm resolution by exposing a layer of hydrogen silsesquioxane (HSQ) resist with a scanning-focused helium ion beam. [31]

Photolithography
Photolithography (PL) contains optical and ltraviolet lithography, using light to transfer a geometric pattern from a photomask to a photosensitive resist on the substrate.PL is a widely used technique in micro-and nanoelectronics manufacturing due to its high-throughput, great pattern fidelity, and economic viability.It uses light to transfer a geometric pattern from a photomask to a photosensitive resist on the substrate.However, PL has reached the diffraction limit, which is determined via Abbe's law by the wavelength of the light source and numerical aperture. [45]To overcome the resolution limit in conventional PL, deep ultraviolet lithography, [46] and extreme ultraviolet lithography [47] with advanced light sources with smaller wavelengths have been developed, but the high equipment costs limit their availability and practicality in actual production.The general schematic of the PL process is illustrated in Figure 1b-i.Various structures ranging from microscale to nanoscale can be fabricated through PL.As displayed in Figure 1b-ii-v, various electrode patterns were formed in the photoresist.The resolution of PL could be enhanced down to hundreds of nanometers by utilizing phase-shift masks. [48]For example (Figure 1b-vi,vii), 300 and 150 nm lines in width were fabricated in a photoresist by exposure through a Cr mask. [34]

Interference Lithography
Interference lithography (IL) is a maskless photolithography method that fabricates high-density periodic nanoscale patterns over a large area by exposing the photoresist to coherent light waves.17a,49] Because of the ability to easily create regular nanostructures over a large area and  [32] Copyright 2019, American Chemical Society.iii,iv) SEM image of the EBL fabricated SU-8 lines and tubes.Scale bar: 1 μm.29a] Copyright 2005, write 3D period structures, interference lithography can be used in many versatile applications, including the manufacturing of plasmonic nanolasers, [50] SERS active substrates, [36] cell-based biosensors, [2a] solar cells, [51] and data storage. [52]The typical interference lithography is illustrated in Figure 1c-i, one coherent beam from a narrow-linewidth laser source is split into two or more waves.A laser beam is split into two coherent beams generated from a narrow-linewidth laser source.These two coherent beams are directed to overlap and interfere with each other, creating a spatial interference pattern consisting of periodically distributed intensity maxima and minima.Two experimental arrangements are widely used for two-beam IL.One uses a Lloyd mirror interferometer, as shown in Figure 1c-ii.Lloyd's mirror is a simple corner cube arrangement with a 90°geometry where half of the beam is reflected from the Lloyd's mirror to overlap with the other half from the original beam.Because the reflecting mirror is attached to the sample, the incidence angles of the two beams can be changed simultaneously by rotating the stage.However, increasing the pattern area by increasing the mirror size can be expensive because the exacting smoothness and flatness tolerances of the large area mirrors are difficult to achieve.As shown in Figure 1c-iii-vi, different structures from 1D nanogratings to 2D nanostructures can be fabricated although IL in the photoresist.17a]

Nanoimprint Lithography
Nanoimprint lithography (NIL) is a facile fabrication technique that relies on the direct mechanical deformation of the resist material. [53]It is widely used for the fabrication of biosensors, [54] photovoltaics, [55] bactericides, [56] and flexible electronics [57] because of its unique advantages, including high-throughput [58] and scalability, the ability to pattern a large variety of geometri-cal features and materials with very small feature sizes down to sub-5 nm, and low fabrication cost. [59]The fabrication process is shown in Figure 1d-i, A stamp mold made by lithography carrying nanopatterns is pressed into a polymer layer which is heated to above its glass transition temperature.After cooling down, the polymer solidifies, peeling off the stamp mold and leaving the imprinted patterns in the polymer.The imprinted patterns in the polymer can be the final functional nanostructure or act as an etching mask to further transfer the patterns into underlying substrates in subsequent etching steps.As displayed in Figure 1dii-v, NIL was used to fabricate uniform arrays of nanopillars that ranged in center-to-center pillar periodicities of 200, 300, 500, and 600 nm on the poly methyl methacrylate (PMMA) surface. [37]

Ultraviolet Nanoimprint Lithography
Ultraviolet nanoimprint lithography (UV-NIL) is a special type of nanoimprint lithography, UV was used to replace heat to replication nanostructures.UV-NIL is employed in the fabrication of electronic, [60] photonic, [12b] multispectral imaging, [61] LED, [62] magnetic and semiconductor devices [63] with high accuracy, various micro/nano-structures, high-resolution and throughput, and low cost of manufacture and operation at room temperature and low pressure.Unlike PL, UV-NIL uses UV exposure of all resist on the substrate and only negative resists are used.The fabrication process is shown in Figure 1e-i, a stamp mold made by lithography carrying nanopatterns is pressed into a polymer layer when UV exposure of all resist on the substrate.After UV curing, the polymer solidifies, peeling off the stamp mold and leaving the imprinted patterns in the UV resist.As shown in Figure 1eii, SEM images of UV-NIL pattern prepared using the photoresist DTDA-7.5 and Figure 1e-iii is a UV-nanoimprint nanograting pattern SEM image.SEM images of meanders and checkerboards fabricated by UV-NIL as shown in Figure 1e-iv,v, the pattern dimensions are 300 nm with a depth of 110 nm.

Nanosphere Lithography
Nanosphere lithography (NSL) is an adaptable micro-and nanofabrication technique for a large variety of nanostructures and well-ordered 2D nanoparticle arrays employing colloidal particles as an etching [64] or material deposition mask [65] with the advantages of being inexpensive, simple to implement, and highthroughput. [43]NSL can be applied to fabricate a great range of periodic nanostructures from hundreds of nanometers to several micrometers on rigid and flexible substrates, [66] which yields defect-free domains that are appropriately 10-100 μm 2 in size. [67]he nanosphere solution is deposited on the desired substrate by spin coating, drop coating, and other methods.The encapsulated colloidal pattern can be transferred to the desired substrate by etching, evaporation, sputtering, or imprinting. [27,28]Lithographic masks from colloids can be used to fabricate triangular patterns, [68] nanorings, [43,69] and pillars. [43,70]Figure 1f-i shows the typical schematic procedure of NSL.The self-assembly of polystyrene (PS) nanospheres can create a close-packed colloidal pattern, and the oxygen plasma etching of PS nanospheres can reduce the diameter of the nanospheres, as shown in Figure 1fii,iii.Triangular copper nanoparticles were obtained by evaporation and liftoff process, as shown in Figure 1f-iv,v.Nanohole, nanodisk, and nanoring cavity arrays were fabricated, as shown in Figure 1f-vi,vii.

Nanotransfer Lithography
Nanotransfer lithography (NTL, also known as nanotransfer printing) is a technique with outstanding simplicity, costeffectiveness, large area, high-resolution, and high throughput.In the nanotransfer lithography process, as shown in Figure 1g-i, metal deposition on the template first, these templates can be fabricated by EBL or other nanofabrication methods, even the flexible template. [71]Then transfer the metal nanostructures on the template to the traget substrate by put the template on the polymer with press or UV curing, polymers from polydimethylsiloxane (PDMS) to UV resist can be chose.In the last step, remove the template from the polymer, which is reusable after cleaning.Nanotransfer lithography successfully achieves different nanostructures [44] with different nanoscales in Figure 1g-iivii, Au line, Au nanomeshes, Au nanomeshes with dimensions of 800 nm (width) × 800 nm (length) × 20 nm (thickness), microcross dots (1.4 μm × 1.8 μm), micro-cross holes (1.4 μm × 1.8 μm), square nanodots (width: 800 nm, pitch: 1000 nm).

Nano-Electrodeposition
Nano-electrodeposition (NED) is a template-based fully solutionprocessed technique, which usually utilizes an inductive mask template to selectively deposit micro-and nanoscale metallic structures from electrolytes, like micromesh transparent electrodes, nanocheckerboard, [21] and nanorod arrays. [72]The advantages of NED include one-step formation and metallization, highthroughput, and high-resolution based on the template's resolution.Although, there is a little controversy about NED as a topdown fabrication method.NED can fabricate designed nanostructures depending on the template from NIL, so we regard it as a top-down fabrication technique.Furthermore, the fabricated metallic nanostructures can be further transferred to arbitrary substrates using an electro-static adhesive sacrificial carrier. [73]The templates of NED is divided into two categories, disposable [21,72] and repeatable. [74]Figure 1h-i,ii shows the fabrication process of disposable and repeatable.A typical disposable NED template fabrication process involves patterning a photoresist as an electroplating mask, followed by electroplating gold inside the exposed trenches.The photoresist is then dissolved in acetone as shown in Figure 1h-i.For the repeatable NED template, the fabrication process is depicted in Figure 1h-ii, NED template was obtained through RIE of an inductive silicon dioxide layer sputtered on conductive glass.After NED process, the gold nanostructures can be transferred to a plastic film, and the NED template can be released for another round of electrodeposition.Figure 1h-iii,iv shows the SEM micrograph of a nanocheckerboard pattern in a photoresist and the complementary gold nanocheckerboard embedded in a cyclic olefin copolymer (COC) film.

Comparison between Various Top-Down Fabrication Methods for Biomedical Applications
The most potential top-down nanofabrication methods with the great capability of controlling the nanostructures' size, distribution, and specific shape in biomedical are briefly introduced in this review.Besides these aforementioned nanofabrication methods, there are many other top-down methods invented and developed in the past decades, like focus ion beam lithography, [75] scanning probe lithography, [76] multiphoton lithography, [77] and their uses in bioapplication are still in investigation.There are some crucial parameters should be taken into consideration, containing materials, complexity, resolution, area, throughput, and cost, to select the most suitable method for fabricating nanostructures with desired properties in biomedical applications, which are reviewed as follows and compared using radar plots.

Material
The versatility of lithography material is preferred.However, there are many limitations in lithography material, like only ebeam exposure-sensitive materials could be used as the resist in the EBL process.On the contrary, any thermoplastic polymers could be used in NIL process.Sometimes, even metal [78] and glass can be used in NIL process.A 5-point scale was used to compare the versatility of materials here, ranging from limited to versatile.

Complexity
Complexity refers to the complex level of operation methods and instruments used, ranging from very high, high, medium, and low to very low.

Resolution
The resolution of these fabrication methods is reviewed and compared using a 5-point scale in the range from <10 to >1 μm.

Area
Area indicates the maximum area on the substrate that can be patterned with typical nanostructures in a reasonable time.The radar plots compared the pattern using a 5-point scale of <1 mm 2 , 1-99 mm 2 , 1-10 cm 2 , 10-100 cm 2 , and >100 cm 2 .

Throughput
Throughput is the speed of fabricating typical nanostructures.The throughput ability of the aforementioned lithography methods is described as very low, low, medium, high, and very high.

Cost
Cost is one of the crucial factors determining whether a technology can be used in mass industrial production.This problem also exists in nanostructure fabrication.Cost includes equipment and material purchase and operation costs, ranging from very high, high, medium, low, to very low.
As Figure 2a shows, EBL and HIBL are characterized as highresolution below sub 10 nm but relatively high cost, relatively low, and patterning area.On the contrary, PL (Figure 2b), IL (Figure 2c), and NSL (Figure 2f) have relatively low resolution and cost, but the throughput and total area are much higher.PL's resolution is limited by the diffraction limit, and the low resolution hinder its application in biomedical.In NSL process, 1 m × 1 m PS monolayer fabricated on a glass substrate is successfully proved by a low-cost micro-propulsive injection method. [66]s shown in Figure 2d,e,g,h, NIL, UV-NIL, NTL, and NED have the advantages of high-resolution, high-throughput, large patterning area, and high material diversity.Moreover, NIL does not require bulky equipment of electron/ion beam source, vacuum chamber, alignment, or focusing compared with IL, EBL, and HIBL.Thus the cost and complexity of NIL are relatively low.However, the resolution of NIL is determined by the pattern size of the mold, not the optical limit.And the cost of the imprinting mold should be carefully considered because it dramatically increases with the decreasing feature size and the increasing area of the mold.
Finally, the scale-up potential is essential when determining the method to fabricate nanoparticles for biomedical applications.And this issue is an overall consideration of the parameters mentioned above.Since top-down methods have great success in the semiconductor industry, there is great potential for top-down fabrication of nanoparticles to get out from laboratory to factory in the future.

Nanophotonic Structures in Biomedical Applications
Nanophotonic structures have been widely employed in biomedical applications.This chapter will briefly review nanophotonic structures' working mechanisms in biomedical applications.Plasmonics is one of the nanophotonic structures' effects since surface plasmons respond to the local refractive index with high sensitivity owing to the evanescent electromagnetic field and thus offer opportunities for label-free molecular sensing [54b,79] and chemical imaging.When incident light resonates with free electron interactions near the surface of metal nanostructures, the structural localization produces enhancement of the optical near field and the electromagnetic field.Furthermore, optical materials for dielectric metasurfaces can operate resonance peaks in the UV, VIS, and IR ranges. [80]he mechanisms of SERS are commonly regarded as the product of two contributions, an electromagnetic (EM) enhancement mechanism, and a chemical enhancement mechanism.Localized electromagnetic field enhancement, as a result of LSPR, also contributes to the enhancement factor of SERS (EF SERS ).In the EM enhancement mechanism, Raman scattering is relative to the incident EM field, E, and approximately scales as E 4 .It has been proven that enormous EM field enhancement can be found in sub-10 nm gaps and sharp nanotips, also called "hot spots." [81]y utilizing the electromagnetic field enhancement caused by plasmonic coupling, the sensitivity and accuracy of SERS can be greatly improved.
The electromagnetic field enhancement caused by plasmonic can also contribute to fluorescence enhancement.Plasmon coupling between the metal and fluorophores can increase fluorescence intensities, photostability, and distances for fluorescence resonance energy transfer, and these effects are referred to as metal-enhanced fluorescence (MEF). [82]So, the combination of fluorescence, plasmonics, and nanofabrication can fundamentally change and increase the capabilities of fluorescence technology.

Plasmonic Resonance Sensing
Plasmonic resonance is a thriving scientific and technological field attracting much attention since surface plasmons respond to the local refractive index with high sensitivity owing to the evanescent electromagnetic field and thus offer opportunities for label-free molecular sensing [54b,79] and chemical imaging.If the local enhancement of light occurs at the metaldielectric interface and propagates continuously, it is called SPR; if electromagnetic field enhancement occurs locally in the nanostructure, it is called LSPR.When LSPR is excited, the metal nanostructures absorb photon energy to produce obvious extinction characteristics and localized electromagnetic field enhancement characteristics and show peaks in the extinction spectrum and absorption spectrum.The spectral position corresponding to the peak is the surface plasmon resonance wavelength ( max ).

Surface Plasmon Resonance
SPR is a charge-density oscillation at the interface of two media with dielectric constants of opposite signs, such as, a metal and a dielectric.83c,84] The traditional method to induce SPR on the metal surface requires a glass prism with precise control of the incident light angle. [85]Compared to the prism coupling method, metallic nanostructures offer a more straightforward way for SPR to achieve chip-based, high-throughput, and cost-effective detection.
Bai Yang's group presented an effective NSL approach to fabricating geometric gradients plasmonic arrays based on ordered micro/nanostructures. [86] The fabrication process of Ag nanowell arrays with a geometric gradient is shown in Figure 3a.First, the packing structure of the PS microspheres was orderly and hexagonally close-packed, and the prepared substrate was mounted at a tilt angle of 45°and etched in an inductively coupled plasma etcher to obtain a geometric gradient.After using graded PS microsphere arrays as depositing masks and vaporing deposition of Ag, attain an Ag porous membrane with a geometric gradient.A tape removed the residual PS masks in Ag nanoholes, and then a second vapor deposition process of Ag was carried out, and then a second vapor deposition process of Ag was carried out.Graded geometric parameters of Ag nanowell arrays as a fine plasmonic "library" with an adjustable spectral range were successfully fabricated by NSL combined with inclined RIE and systematically investigated. [86]The SPR peaks along the Ag  c) The plot for the correlation between the positions and plasmon resonance wavelength on the FPLs with a lattice constant of 685 nm.Adapted with permission. [86]Copyright 2017, The Royal Society of Chemistry.
nanowell arrays with a geometric gradient shifted linearly 5 nm in the short range of 5 mm (Figure 3b,c), with a step size of 0.5 mm. [86]hilin Yang's group presented a series of periodic 2D Al plasmonic arrays by a low-cost and facile LIL technology, the plasmonic resonant modes covering in the visible-UV wavelength.The fabrication process of 2D Al plasmonic arrays is shown in Figure 4a and the SEM image is in Figure 4b.They achieved an ultranarrow linewidth of 14 nm in the near UV wavelength in experiment, demonstrated in the case of the plasmonic arrays with a period of 400 nm.Sensing performances for aqueous solutions with a low concentration of sodium citrate show a high sensitivity of 485 nm RIU −1 (Figure 4c,d), which is promising for exploring the degeneration process of the biomolecules in the UV region.

Localized Surface Plasmon Resonance
Compared with SPR biosensor, LSPR biosensor is sensitive to molecule binding to the LSPR surface but is less sensitive to bulk refractive index changes caused by the composition of the surrounding medium or temperature fluctuations. [87]Additionally, LSPR can be excited by free propagating light, but SPR cannot.LSPR biosensing can be conducted using simpler and more compact optical setups and thus can be more easily integrated with microfluidics for portable optofluidic sensing. [88]For this reason, LSPR can be employed for real-time monitoring of biomolecule binding to receptors on the metal surface and, consequently, is suitable for label-free, rapid, sensitive, and multiplexed detection of molecules. [3b,54b,79a-c,87c,89]  [50] Copyright 2019, The Royal Society of Chemistry.Tatsuro Endo's group presented a core-shell-structured gold nanocone array (AuNCA) fabricated by NIL for label-free DNA sensing in the visible wavelength spectrum (Figure 5a).79c] It achieved a very low limit of detection of 161 fM in DNA hybridization detection (Figure 5d), and 1-base mismatch DNA was successfully discriminated by using AuNCA.
12b] To extend the AFP detection limit below femtomolar concentrations, a scheme involving self-controlled detection was employed to lower the limit.12b] Wen-Di Li's group reported a novel electrodeposition-based fabrication strategy for a metasurface-based LSPR sensor with excellent performance that features a gold nanocheckerboard em-bedded in a thermoplastic COC film.Refractometric sensitivities of 435.1 RIU −1 and FoM values of 7.38 were demonstrated for the prototype LSPR sensors at 570-610 nm [21] within the visible range, which allows low analytical instrumentation cost.
As we discussed before, NSL, NIL, IL, and NED with excellent shape control and high throughput can be employed for constructing nanoscale structures in bioapplications.However, NSL does not provide nanostructures with high fidelity over a large area.The potential of NED and IL used in plasmonic sensors had been proven.However, the report of templated electrodeposition applied in plasmonic sensors still needs intensive investigation to be successfully employed.Compared to NSL, NIL, and NED, IL is less economic.So, we regard NIL is a suitable approach for fabricating uniform nanostructures over large areas.

Surface-enhanced Raman Scattering
Since SERS discovery in the 1970s as an analytical tool for the sensitive and selective detection of molecules adsorbed on The peak wavelength was redshifted with an increase in the target DNA concentration.d), A calibration curve of hybridization detection of target DNA.The peak shift was defined as the wavelength shift from the peak wavelength of probe-immobilized AuNCA.The limit of detection (3) was 161 fM.79c] Copyright 2019, American Chemical Society.
noble metal nanostructures that can achieve the single-molecule level, it has attracted a great deal of research interest. [90]In the biosensor area, various biomarkers have been reported for small molecule SERS detection, such as dopamine, [91] folic acid, [92] toxin, [93] and glucose, [87d,94] and macromolecules, such as vascular endothelial growth factor, [95] prostate-specific antigens, [96] alpha-fetoprotein, [95,97] and DNA. [98]aturally, how to prepare effective metallic nanostructures as SERS-active substrates has become a hot issue.One of the best approaches to generating efficient plasmonic surfaces relies on the colloidal synthesis of nanoparticles resulting in aggregate morphologies. [99]However, the nanosphere size dispersion and position randomness limit the reproducibility of plasmonic surfaces and prevent the full power of SERS from being realized.With the development of nanofabrication techniques, numerous assembly methods that have been used in the IC industry, such as NIL, EBL, and NSL, have been used to prepare metallic SERS substrates with various geometries and morphologies, with the ultimate detection capability achieved at the single-molecule level by optimizing their sizes, shapes, compositions, intergap distances, and dielectric environments.Moreover, many unique composite nanostructures have been reported, such as quasi-3D gold nanoring cavities, [43,69] hexagonal-packed Si nanorod (SiNR) arrays, [100] 3D hierarchical MoS2-NS@Ag-NP nanocomposites, [101] and nanopyramidal arrays [102] to provide more hot spots for higher EF SERS .
Nanostructures for SERS applications are mostly fabricated through EBL.EBL is an optimal method for fabricating engineered plasmonic substrates with high resolution below sub 10 nm, and sub 10 nm gap is proven achieve the enormous EM field enhancement of SERS.Using EBL, it is possible to fabricate uniform nanopattern substrates by controlling each particle's shape and position at the nanoscale.This approach enables the fabrication of designed plasmonic properties and a high density of hot spots nanopatterns.In addition, the main limitations of the EBL approach, resulting from the lack of cost-and time-effective schemes for device fabrication and scalability, can be overcome by efficient nanoimprint techniques with nanometer resolutions.Comparison between the spectra of the Ab + Stx2a complex and the antibody alone.Two spectral bands (460-590 and 1616-1660 cm −1 ) highlighted in yellow correlated with the interaction between the antibody and toxin.Adapted with permission. [103]Copyright 2017, American Chemical Society.
L. Petti's group proposed a 2D hybrid metallic polymeric nanostructure based on an octupolar framework (Figure 6a) with enhanced sensing properties. [103]The nanostructures' plasmonic features numerically and experimentally demonstrate the higher values of their relevant figures of merit: a SERS enhancement factor of 9 × 10 7 and an SPR bulk sensitivity of 430 nm per RIU (Figure 6b). [103]A dual resonance is exhibited in the visible and near-infrared region, enabling multispectral plasmonic analysis. [103]It is the first time to demonstrate a SERS fingerprint of Shiga toxin 2a due to the outstanding performance of a SERS-based biosensor fabricated by EBL that targets Shiga toxin 2a(Figure 6c). [103]SL is an adaptable micro-and nanoscale fabrication technique for a large variety of nanostructures and well-ordered 2D nanoparticle arrays by employing colloidal particles as etching or material deposition masks.The advantages of being inexpensive, simple to implement, and generally, high-throughput make NSL a potential candidate for SERS.
Xinju Yangu group reported large-area hexagonal-packed SiNR arrays in conjunction with AuNPs fabricated for SERS [100] as shown in Figure 7a.NSL achieved ultrasensitive molecular detection with high reproducibility and spatial uniformity. [100]A finitedifference time-domain simulation suggests that a wide range of 3D electric fields are generated along the surfaces of the SiNR array. [100]By tuning the gap and diameter of the SiNRs, the enhanced electric field's long decay length (>130 nm) makes the SERS substrate a zero-gap system for the ultrasensitive detection of large biomolecules. [100]In detecting R6G molecules, this SERS system achieved an enhancement factor of 3.3 × 10 7 .The sensitivity of the SiNR@AuNP SERS substrate was further investigated by varying the concentration of R6G molecules from 10 −6 to 10 −10 m (Figure 7d).Distinguishable signals at the concentration of 10 −10 m can still be observed, which reveals excellent Raman sensitivity in the AuNP-related SERS system.More significantly, the SERS substrate yielded ultrasensitive Raman signals on long amyloid- fibrils at the single-fibril level, which provides a promising potential for the ultrasensitive detection of amyloid aggregates related to Alzheimer's disease.Xinju Yangte group's study demonstrates that SiNRs functionalized with AuNPs may serve as excellent SERS substrates in chemical and biomedical detection.
NIL is characterized by the ability to create nanometer-sized patterns in parallel over a large area.It relies on using a mold with a nanometer-sized relief surface to create replica patterns in a substrate surface through molding. [104]The molding process of NIL is mild and compatible with various biomaterials.NIL is one of the most promising candidates for scalable, size-and shapecontrolled fabrication of biocompatible nanoparticles.However, the inherent interconnecting residual layer on the bottom of the imprinted structures is a significant challenge to forming isolated nanoparticles. [105]Several ways have been proposed to avoid this problem.
Rakesh S. Moirangthemem group reported a flexible SERS substrate with an array that NIL can fast replicate in a lowcost and reproducible fashion. [102]The fabrication method is illustrated in Figure 8a.The general fabrication process involves transferring well-aligned nanopyramid patterns onto a poly ethylene terephthalate (PET) substrate by two NIL processes.First, pouring PDMS onto a Si master to obtain a PDMS mold re-sulted in a positive replica of inverted nanopyramid arrays. [42]hen, a two-step replication process was carried out using a NIL process. [42]The PDMS mold was carefully pressed over a laminated plastic substrate using a customized T-NIL setup in the first step.In the second step, lamination plastic was used as a molding template to transfer nanopyramid patterns on UV curable resin coated on PET substrate via the UV-NIL technique.A 50 nm gold film was coated on the polymer nanopyramid array to fabricate a flexible SERS substrate.The potential capabilities of nanopyramids as SERS substrates were confirmed using Rd6G as a probe molecule (Figure 8c).The experimentally calculated SERS enhancement factor is 1.9 × 10 4 .Finally, the detection of Hb proteins down to 10 −6 m proves the biosensing potential of this proposed SERS substrate (Figure 8d).The gold-coated polymer nanopyramid arrays fabricated by NIL open the path of low-cost SERS platforms for detecting hazardous biological and chemical compounds at ultralow concentrations in practical applications.
81a] They developed and applied a second-generation S-NTL technique based on a multipurpose single-layer replica without using an additional transfer-medium, thereby significantly simplifying the overall NTL steps and enabling the prompt repetition of NTL for multilayer stacking of nanowires, which is similar to recent memory device architectures, as shown in Figure 9a,b.Compared to the average enhancement factor (AEF) of 5.2 × 10 3 and 1.7 × 10 6 were obtained from a monolayer 2D Au nanowire array and monolayer Ag head-to-tail nanorods, they report that 3D cross-point structures have increased an AEF to 4.9 × 10 4 and 1.5 × 10 7 for Au nanowires and Ag nanorods, respectively.And the maximum AEF of 4.1 × 10 7 , which allows single-molecule detection, was obtained for two-layer Ag nanorods on a continuous Ag film, as shown in Figure 9c,d.
As shown in the above works, EBL, NSL, NIL, and NTL have been employed in fabricated SERS-active substrates and have shown good performance in biomolecule detection.Among these top-down fabrication techniques, EBL achieves the highest SERS enhancement factor based on its precise shape control.NIL is a powerful technique to fabricate nanostructures over large-scale surfaces with simple, economic, and high-resolution capabilities down to 10 nm.Despite several challenges, the excellent control over nanostructure shape and size during fabrication, and substrate flexibility with fabrication reproducibility, still make them attractive and promising alternatives for designing SERS substrates.Even do not have high-resolution, the above researches proved NSL and NTL can be developed and improved to attain great performance in SERS-active substrate, making them potential methods for SERS-active substrate fabrication.

Fluorescence Enhancement
Fluorescence technology is widely applied in all aspects of biological research.However, fluorescence technology still has some detection limits in biomedical applications.For instance, the sensitivity of many clinical assays is limited by sample autofluorescence, single-molecule detection is limited by  [102] Copyright 2022, Tsinghua University Press.fluorophore brightness and photostability, and the spatial resolution of cell imaging is limited to approximately one-half of the wavelength of the incident light.Scientists found that the combination of fluorescence, plasmonics, and nanofabrication can fundamentally change and increase the capabilities of fluorescence technology.MEF-based methods have been employed in many fluorescence-based biomedical applications, such as DNA [106] and RNA [3a,107] sensing, immunoassays, [108] and fluorescencebased imaging. [109]onventional MEF substrates were fabricated by electrochemical roughening and thermal evaporation, a simple fabrication with a low enhancement factor.Several surface modification techniques [110] have been developed by carefully controlling the properties of nanoparticles deposited on the substrate.Although highly monodisperse or regular nanoparticles are used in 2D MEF platforms, the reproducibility of these complex platforms is poor due to the random distribution of metal nanoparticles.To overcome these obstacles, more sophisticated patterning techniques with precise size and shape control are adopted in 2D MEF platform fabrication, such as EBL, NSL, and NIL.By combining the unique optical properties of nanostructured gaps and nanostructures (e.g., nanotriangles, nanoholes, [111] bowtie nanoapertures [13b] ), a remarkable increase in fluorescence emission is achieved.22a] They used EBL on negative tone HSQ resist to achieve a high-patterning resolution of 10 nm.After Au evaporation, planarization, etching, HSQ removal, and template strip approaches, as shown in Figure 10a, the gold nanostructures are transferred and flipped onto a microscope coverslip to make the probe solution conveniently access the narrowest and brightest region of the nanogap.22a] Fang Xie's group presented a low-cost NSL fabrication method to produce tunable 3D gold nanohole-disc arrays (Au-NHDAs) for large near-infrared fluorescence enhancement [70] (Figure 11a).  22a] Copyright 2017, American Chemical Society. .Adapted with permission. [70]Copyright 2019, American Chemical Society.
They illustrated that Au-NHDAs with controlled structural properties and tunable optical features in the NIR windows allow a considerable NIR fluorescence enhancement (more than 400 times) due to the 3D plasmonic structural arrays that allow strong surface plasmon polariton and localized surface plasmon resonance coupling through glass nanogaps. [70]Computational electromagnetic modeling was used to provide insights into the excitation enhancement of electric field enhancement at 790 nm, which occurs due to an increase in the intensity of the electric field (Figure 11d,e).Fluorescence lifetime measurements indicate that the total fluorescence enhancement may depend on controlling excitation enhancement and therefore the array morphology. [70]Overall, this protocol generated Au-NHDAs with tunable optical features in the NIR/NIR-II windows.These optical properties could allow Au-NHDAs to be spectrally coupled with several different fluorophores throughout NIR/NIR-II, which could be crucial for developing multiplexed and multicolor biosensing applications.The extinction spectra of Au-NHDA-215 and Au-NHDA-148 are presented in Figure 11f.These arrays were selected to provide maximum spectral overlap with a commercially available NIR dye (AlexaFluor 790, Abs 782 nm/Em 805 nm;).An AlexaFluor dye was selected because, compared with other available dyes with similar excitation/emission, it is hydrophilic, more photostable, and less pH-sensitive, making it suitable for biosensing applications, and cell and tissue labeling.
For MEF to occur, fluorophores need to be positioned close to the surface of metallic nanostructures (typically in the range of 5-30 nm).In addition to the electric field enhancement, the proximity of the metal nanoparticles leads to electromagnetic coupling between the fluorescent emitter and the nanoparticle, which will modify the radiative decay rates, change the fluorescence lifetime and quantum yield, and improve the photostability of the fluorophore.The magnitude of fluorescence enhancement critically depends on the design of the plasmonic substrates.EBL can fabricate sharp edges of nanostructures or small gaps between them with a large MEF.However, the low throughput and high cost limit its use in biomedical applications.NSL is a low-cost method that allows the production of regular arrays over large surface areas (on the order of 2 × 2 cm 2 ) with flexible tuning parameters.The high-throughput feature makes it possible to fabricate tunable plasmonic arrays for considerable near-infrared fluorescence enhancement.

Summary
In conclusion, top-down nanofabrication techniques, including electron beam lithography, focused ion beam lithography, photolithography, interference lithography, nanoimprint lithography, nanosphere lithography, and nano-electrodeposition, can fabricate highly ordered nanophotonic structures on various substrates for plasmonic, surface-enhanced Raman, and fluorescence-based biomedical applications, realizing the detection of trace protein, nucleic, and other biomolecular signals.However, these nanofabrication techniques need to improve for being complex, expensive, and time-consuming for practical applications.Therefore, more convenient, cost-effective, and highthroughput nanofabrication methods should be developed to optimize nanophotonic structures and realize their potential in bioapplications, such as high-sensitivity biomolecular sensing and wearable detection for healthcare monitoring and early detection of disease.Moreover, combining spectral information of nanophotonic structures with emerging techniques, such as artificial intelligence, can develop more spectrometer-free, pointof-care, and high-throughput biomolecular detection techniques.With the development of new top-down nanofabrication techniques and high-performance nanophotonic structures, enormous wearable, affordable, and sensitive biosensors can be fabricated to improve human quality of life and well-being.

Figure 2 .
Figure 2. Comparison among the major reviewed nano-and microfabrication methods, in terms of material, complexity, resolution, area, throughput, and cost.

Figure 3 .
Figure 3. a) Schematic illustration of the fabrication of Ag nanowell arrays with a geometric gradient.b) The normalized measured reflectance spectra of ten continuous and homogeneous points in the range of 5.0 mm on FPLs with a lattice constant of 685 nm.c)The plot for the correlation between the positions and plasmon resonance wavelength on the FPLs with a lattice constant of 685 nm.Adapted with permission.[86]Copyright 2017, The Royal Society of Chemistry.

Figure 4 .
Figure 4. a) Schematic illustrations to fabricate Al plasmonic arrays by the LIL technology.b) Typical SEM image of Al plasmonic arrays with a 400 nm period.c) Reflectance spectra of plasmonic arrays that is immersed in aqueous solution with different concentrations of sodium citrate.d) The magnified spectrum of the (−1, 0) mode.The insets in (d) denote the corresponding SPR mode shift at different concentrations of sodium citrate.Adapted with permission.[50]Copyright 2019, The Royal Society of Chemistry.

Figure 5 .
Figure 5. Conceptual illustration.a) Schematic illustration of LSPR excitation by irradiation of white light on AuNCA and simulated enhanced electric field distribution.b) Absorption spectra of bare AuNCA (d = 80 nm) (black), after immobilization of probe DNA and after hybridization of each concentration of target DNA.c) Enlarged figure of a section of (b) (dotted square part).The peak wavelength was redshifted with an increase in the target DNA concentration.d), A calibration curve of hybridization detection of target DNA.The peak shift was defined as the wavelength shift from the peak wavelength of probe-immobilized AuNCA.The limit of detection (3) was 161 fM.Adapted with permission.[79c]Copyright 2019, American Chemical Society.

Figure 6 .
Figure 6.a) SEM image of morphological analysis.b) SERS spectrum achieved for a SAM of the 4-MbA molecule (red line) and the Raman spectra of 4-MbA in the bulk (blue curve), further magnified in the inset.c) Spectroscopic characterization of the hybrid nanostructure based on octupolar geometry:Comparison between the spectra of the Ab + Stx2a complex and the antibody alone.Two spectral bands (460-590 and 1616-1660 cm −1 ) highlighted in yellow correlated with the interaction between the antibody and toxin.Adapted with permission.[103]Copyright 2022, The Authors, published by American Chemical Society.

Figure 8 .
Figure 8. a) The fabrication process of pyramid arrays.b) SEM image of a gold-coated UV-cured polymer pyramid replica.c) Raman signal of Rd6G collected from Au film and Au-coated polymer nanopyramid arrays.d) SERS spectra of Rd6G absorbed on a nanopyramid substrate showing the minimum detected concentration as 10 −9 m.Adapted with permission.[102]Copyright 2022, Tsinghua University Press.

Figure 10 .
Figure10.a) The antenna fabrication process flow is performed on silicon nitride on a silicon thin film.The HSQ resist is patterned by EBL (I) followed by gold evaporation (II), a flowable oxide is spun for planarization (III) followed by etch back by Ar ion beam etching (IV), wet etching of the remaining HSQ (V), and final template stripping by UV curable adhesive (VI).b) TEM image of a 5 × 5 antenna array with a 10 nm nominal gap width.The scale bar is 500 nm.c) Scatter plot of the fluorescence enhancement versus the nanoantenna's detection volume as deduced from FCS analysis on 83 different nanoantennas.The black line fit follows a power law dependence with a fixed −2/3 exponent.d) Distribution of fluorescence enhancement factors deduced from the data.e) Distribution of the nanoantenna detection volume.Adapted with permission.[22a]Copyright 2017, American Chemical Society.

Figure 11 .
Figure 11.a) Cross section of Au-NHDA, showing the nanodiscs (NDs) deposited on the surface of the glass nanopillars and the nanohole array (NH) formed on the base of the pillars.Fluorescence enhancement was measured by immobilizing monolayers of a streptavidin (S)-functionalized nearinfrared dye (AlexaFluor 790: AF790) with self-assembled monolayers of biotin (B)-labeled bovine serum albumin (BSA) through biotin-avidin binding.b,c), SEM images of Au-NHDAs fabricated through nanosphere lithography using 280 nm polystyrene (PS) spheres with nanodisc diameters of 215 nm (Au-NHDA-215) (a) and 148 nm (Au-NHDA-148).Scale bar 400 nm.d,e), The E field enhancement was calculated at 12 nm above the surface of the Au nanodiscs for a 790 nm incident wave.f) Normalized extinction spectra of Au-NHDA-215 and Au-NHDA-148 (solid lines), and absorption and emission spectra of AlexaFluor 790 (AF790; dashed lines).Fluorescence lifetime spectra of streptavidin-conjugated AF790 monolayers immobilized on Au-NHDAs and glass substrates as a control.The dotted line shows the instrument response function (IRF).Adapted with permission.[70]Copyright 2019, American Chemical Society.