Instant formation of nanopores on flexible polymer membranes using intense pulsed light and nanoparticle templates

ABSTRACT The development of simple and high-throughput approaches to yield solid-state nanopores on large surface membranes may facilitate the prevalence of nanopore analysis technology and in-vitro diagnosis using portable devices. However, solid-state nanopores are typically realized by complex and high-end nanofabrication equipments. Here, we present a method to achieve nanopores on polymer membranes using silver nanoparticles (AgNPs) as templates and intense pulsed light (IPL) as a heating source. The density and size of nanopores are controllable by adjusting the spin coating rate, the concentration of nanoparticle suspension, and the size of nanoparticles (NPs). The temperature of the AgNPs can rapidly reach 1132 K under instant heating of photothermal effect through light irradiation in 2 ms, resulting in localized melting and decomposition of an underneath polycarbonate (PC) membrane to yield nanopores with sizes ranging from 10 to 270 nm. After removing the nanoparticle residues, the flexible membrane with nanopores can be integrated into a flow cell to achieve a nanopore sensor that has been used to measure the translocation behaviors of bovine serum albumin (BSA). The results have demonstrated the capability of the sensor in protein denaturation identification. This low-cost and high-throughput technique to fabricate solid-state nanopores on flexible polymeric membranes may facilitate the development of more nanopore-based flexible sensors that can be integrated with other flexible components for wearable diagnosis. GRAPHICAL ABSTRACT


Introduction
Flexible wearable sensors for chemical detection are currently primarily achieved by amperometric [1,2], potentiometric [3,4], voltammetric [5,6], fluorescent [7][8][9], colorimetric [10,11], and surface-enhanced Raman scattering [12,13] approaches.In comparison with many analytical methods such as mass spectrometry, chromatography, spectroscopy, and nuclear magnetic resonance that are typically achieved by bulky equipment, nanopore detection, which is a rapid and high throughput approach to characterize nanomaterials through their translocation behaviors holds the promise to be miniaturized with assistance from nanofabrication technology and advanced application specific integrated circuits [14].The changes in ionic current between the two sides of the nanopores can be used to reflect the volumes and charges of the nanomaterials, while the duration time of translocation can determine the length of the nanomaterials.The specificity in the nanopore signals can be applied to conduct DNA sequencing [15,16], protein recognition [17][18][19], nanoparticle size determination [20,21], and surface charge detection [22] using either biological nanopores derived from natural proteins in lipid membranes or solidstate nanopores based on synthesized materials.Compared with the biological nanopores that are prevalent in commercial nanopore detection systems, the solid-state nanopores hold the promise of high mechanical and chemical stability and possess a wide range of candidate materials, such as silicon dioxide [23], silicon nitride [17,22,[24][25][26], alumina [19,27], graphene [15,28,29], and polymer [30].In addition, the specificity and sensitivity of the solid-state nanopores can be further improved by decorating with surficial functional materials or proteins with hollow interiors [29,31], showing great potential to replace biological nanopores for DNA sequencing and nanomaterial detection based on miniaturized wearable systems.
Nanofabrication techniques employing focused ion beam and electron beam enable real-time monitoring of nanopore morphology and sizes during the manufacturing process [17,24], facilitating the production of ultra-small nanopores with dimensions as minute as 0.13 nm [32].Conversely, a more cost-effective controllable dielectric breakdown can generate multiple nanopores on thin dielectric films below 10 nm in thickness, with pore size roughly controlled by an applied voltage.However, precise manipulation of nanopore position and quantity necessitates the use of auxiliary tools such as atomic force microscopes or lasers [33,34].Compared with the above methods, chemical etching methods can achieve low-cost and large-area fabrication of conical nanopores with a size less than 2 nm by redox reactions of chemical solution and membranes, but accurate etching profiles by heavy ion irradiation or mask deposition are needed to determine the position of the nanopores [28,35].In addition, nanoimprint lithography has the capability to produce nanopore arrays in large quantities, but its application is confined to polymer films, limiting its versatility [36,37].DNA nanotechnology can generate nanopores with precise sizes and shapes but requires an additional process before the nanopores being employed as a nanopore sensor.Nanopore fabrication methods are gradually driving toward achieving lower cost, shorter processing time, and smaller dimensions of the nanopores.
Nanopore formation simply through the gravitation effect of heated gold nanoparticles (AuNPs) has been realized by global and nonselective heating in conventional ovens under extended periods of heating on silicon nitride and silicon dioxide substrates [38,39].On the other hand, rapid, localized, and selective nanoparticles (NPs) heating through intense pulsed light (IPL) [40][41][42] as an important photonic sintering approach has been demonstrated in printing electronics.As these two techniques have not yet been combined to utilize their unique advantages, an effective and simplified approach to yield nanopores through melting templates of NPs heated by IPL has seldom been demonstrated.Here, we propose a method to achieve nanopores on polymer membranes using silver nanoparticles (AgNPs) and IPL.The temperature of the AgNPs can rapidly reach 1132 K under instant heating of photothermal effect through light irradiation with an energy density of 962.07 J in 2 ms, resulting in melting and decomposition of a surrounding polycarbonate (PC) membrane in a highly confined region.The AgNPs can then penetrate through the membrane due to the gravitation effect and phase changes of the membrane to generate nanopores with sizes ranging from 10 to 270 nm.After removing the NP residues, the membrane with nanopores can be integrated into a flow cell to yield a nanopore sensor that has been used to measure the translocation behaviors of bovine serum albumin (BSA).The results have demonstrated the capability of the sensor in protein denaturation identification.The high throughput approach to yield nanopores at low cost and in large areas on flexible membranes may facilitate the development of nanopore-based flexible sensors and diverse wearable analytical devices, offering a promising candidate for chemical analysis using flexible electronics and wearable systems.

Results and discussions
The concept for the fabrication of nanopores by IPL irradiation of NPs on a polymer membrane is shown in Figure 1.AgNP dispersion with a proper density of NPs is first spincoated on the surface of a PC membrane on a Cu backing layer, resulting in randomly distributed NPs on the membrane after evaporation of the solvent in the suspension.The histogram illustrates that the PC membrane thickness gradually decreases with the increase of the spin coating rate (Figure S1).The thickness of the PC membranes used in this work is 75 nm.Each remaining NP can absorb the energy of IPL and convert the photonic energy into heat due to the photothermal effect [43][44][45].The pulsed light with a wavelength from 370 nm to 760 nm at 2 ms and 2300 V outputs 962.07 J of energy.More specifically, light absorption by the AgNPs induces localized surface plasmon resonance and electron oscillation followed by electron-electron scattering and electron-phonon coupling within the metal lattice, leading to energy conversion into a format of heat.The generated heat can be further dissipated from the metal lattice to the surrounding polymer medium, causing the rapid melting of the membrane and material decomposition to form a hole when the temperature exceeds its thermal decomposition temperature.Under the gravitation effect, the heated NPs gradually fall through nanochannels formed by ablation until reaching the Cu backing layer.The NP and the Cu backing layer can be subsequently removed through chemical etching, resulting in a free-suspended polymer membrane with nanopores.The polymer membrane can then be picked up from the Cu etchant through the top chamber of a plastic flow cell and then released from the top chamber in a water bath.Afterward, the membrane is aligned and transfer-printed to the top chamber again for later assembly of the flow cell.Despite that, the PC with a melting point (T mp ) of 230°C and a high elastic modulus (δ) of 2.3 GPa is chosen as the membrane material, other polymers such as polyimide (T mp = 340°C, δ = 2.1 GPa), polyethylene terephthalate (T mp = 260°C, δ = 4 GPa), polyethylene naphthalate (T mp = 269°C, δ = 2.4 GPa) may also be used as candidates due to their moderate melting points that accommodate the temperatures achievable by the photothermal effect and high elastic modulus that allow suspension across large surface.Similarly, other NPs made of Au [46], Cu [47], Ni [48], ferric oxide [49], and vanadium dioxide [50], which have been confirmed to possess strong photothermal effects, may also serve as replacements for the AgNPs used in this research.
The distribution and agglomeration states of AgNPs can influence the properties of the nanopores.Despite the uniform sizes of AgNPs observed by the transmission electron microscope (TEM) (Figure 2(a)), single or agglomerated AgNPs from 0.1 µg/mL AgNP dispersion exhibit random distribution on the PC membrane after being spin-coated on a PC membrane.After repeated IPL under an energy level of 962.07 J for 10 times at a pulse width of 2 ms and an interval of 1 s, the resulting nanopores with a size less than 100 nm can be achieved on the PC membrane (Figure 2(b,c)).A 3D AFM image of the PC membrane with a nanopore shows a detailed visualization of the nanopore's profile, revealing a subtle disparity in the size of the upper and lower apertures.This discrepancy can be attributed to the influence of gravity on the molten PC material during the ablation process (Figure S2).On PC membranes with the same areas, the number of nanopores obtained by depositing AgNPs at a spin-coating speed of 1000 rpm is significantly higher than those obtained by depositing AgNPs at speeds of 2000 rpm, 3000 rpm, and 4000 rpm (Figure 2(d-f)).The relationship between spin coating rate and nanopore density has suggested that few or no NPs can deposit on the surface of the PC membrane at excessively high speeds due to large centrifugal force.Therefore, a spin-coating speed of 4000 rpm is chosen in this work.Compared with the nanopores with diameters lower than 100 nm obtained using 0.1 µg/mL AgNP dispersion, large sizes of nanopores are observed for membranes spin-coated with AgNP dispersion 1 µg/mL and 10 µg/mL in concentrations, likely due to the existence of more agglomerated AgNPs in the dispersion and their induced thermal ablation effect (Figure 2(g,h)).A total of 100 nanopores from more than 45 SEM images were used to statistically analyze by using Image Pro Plus software.Despite that, the diameters of 46.4% AgNPs are below 3 nm, and the maximum diameter of AgNPs is only 5 nm (Figure 2(i)), the induced nanopores possess size distribution ranging from 10 nm to 270 nm, further suggesting the agglomeration effect of AgNPs that lead to combined particles after sintering with IPL [51].The size distribution of nanopores is concentrated at 32.52 nm, which is 10.84 times the diameter of the NPs.The new combined particles ablate the PC membrane to form large-sized nanopores.The hydrophobic nature of polycarbonate may also contribute to the aggregation of AgNPs.In addition, the heated AgNPs may influence regions larger than their sizes, indicating the necessity to further adjust the sintering parameters to achieve more localized heating conditions.The 40 nm AuNPs with photothermal effects have also been used as templates to fabricate nanopores of approximately 100 nm in size on PC membranes (Figure S3), indicating that the diameter of nanopores can be controlled through template NPs.In addition, 5 nm AgNPs can also penetrate through the polyimide membranes under IPL (Figure S4).
The underlying mechanisms of nanopore formation through IPL heated NP templates have been investigated by numerical computation and finite element simulation.To simplify the analysis process, the melting of AgNPs was not considered [52], and only the sublimation of AgNPs under IPL irradiation was studied.To understand the process of nanopore formation, the sublimation of AgNPs under IPL irradiation was studied.The radius changes of NPs in the sublimation process follow the Kelvin equation: where P r is the vapor pressure above an Ag particle with a curvature radius of r, P is the vapor pressure over the plane surface, γ is the surface energy, ρ is the density, R is the gas constant, M is the molecular weight, T is the temperature.It supposes that the density and the surface energy of solid Ag do not change with the radius.The sublimation volume dV/dt can be related to the number of molecules leaving the surface per unit time dN/dt, where V α is the molecular volume of AgNPs, and N is the number of sublimated Ag atoms.According to the Maxwell velocity distribution law, dN/dt can be determined by Equation 3, in which v is the mean speed of the vapor, n s is the number of molecules per unit area.Substituting Equation 3 into Equation 2, the change rate of AgNP radius with time can be written as: Assuming that the vapor is a single molecule ideal gas, where k is Boltzmann constant.Substituting Equation 5and Equation 1 to Equation 4yields Equation 6For the AgNPs with radii of 1.5 nm and 2.5 nm, their radius as a function of time at constant temperature was calculated by integrating Equation 6(Figure 3(a)).As the temperature increases from 700 K to 1500 K, the sublimation rates of AgNPs have been accelerated due to the increased velocity of the Ag atoms to leave the surface of AgNPs.The time for complete sublimation of AgNPs varies from 10 −5 and 10 6 s, which is significantly longer than the duration of IPL (2 ms).This analysis implies that complete sublimation of AgNPs can seldom happen in practical situations.
Due to the absorption of the AgNPs with a diameter of 5 nm at 400 nm, the energy generated by IPL in the wavelength range of 370 nm to 760 nm cannot be fully absorbed and converted into thermal energy by AgNPs.According to the simulation results of COMSOL by considering 35% of heat flow generated by repeated IPL can be absorbed and converted into heat by the AgNP [47], a temperature change of the AgNP to 1132 K can be obtained (Figure 3(b)).Under this temperature and according to Equation 6, the time for complete sublimation of AgNPs with initial radii of 1.5 nm and 2.5 nm can be determined to be 0.018 s and 0.082 s, respectively, significantly longer than the single pulse duration of IPL (Figure 3(c)).As a result, the volume change of AgNPs due to sublimation can be neglected due to the short period of IPL.The formation of nanopores on PC membranes using the AgNPs with radii of 1.5 nm and 2.5 nm was simulated.Based on the geometric model of a two-dimensional structure, the temperature change and phase transition are also simulated using COMSOL by considering no volume change of the AgNPs during the entire process (Figure 3(d)).The results indicate that temperature increases in the PC membranes and resulting phase changes of the membranes lead to material melting and decomposition to form nanopores.The AgNP with a radius of 2.5 nm can lead to larger influencing areas than the one with a radius of 1.5 nm, leading to a large nanopore.The simulated nanopore size at 3 nm nanoparticle is 30.7 nm that closely approximates the actual nanopore size.The nanopore profile is mainly influenced by the shape of the NPs.The simulation temperature distribution and phase change of the cubic NPs with a side length of 5 nm show that the upper and lower apertures are inconsistent (Figure S5).The simulation results also indicate that the achieved nanopores are larger than the NP templates due to different thermal properties of materials and heat induced by IPL, suggesting the necessity to fine tune the properties of polymer membranes and IPL conditions in future research.
The PC membrane can be sandwiched between two chambers by two silicone gaskets to achieve the flow cell (Figure 4(a)).Two Ag/AgCl electrodes can be fixed on the top and the bottom chambers to supply an electrical field for electrophoresis and monitor ionic current.Both chambers contain an inlet and an outlet to allow samples to flow in and out.After completion of assembly, the currentvoltage curves of the PC membranes with nanopores exhibit increased slopes with the concentrations of KCl solutions and high linearity with increased bias voltages (Figure 4(b)), implying the existence of nanopores and a relatively symmetric internal electric potential in nanopores [53].The presence of an electric double layer formed by excessive counter ions near the nanopore surface leads to access resistance (R acc ) during ion entry or exit.Pore resistance (R pore ) represents the resistance within the nanopore itself.Hall proposed that the access resistance of a pore can be modeled by considering a semispherical cupola as an effective electrode [54][55][56].The resistance (R) can be described using Maxwell's model, which combines the constant resistivity of the bulk salt solution (ρ) with a summation of R pore and R acc of the nanopores.In the case of a cylindrical nanopore of length L and diameter d; R pore = 4ρL/πd 2 and R access = ρ/2d.As is noted before, the total resistance of a nanopore is as follows, So, a nanopore conductance (G) behaving as where κ is the ionic conductivity of the solution.Assuming that n nanopores on the membrane are in parallel, Assuming that all nanopores on the film have the same size, where G 0 is the ionic conductance of nanopores with a diameter of d 0 .According to the nanopore size range in Figure 2(i), the range of n is from 38 to 12,661.
The characterization of spatial configuration through the nanopores has been demonstrated using BSA before and after denaturation with urea.Both BSA and denatured BSA have exhibited increased currents in the translocation events (Figure 4(c)).As BSA possesses an isoelectric point of 5.5, both BSA and denatured BSA exhibited negatively charged surfaces in solutions with pH levels of 8. BSA and denatured BSA can induce K + ions to generate the electrical double layers, which can lead to more positive charges entering the nanopores, resulting in an increase in current as observed [22,23].The signals of BSA translocation show consistent current changes and translocation time, while the denatured BSA possesses large variation in current and excessively long translocation time, likely due to larger effective size and more charges adsorption of the denatured protein through peptide chains unfolding.In Figure 4(d), the Gaussian fitting peak values of duration time for BSA and denatured BSA are 1.0 ms and 3.1 ms, respectively.The fitting peaks of the current change for BSA and denatured BSA are quite different, where one peak at 707.7 pA for the former and two peaks at 1637.7 pA and 2980.0 pA for the latter.The obvious difference between the translocation behavior of BSA and denatured BSA suggests the nanopore sensors can also be used to identify the denaturation of other proteins.

Conclusion
The rapid and large-area fabrication of nanopores by IPL irradiation using metal NPs as templates has been presented.The AgNPs can rapidly reach 1132 K by photothermal conversion under IPL to melt and decompose PC membranes, leading to the formation of nanopores 10 to 270 nm in diameters.The nanopore membranes have been integrated with the flow cell to measure the translocation of various substances, indicating the potential use of such a nanopore sensor in determining the properties of proteins.In addition, the combination of the IPL method with other techniques such as selective wetting of the membrane surface, printing of NPs, and self-assembly of NPs may be adopted to control the spatial position and number of nanopores.Furthermore, biological proteins may be attached to the polymer nanopores to yield hybrid nanopores that offer even higher sensitivity and resolutions to a variety of samples.The integration of ordered biohybrid nanopore arrays with other flexible sensors and flexible microfluidic systems to achieve a fully wearable analytical system with single-molecule sensing resolution to facilitate high precision analysis in daily life.

Experimental section
Fabrication and Characterization of PC nanopores: Firstly, PC granules (Mw ~ 26,000, Shanghai Macklin Biochemical Co., Ltd.) were dissolved by 90% dichloromethane and 10% cyclohexanone.The mixed solution was spin coated on Cu foil (MT18Ex, Suzhou Mitsui Kinzoku Co., Ltd.) at 5000 rpm to form the PC membrane.AgNP suspension (diameter 5 nm, 0.1 mg/mL, Nanjing XFNANO Materials TECH Co. Ltd.) was diluted to 0.1 µg/mL by deionized water and ethanol (1:1) and dispersed on Cu foil at 4000 rpm.After solvent volatilization, the PC membrane with AgNPs was irradiated by an IPL system (Model S-2100, Xenon Co., Ltd.).Finally, the PC membrane with AgNPs was immersed in hydrogen peroxide (H 2 O 2 ) (30%, Tianjin Jiangtian Chemical Technology Co., Ltd.) to selectively remove AgNPs after IPL irradiation.The morphology of AgNPs was investigated using TEM (JEM-2100F, JEOL Ltd.), and the surface morphology of PC membrane with AgNPs and PC membrane were investigated using SEM (Apreo S LoVac, FEI Ltd.).The surface morphology of PC membrane with nanopores was investigated using TEM (MultiMode 8, Bruker Nano Inc.).The thickness of the PC membranes was investigated using spectroscopic ellipsometry (RC2-XI J.A. Woollam, Co., INC.).The size of AgNPs, nanopore sizes, and nanopore density were subjected to statistical analysis using Image Pro Plus software.
Assembling the nanopore sensor: Before assembling the sensor, the PC membrane with nanopores was cut to a dimension of 0.5 cm × 0.5 cm, and treated with Cu etchant (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.) to remove the backing Cu foil.The resulting PC membrane was washed three times with pure water to remove the residual Cu etchant.Then, the PC membrane was attached to the designated location on the flow cell through a water transferring process and then clamped by two silicone gaskets.The top chamber and the bottom chamber of the flow cell were firmly connected by screws.Two Ag/AgCl electrodes were at last inserted into both chambers for voltage application and current monitoring.
Sample preparation: KCl (Tianjin Dingshengxin Chemical Co., Ltd.) was dissolved in pure water and then KCl solution was filtered through a pinhole filter.The white powder of BSA (Beijing Solarbio Technology Co., Ltd.) was weighted and dissolved in PBS buffer (Beijing Solarbio Technology Co., Ltd.) to prepare 1 mg/mL BSA solution containing 1 M KCl at pH = 8.Urea (Tokyo Chemical Industry Co., Ltd.) at a concentration of 8 M was added into the prepared BSA solution to obtain denatured BSA.
Translocation event measurement and analysis: A current amplifier (Axopatch 700B, Molecular Devices, LLC.) holds a command bias voltage across the membrane while amplifying and recording the ionic current during nanomaterial translocation with a 250 kHz sampling rate (Figure S6).The amplified electrical signals from the current amplifier were converted into digital signals using a 1440A digitizer (Molecular Devices, LLC.).The translocation data analysis was carried out using Pclamp.The temperature increase in AgNPs due to IPL was simulated using COMSOL Multiphysics software with a model that AgNP with an actual radius of 1.5 nm or 2.5 nm in the air domain using a physical model of solid heat transfer.For the simulation of the temperature distribution and PC phase change, a model that included a PC membrane with a thickness of 75 nm and an AgNP with a radius of 1.5 nm or 2.5 nm and a physical model of solid and fluid heat transfer were used.To simplify the analysis process, the temperature of AgNPs was maintained at a constant temperature of 1132 K during the process of PC membrane penetration.The detailed parameters for the simulation can be found in Table S1.

Figure 1 .
Figure 1.Concepts to yield and utilize nanopores on polymer membranes.A schematic of the fabrication processes of nanopores on a PC membrane by IPL irradiation of an AgNP.

Figure 2 .
Figure 2. Characterization of the morphology of nanopores.(a) a TEM image of AgNPs.(b) a scanning electron microscope (SEM) image of spin-coated AgNPs on the PC membrane before IPL.(c) a SEM image of the PC membrane after IPL and removal of AgNPs.(d) a SEM image of the PC membrane with nanopores fabricated by spin-coating at 1000 rpm.(e) a SEM image of the PC membrane with nanopores fabricated by spin-coating at 2000 rpm.(f) a SEM image of the PC membrane with nanopores fabricated by spin-coating at 3000 rpm.(g) a SEM image of the PC membrane with nanopores fabricated by 1 µg/mL AgNP suspension.(h) a SEM image of nanopores fabricated using 10 µg/mL AgNP suspension.(i) Size distribution of AgNPs and nanopores.(j) Density distribution of nanopores obtained by depositing AgNPs at different spin-coating speeds.

Figure 3 .
Figure 3.The simulation and calculation results for the formation of nanopores.(a) the radius changes of AgNPs with radii of 1.5 nm and 2.5 nm as a function of time from 700 K to 1500 K. (b) the temperature of AgNPs during IPL.(c) Time variation of radius of AgNPs with radii of 1.5 nm and 2.5 nm at 1132 K.(d) the simulation for temperature distribution and phase transformation of an AgNP and a PC membrane during IPL irradiation.

Figure 4 .
Figure 4. Assembly and measurement of nanopore sensors.(a) Images and a diagram of the nanopore sensor integrated with a PC membrane and a flow cell.(b) Current-voltage characteristics of PC membranes with and without nanopores in different concentrations of KCl solutions.(c) Typical ionic current signals of BSA and denatured BSA across nanopores at a bias voltage of 400 mV.(d) scatter plots of ΔI versus duration time of BSA and denatured BSA, and their corresponding event distribution.