Plasmonic nano-bowls for monitoring intra-membrane changes in liposomes, and DNA-based nanocarriers in suspension

Programmable nanoscale carriers, such as liposomes and DNA, are readily being explored for personalized medicine or disease prediction and diagnostics. The characterization of these nanocarriers is limited and challenging due to their complex chemical composition. Here, we demonstrate the utilization of surface-enhanced Raman spectroscopy (SERS), which provides a unique molecular fingerprint of the analytes while reducing the detection limit. In this paper, we utilize a silver coated nano-bowl shaped polydimethylsiloxane (PDMS) SERS substrate. The utilization of nano-bowl surface topology enabled the passive trapping of particles by reducing mobility, which results in reproducible SERS signal enhancement. The biological nanoparticles’ dwell time in the nano-trap was in the order of minutes, thus allowing SERS spectra to remain in their natural aqueous medium without the need for drying. First, the geometry of the nano-traps was designed considering nanosized bioparticles of 50-150 nm diameter. Further, the systematic investigation of maximum SERS activity was performed using rhodamine 6 G as a probe molecule. The potential of the optimized SERS nano-bowl is shown through distinct spectral features following surface- (polyethylene glycol) and bilayer- (cholesterol) modification of empty liposomes of around 140 nm diameter. Apart from liposomes, the characterization of the highly crosslinked DNA specimens of only 60 nm in diameter was performed. The modification of DNA gel by liposome coating exhibited unique signatures for nitrogenous bases, sugar, and phosphate groups. Further, the unique sensitivity of the proposed SERS substrate displayed distinct spectral signatures for DNA micelles and drug-loaded DNA micelles, carrying valuable information to monitor drug release. In conclusion, the findings of the spectral signatures of a wide range of molecular complexes and chemical morphology of intra-membranes in their natural state highlight the possibilities of using SERS as a sensitive and instantaneous characterization alternative.


Substrate fabrication details
To create a monolayer polystyrene (PS) stamp on Si wafer, the spin coating technique was utilised at the first step.The Si wafer was cleaned ultrasonically using acetone, propanol, and DI water subsequently.The cleaned wafer was further dried using nitrogen gun.To make the Si surface hydrophilic, the wafers were placed inside a nitrogen plasma for 15 mins.Next, the PS beads suspension in deionized (DI) water having 5% concentration was immediately pipetted on the plasma treated wafer for spin coating.The beads solution was spin coated at a speed of 2000 rpm (500 rpm/s acceleration) for 40 s followed by 5000 rpm (1000 rpm/s acceleration) for 20 s.The spin coating step enabled a partial monolayer formation of beads.Subsequently, the Si wafer with the coating was gradually submerged at an inclined position in a petri dish filled with a solution of 4% sodium dodecyl sulphate (SDS) in deionized water.The inclusion of SDS renders the solution hydrophobic.Consequently, the beads experience a repulsive force from the solution and adhere to other beads, creating a closely packed monolayer at the interface between air and water.The formed monolayer was further scooped using a fresh Si wafer.For better attachment of the monolayer PS beads to the Si surface, the coated Si wafer was kept at 110°C for 20 min on a hot plate.
The creation of a nano-bowl pattern was generated using the monolayer PS templated Si wafer.To create the nano-bowl PDMS stamp, a bubble-free PDMS solution was spin-coated on the Si wafer containing PS beads template at a speed of 800 rpm for 20 s for soft lithography.The PDMS-coated wafers were thereafter put on a hotplate and subjected to a curing process at a temperature of 70°C for a duration of 2 hours.Finally, the cured PDMS film was lifted off.The process developed a pattern of honeycomb-like nano-bowls on the PDMS film (Fig. 2).The structured PDMS was further washed using dichloromethane and DI water to remove any bead residues from its surface and subsequently dried with a nitrogen gun.To fabricate plasmonic PDMS nano-bowls, the nano-bowl substrates were sputter coated with Ag considering different coating parameters using a tabletop sputter coater (Cressington 208HR, 20 mA current).The SERS activity of coated nano-bowls was investigated using a 1 µM concentration of Rhodamine 6G (R6G) as a molecular probe.

Optimization of Ag coating on patterned PDMS Optimization with Ag coating
The optimization of Ag coating on PDMS nano-bowl for maximum SERS intensity was performed with different Ag deposition thickness, considering the 1 mM R6G molecule as a probe.The SERS intensity of R6G for 40 nm Ag coated PDMS nano-bowl exhibited maximum Raman signal intensity, compared to other PDMS substrates, as shown in Fig. S2.The reason for the maximum intensity in the Raman signal is due to the increased surface area and full coverage of Ag coating onto the nano-bowl structure.A further increase in sputtering time reduced the SERS signal intensity, as the bowl radius of curvature was reduced by filling up with Ag.Therefore, the maximum SERS activity for a particular Ag thickness is attributed to the optimum curvature induced upon deposition of Ag, as established from the previous studies [31].In brief, if the thickness of Ag deposition too small, the number of hotspots will be less, since the deposition caused discontinuous small Ag islands, having large gap distance.Due to the decreased number of hotspots, the enhancement factor would decrease.If the Ag film is too thick, the surface will be flat, and curvature will be reduced, lowering the enhancement factor.Therefore, when the thickness of the Ag film is within the optimal range (~ 40 nm), the SERS enhancement factor is at its maximum.Based on the results, the 40 nm Ag coating on PDMS nanobowl SERS film was used for all measurements (Fig. S2).

Calculation of enhancement factor (EF)
The Raman signal enhancement factor (EF) was calculated considering Raman shift at 1649 cm -1 of 1 µM concentration of R6G molecule placed on Ag coated nano-bowl film and on Ag coated flat PDMS film.The EF was calculated using the following formula: 16 Here,   and  0 denote the peak intensity for Ag coated structured SERS substrate and on flat film. 0    denotes the molecules present on structure film and on flat film, respectively.The formula to calculate  0 used is following: Here    the excitation laser volume, N: Avogadro number,  6and  6 denotes density and molecular weight of the analyte sample.
To calculate  , following formula was used: Here   : density of nano bowl,  : Area of one nano bowl,   :  ,  6 : Area of analyte molecule.Fig. S3.SERS spectra of 1 µM R6G dropped on 40 nm Ag coated flat PDMS film and structured PDMS film (cured from PS templates of 190 nm, 600 nm, and 1 µm-diameter beads).The 40 nm Ag coating led to optimized enhancement of nano-bowls PDMS film cured using 1 µm PS template.

Reproducibility test
The reproducibility of the optimised SERS substrate was investigated considering random locations on a particular SERS substrate.
The optimized SERS substrate produced reproducible Raman results for 1 µM R6G considering eight random spots on a single substrate, as shown in Fig. S3 (a).Peaks occurred at the same wave shift value, and the intensity variation was calculated to be less than 5%.Further, the substrate can identify the peaks down to a 10 -15 M concentration of R6G (Fig. S3 (b)).The peaks at 608 cm -1 represent the bending of C-C-C ring, peak at 773, 1126 cm -1 for in plane bending of C-H group, 1309 cm -1 for in plane bending mode.The peaks at 1361, 1505, 1574, and 1649 cm -1 are assigned for aromatic C-C stretching.

Fig. S1 .
Fig. S1.Methodology for Raman measurements of bioparticles Firstly, 5 µl of sample were placed inside the PDMS chamber, placed on a nano-bowl SERS chip, and then sealed with a glass coverslip.The sealed solution was further placed under the Raman microscope for spectrum acquisition.

FigFig
Fig. S3 (a) Spectra reproducibility of the SERS substrate considering the acquisition of 1 µM R6G SERS spectra over eight random spots.The substrate showed reproducible spectrum intensity with a variation of less than 5%.(b) SERS spectra of R6G for different concentrations (10 -6 M -10 -15 M).The nano-bowl substrate detects enhanced and distinct peaks of R6G down to a concentration of 10 -15 M. The shaded regions indicate the Raman peaks of the PDMS substrate.