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Biological and Medical Applications of Materials and Interfaces

SERS Active 3D Interconnected Nanocarbon Web towards Nonplasmonic In-Vitro Sensing of HeLa Cells and Fibroblasts. A K M Rezaul Haque Chowdhury, Bo Tan, and Krishnan Venkatakrishnan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10308 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

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ACS Applied Materials & Interfaces

SERS Active 3D Interconnected Nanocarbon Web towards Nonplasmonic In-Vitro Sensing of HeLa Cells and Fibroblasts. A K M Rezaul Haque Chowdhurya, Bo Tanb, Krishnan Venkatakrishnanc,d, * a

Nanocharacterization Laboratory, Department of Aerospace Engineering, Ryerson University,

350 Victoria Street, Toronto, ON, Canada M5B 2K3 Email: [email protected] b

Nanocharacterization Laboratory, Department of Aerospace Engineering, Ryerson University,

350 Victoria Street, Toronto, ON, Canada M5B 2K3 Email: [email protected] c

Micro/Nanofabrication Laboratory, Department of Mechanical and Industrial Engineering,

Ryerson University, 350 Victoria Street, Toronto, ON, Canada M5B 2K3. d

Affiliate Scientist, Keenan Research Center for Biomedical Science, St. Michael's Hospital,

Toronto, Ontario M5B 1W8, Canada * Corresponding author Email: [email protected] Phone: +1 (416) 979-5000 Ext. 4984 Fax: +1 (416) 979-5056

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Abstract A noninvasive intracellular component analysis technique is important in cancer treatment and the initial identification of cancer. Carbon nanomaterials/nanostructures, such as CNTs and graphene, have little to no SERS (surface enhanced Raman scattering) ability. Because of these structures’ low Raman responses, they are conjugated with gold or silver to attain the SERSactive ability to detect normal fibroblasts and HeLa cancer cells. To the best of our knowledge, the effectiveness of the individual use of carbon-nanomaterials as a nonplasmonic SERS-active platform for in-vitro cancer/normal cell detection has not been investigated to date. Here, for the first time, we introduce a unique nonplasmonic SERS-based biosensing platform that uses a biocompatible self-assembled 3D interconnected nanocarbon web (INW) for in-vitro detection and differentiation of HeLa cells and fibroblasts. The sub-10-nanometer morphology of the INW facilitates the endocytic uptake of INW clusters to the cells, and its SERS functionality introduces live cell Raman sensing. The INW platform has achieved an enhancement factor (EF) of 3.66×104 and 9.10×103 with crystal violet and Rhodamine 6G dyes, respectively, significant in comparison to the EF of graphene surfaces (2 to 17). The results of the time-based Raman spectroscopy of live HeLa cells and fibroblasts revealed chemical fingerprints of intracellular components, such as DNA/RNA, proteins and lipids. The components’ spectroscopic differences facilitate and elucidate the specification of each cell. The highest Raman enhancement achieved was four-fold for fibroblasts (protein) and six-fold for HeLa cells (DNA). Furthermore, the SERS spectra along with SEM and FM analysis of the immobilized cells after 24 and 48 hours shed light on the health of fibroblasts and HeLa cells. A photon energy-induced ionization achieved with the femtosecond laser fabricated a biocompatible INW platform with the


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designated unique attributes. This simple, label-free, in-vitro diagnosis approach for HeLa cells and fibroblasts has strong potential for cancer research. Keywords: interconnected nanocarbon web, biosensitive, SERS-active, Raman signature, HeLa, fibroblasts 1. Introduction Carbon-based nanomaterials have attracted substantial attention in the field of diagnostics and biosensing1,2. It is important to understand the behavior of living cells and microorganisms immobilized on a substrate while designing the biosensors3. To date, several optical biocompatible methods, such as infrared spectroscopy4, surface plasmon resonance5, and bioluminescence imaging6, have been applied for the investigation of cellular behavior in cultures. However, there is a requirement for a noninvasive method to investigate the cells with minimal external perturbation. A label-free, rapid, powerful and noninvasive investigation method employing Raman spectral analysis has been used extensively by researchers for biological analysis7. The Raman analysis of live cells could provide valuable biological data for the cells that plays an important part in the identification of diseases, apoptosis and the interactions between toxins and drugs with the cells after treatment8–10. One of the major limitations of the Raman scattering process is that it exhibits weak intensity11. This weak intensity problem may be solved by using intense light sources, such as a laser. The use of an intense light source, however, has an unfavorable consequence in cell investigations as the light source degrades the analytes. Conversely, the SERS method utilizes nanostructured surfaces12 and increases the conventional Raman spectral analysis sensitivity through the reduction of collection time and laser power13. The application of SERS can extend



to such applications as the detection of chemical molecules/ions and the clinical discrimination of cancer tissues 14,15. The use of noble metal nanostructures/nanomaterials in SERS is common because of their property of exhibiting LSPR (localized surface plasmon resonance) in the NIR (near infrared) and visible spectra16. For various biosensing applications, both semiconductor hybrid nanostructures coated with monolayer noble metals17 and nanomaterials (semiconductor-based), such as titanium dioxide13, zinc oxide, graphene18 and copper telluride19, have been used as SERS platforms. Nanomaterials conjugated with noble metals have showed an increase in SERS sensitivity. Therefore, researchers currently focus on either noble metals or semiconductor nanomaterials coated with noble metal for the development of SERS-active substrates20. However, different synthesis processes (physical deposition, oxidation or chemical reduction) for creating hybrid nanomaterials (semiconductor nanomaterials coated with noble metal) and noble metal nanomaterials have led to poor biocompatibility. Consequently, these nanomaterials are less desirable for various biomedical applications, such as biomolecular sensing21. Researchers have explored different nonmetallic nanomaterials for carbon, such as CNTs (carbon nanotubes)22, graphene23 and graphene oxides24, for biosensing applications. Carbon-based nanomaterials have the capability to transfer electrons directly from the carbon-based nanomaterial to functional bioreceptor sites without the engagement of a mediator and will thus amplify the signals and provide sensing without the use of isotopic labels25. The cytotoxicity of pristine CNTs, which is due to the presence of catalytic particle residue and amorphous carbon in manufactured CNTs, influences the efficacy of biosensing26. The functionalization of CNTs is completed by having various chemical groups bind together, covalently or noncovalently, making them biocompatible. Further conjugation with biomolecules makes CNTs a suitable


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candidate for biosensing25. Sun et al.27 reported the fabrication of a SERS substrate loaded with a super-aligned CNT grid with densely packed Ag nanoparticles (NPs). These researchers showed that in the presence of R6G dyes, the CNT grid alone did not depict any Raman enhancement. Lee et al. 28 also described the synthesis of the SERS substrate (3D) of CNTs (vertically aligned) with structural adjustability and the hybrid SERS substrate of CNTs (vertically aligned) with gold NPs, which was conjugated on the wall of the CNTs. Ashwinkumar et al.29 reported a technique that used two types of CNTs, namely, SWCNTs (single-walled carbon nanotubes) and MWCNTs (multiwalled carbon nanotubes), coated with a lipid-polymer along with movable Raman devices to detect ovarian cancer cells and their photothermal ablation. The biosensors derived from graphene were presented to sense hydrogen peroxide (H2O2), dopamine, different proteins and reduced NADH (b-nicotinamide adenine dinucleotide)23,30. M. Manikandan et al.31 prepared two different SERS substrates by synthesizing gold nanohexagons in situ onto the graphene templates and by decorating the graphene sheets with gold NPs via electrostatic interaction using ultrasonication. These SERS substrates were used to intensify the Raman signal and differentiate three different human breast cells, namely, normal breast cells, breast cancer cells and breast cancer stem cells. Shijiang He et al.32 developed a functioning SERS substrate made of Au NPs ornamented with graphene (chemical vapor deposition growth), which was used to detect DNA. In addition, different platforms for biosensing, created through the conjugation of graphene and its derivatives (especially graphene oxide) with noble metals (gold or silver), proteins or polymers, were used to detect platelet-derived microparticles24, Escherichia coli (E. coli)33 and the human papillomavirus DNA34. To date, the researchers have used carbon nanomaterials as a component of the hybridized biosensing platform along with NPs characterized as noble metals and attained SERS-active



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detection of different normal and cancer cells. However, carbon nanomaterials have not been reported as an individual biocompatible nonplasmonic SERS-active platform for biosensing and detecting

fibroblast

(normal)

and

HeLa

(cancer)

cells

in

vitro.

Overall,

carbon

nanomaterials/nanostructures play a supporting role with noble metal nanoparticles in the SERSactive biosensing platforms. To the best of our knowledge, carbon nanomaterials/nanostructures were not individually applied as a SERS-based platform for in vitro live cell diagnosis and differentiation due to their little to no SERS ability and/or toxicity. Therefore, a biocompatible nonplasmonic SERS active nanocarbon biosensing platform is a requirement for SERS based in vitro detection and differentiation of live HeLa and fibroblast cells. In this study, we developed a SERS active, biocompatible 3D interconnected nanocarbon web structure and introduced a label-free, nonplasmonic SERS-based biosensing platform to detect and differentiate HeLa (cancerous) and fibroblast (mammalian) cells in vitro. In our previous study,35 we observed that the nanocarbon platform that was created possessed great biocompatibility and cell adhesion capacity. The SERS activity of the INW platform was recognized with both crystal violet (CV) and Rhodamine 6G (R6G) chemical dyes. The INW platform yields an enhancement factor (EF) of 3.66×104 and 9.10×103 for CV and R6G dyes, respectively. This EF was significant in comparison to the EF exhibited by the graphene surface (2 to 17) obtained in other literature36. The sub-10-nanometer physical morphology of the INW platform facilitated the endocytic uptake of INW clusters by both HeLa and fibroblast cells. The SERS functionality of the internalized INW clusters inside the cells provided the basis for SERSbased Raman sensing of live cells. The SERS spectra collected every 6 hours within a 24-hour incubation time from the live fibroblast and HeLa cells seeded on the INW platform depicted peaks related to intracellular biochemical components, such as proteins, DNA/RNA and lipids.


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The DNA/protein peak prominence resulting from the live HeLa and fibroblast cells over the incubation period suggested a guideline in detecting and differentiating fibroblast and HeLa cells. The highest Raman enhancement achieved for fibroblast (protein) was four-fold and that for HeLa (DNA) was six-fold. The spectral profile of various intracellular components, such as the DNA, protein and lipid, of the fibroblasts and HeLa cells, as well as their intensity variations over 24-hour and 48-hour incubation periods, has shed light on cell health. The SEM and fluorescence micrograph of the fibroblast cell after 24 hours of incubation showed a wellelongated healthy cellular morphology with overextended filopodia and actin filaments. The corresponding 24-hour Raman spectra of immobilized fibroblast cells contained DNA, protein and lipid peaks with a moderately strong intensity. These intensities were enhanced, showing more prominent DNA, protein and lipid peaks in the 48-hour Raman spectra of the fibroblast cell. The supporting SEM and fluorescence micrograph depicted an interconnected, overlapped tissue, similar to the body of the fibroblast. However, the 24-hour spectra of HeLa cells had prominent peaks of DNA/RNA, proteins and lipids. The corresponding SEM micrograph showed healthy, well spread-out HeLa cells with short filopodial extensions. However, there was a huge difference in the spectral profile of the 48-hour HeLa cell sample with a reduction of intensity of the DNA/RNA, protein and lipid peaks. The SEM and fluorescence micrograph supported these changes, as it indicated that the number of cells on the nanocarbon platform was reduced and that the elongated HeLa cells ceased in growth, appearing to shrink instead, developing a rounded cocoon-like body. The unique attributes of the self-assembled 3D INW structure were introduced by adopting the bottom-up approach using photon energy-induced ionization of the high energy femtosecond laser. The ultrafast pulse laser ion-plume converted the carbon-carbon bonds of graphite to SERS-active carbon-carbon and carbon-oxygen bonds bearing an interwoven INW



structure. The change in nanomorphology and nanochemistry of the created nanostructure is a function of the adjustment of laser ionization parameters. The simplicity and versatility of the proposed SERS-based INW-aided Raman biosensing technique for HeLa (cancer) and fibroblast (mammalian) cell detection can be used for cancer diagnostics and/or as a tool for evaluating new cancer therapy. 2. Materials and Method 2.1 Synthesis of the interconnected nanocarbon web platform The 3D interconnected nanocarbon web platform was created on an isomolded, very fine grain, high strength graphite plate substrate (3-mm thickness) (Graphtek LLC, USA) through ablation with an ultrashort laser (femtosecond, diode-pumped, Yb-doped) (Clark-MXR, Inc.; IMPULSE Series) at laser pulse repetitions of 26, 8 and 4 MHz and atmospheric condition. Before the photon energy induced ionization processing, the plates were cut into 4 cm2 squares, polished with sand papers (3M Canada, 3000, 2000 and 1000 grit), subjected to ethanol and acetone-based cleaning by ultrasonication (Ultrasonic cleaner-Cole-Parmer 8890) and dried. An array of lines with varying spacings were machined into samples mounted on a fixed stage with a translation scanner (2-D, high precision and computer controlled) that guided the incident laser beam. The ultrashort pulse laser fluence (the amount of energy per pulse/unit area transferred to the substrate) was set at 0.68, 2.22 and 4.43 J/cm2 (referred to as low, medium and high fluence, respectively) to alter the nanotopography and chemistry of the created interwoven nanocarbon network. However, the width of the laser pulse (214 fs), the incident laser beam power (15W), the laser beam scanning speed (1 mm/s) and irradiation focal spot area (84.62 × 10 cm ) remained constant.


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2.2 Morphological characterization of INW platform The surface morphology of the 3D INW structure that was made through self-assembly was evaluated initially with a Hitachi-SU-1500 SEM (scanning electron microscope). The effects brought out by the photon energy induced ionization on the fabricated 3D nanonetwork were studied and evaluated by the Hitachi-SU-8200 FE-SEM (field emission scanning electron microscopy). The nanocarbon width distribution was determined from the images obtained from the FE-SEM through an image analysis by ImageJ (a software for image processing). 2.3 Physicochemical characterization of nanocarbon platform Physicochemical properties of the fabricated biosensitive 3D INW platform were determined through the application of an XPS (X-ray photoelectron spectroscopy) and an MRS (MicroRaman spectroscopy). Both the XPS and MRS analysis deduced the chemical composition of the INW platform. In addition, the MRS also inferred the crystallinity data of the created nanostructure. To examine both the graphite substrate and the developed interwoven 3D INW structure, a Raman system (handheld, NanoRam®, B&W Tek, Inc) (785 nm wavelength, 350 mW-power) was employed. Both the changes of morphology and intensity of the characteristic Raman spectra of the created INW structure, due to the ionization process, were identified and compared to the unablated substrate. The SERS analysis of the 3D INW platform was performed with two dyes, namely, CV and R6G to identify the SERS enhancement factor of the created INW network. These dyes are popular for SERS analysis because their cross-section is large. Before the Raman analysis, each dye was used to coat the individual nanostructure areas at a concentration of 1×10-3 M. The resulting Raman spectrum was obtained three times each in the 3 s timeframe and averaged.



An XPS system (Thermo Fisher K-Alpha) which used a monochromated Al Kα X-ray source calibrated at a 2:1 ellipse and spot size-400 nm was employed to collect the XPS data for analysis from the synthesized samples of 1 cm × 1 cm size. A higher energy resolution and peak clarification was achieved by running a scan, calibrated at 0.1 eV point spacing and 50 eV pass energy, by region. The Avantage software was used for elemental quantification of the created nanocarbon structure.


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2.4 Cell culture The functionality of the fabricated 3D INW platform in sensing different biological cells with the aid of the microRaman system was tested with the embryonic fibroblasts of mice (ATCC, USA, NIH-3T3) and cancer cells from the cervix (ATCC, USA, HeLa). The cells were grown in DMEM/F12 (Dulbecco’s modified Eagle’s medium) including 10% FBS (fetal bovine serum) and 1% streptomycin/penicillin. Incubation of this culture was undertaken at 37 °C with 5% atmospheric CO2. 2.4.1. Cell seeding (both HeLa and fibroblast) on nanocarbon platform Before the in vitro investigation occurred, the INW platforms were sterilized for 20 minutes through UV exposure. Petri dishes prepared each with 3 mL of DMEM/F12 and FBS with 10% concentration were then used to place the INW platforms inside. Within the Petri dishes, the seeded cell density was 105 cells/mL. The cells were incubated for 24 and 48 hours. During the incubation period, Raman spectra of the live cells along with the nanostructures were collected every 6 hours (6, 12, 18 and 24 hours) using a Raman system (handheld, NanoRam®, B&W Tek, Inc). The spent medium was then removed from the cells; the cells were then fixated in glutaraldehyde (2% concentration). Next, with the temperature set at 4°C, 1% buffer (sodium cacodylate at a pH of 7.3) was used to wash the cells twice. Dehydration of the cells then followed for 15 minutes via a graded series of ethanol from concentrations of 10 to 100%. Next, the cells were subjected to critical point drying. The 24-hour and 48-hour Raman spectra of the fixed cell samples were collected with the same Raman system. The Raman spectra at the single cell (fixed) level were collected with a Renishaw Raman microscope system (785 nm wavelength, 40X objective lenses). Gold-sputtered fixed samples were examined with an SEM machine to observe the morphology of cells on the INW structure. An Oxford EDX system was



employed to characterize the endocytic uptake of nanocarbon clusters by the cells with EDX (energy-dispersive X-ray) mapping. 2.4.2. Fluorescence microscopy of cells. To perform the fluorescence microscopy, the cell samples were initially fixed with methanol-free paraformaldehyde. The process was then followed by incubation with skimmed milk for prevention of nonspecific binding. Next, an Alexa Fluor 488 phalloidin (Life Technologies) mediated incubation was performed for the staining of the cytoskeleton and actin. Finally, a DAPI (Life Technologies) mediated incubation was done for staining of the nucleus. A Nikon E400 microscope (epifluorescent) with an FITC and DAPI filter was used to study the samples. A DS-5M-U1 color digital camera (Nikon, Canada) recorded the data for the samples. 2.5 Statistics The mean ± standard deviation was represented by the data obtained from the triplicate experiments. Cells were counted from the images obtained by the SEM using software for image processing. The statistical significance was evaluated using a one-way ANOVA (analysis of variance). The *p and **p values were less than 0.05 and 0.01, respectively, which suggested a significant difference. 3. Results 3.1 Fabrication of 3D interconnected nanocarbon web (INW) platform The schematics in Figure 1 illustrate the synthesis of the 3D interconnected nanocarbon web platform. Under an ambient atmosphere, the surface of the graphite plate was processed by the femtosecond laser, and the photon energy-induced ionization was brought onto the graphite plate, which synthesized the self-functionalized 3D INW structure. With the precise guidance of a programmable galvanoscanner, which has a laser beam capable of 2D movement, a


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predetermined array of lines was transferred onto the surface of the graphite. The surface of the graphite samples was ablated, and a vapor plume containing nanoparticles was formed with the said laser processing.

Figure 1. Single-stage 3D INW platform synthesis illustrated schematically. The carbon-oxygen bond ratio in the INW nanostructure and its morphology were modulated accurately in correspondence to the ionization energy changes.



The vapor plume contained carbon in its various forms, i.e., neutral, ions and radicals37. Within the air, oxygen ions were present. The plume expanded into the surrounding air, and its temperature dissipated. Thus, the process of nucleation occurred and created self-assembled carbon NPs containing both carbon-carbon and carbon-oxygen bonds in the plume. Later, upon collision and aggregation of these NPs, interwoven nanostructures of the 3D interconnected nanocarbon web was formed. Complex dynamics of plume expansion as well as the substrate property, density of the air vapor and the energy for ionization influenced the nanotopography and altered the nanochemistry of the fabricated platform. The parameters of the laser, namely, scanning speed, laser fluence, repetition and power, have a direct correlation with the ionization energy. The nanocarbon dosage ( ) has a direct proportionality to the number of ionized carbon nanoparticles ( ) and the femtosecond laser fluence ( [⁄ ] = []⁄ [ ]. = laser pulse energy and = effective laser beam area). The relationship between the said dosage to the machining speed38 of the laser has an !

inverse proportionality ( ∝ ()!/ ). Three different laser fluences, namely, 0.68, 2.22 and " 4.43 J/cm2, and a 1-mm/s scanning speed were used in this study for the elucidation of various physicochemical properties of the INW structure. 3.2 Morphology and physicochemical characterization of 3D INW platform The 3D web-like interconnected nanocarbon structure was generated on the graphite surface with the help of the ionization energy attained through femtosecond laser fluence irradiation (Figure 2A). The compactness of the synthesized nanofibrous structure of the created nanocarbon varied from less to more with the gradual increase in the energy of ionization and was evident from the SEM micrographs (Figure 2A). The FE-SEM micrographs of the fabricated INW platform


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(Figure 1) also confirmed the interconnected nature of the nanoparticles. The graphite substrate largely contains carbon-carbon bonds. Both physical morphology and alteration of chemical structure of the created nanostructure have been achieved through careful adjustments of the laser processing parameters. The nanocarbon width and the carbon-oxygen to carbon-carbon bond ratio define the morphology and nanochemistry of the 3D biosensitive nanocarbon platform (Figure 2B). There was an opposite trend for these two parameters. Gradual increments of the energy of ionization (low to high) resulted in a nanocarbon width reduction. However, for the same gradual increment of ionization energy, the carbon-oxygen to carbon-carbon bond ratio increased (Figure 2B). A denser plume generated from a higher ionization energy prompted a more compact nanocarbon structure. The average nanocarbon width varied from 6.67 to 5.27 nm when the energy of ionization was increased from 0.68 to 4.43 J/cm2. The carbon-oxygen to carbon-carbon bond ratios also changed from 0.23 to 0.33 with the same energy of ionization increment. The INW structures containing the said bond ratios were known as low carbonoxygen (C-O) concentration, medium carbon-oxygen (C-O) concentration and high carbonoxygen (C-O) concentration INW structures. The nanocarbon distribution of width tends to be narrower with the increase in ionization energy, as shown by the frequency histogram (Figure 2C). These results are consistent with our Si nanostructure that was previously39 synthesized with a femtosecond laser. The graphite substrate and the created 3D INW platforms (of various C−O concentration phases) characteristics determined by the XPS spectra along with the numerical assessments of the major elements are shown in Figure 2D. The nanocarbon platform and the native graphite substrate, as presented by the spectra, exhibited characteristic peaks at 285.08 and 533.08 eV, which could be credited to carbon and oxygen 1s atomic orbital (Figure 2D). Both N2 and O2 gases (neutral and reactive, respectively) were present during the interaction between the



laser and matter within the ambient atmosphere. Hence, there was a carbon-oxygen bond formation through chemically reactive oxygen and carbon ions participating in a chemical transformation.

Figure 2. (A) Synthesized 3D interconnected nanocarbon web platform with (B) different nanocarbon widths and C−O concentration phases with a gradual increment of ionization energies. (C) Histogram showing the nanocarbon width frequency (D) The nanocarbon (of


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various carbon-oxygen concentration phases) XPS spectra along with (E) the numerical assessment of the main element. Thus, the carbon-carbon and carbon-oxygen bonds are the two main bonds existing in the created nanoplatform. With the gradual increment of energy of ionization, the amount of oxygen present in the INW platform increased. From numerical assessments of the main elements (Figure 2E), the carbon-oxygen to carbon-carbon bond ratios were measured as 0.23, 0.32, and 0.33 in accordance with the energies for ionization set at 0.68, 2.22, and 4.43 J/cm2, respectively. The interpretation of the 285.08 eV peak (first peak position) is ascribed to the sp3 hybridization of carbon40, while the interpretation of the 533.08 eV peak (other peak position) is recognized as the carbon-oxygen bond corresponding to chemisorbed oxygen41. 3.3 SERS efficiency of 3D INW platform and EF calculation Figure 3A depicts the Raman spectra for both the synthesized INW platform at three different energies of ionization and the graphite substrate.



Figure 3. (A) Raman spectra of the INW structure (which contains various carbon-oxygen concentrations) and the graphite substrate (B) ISERS/Isubstrate ratio at different nanocarbon width for D and G band


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The graphite substrate shows a peak at ∼1575 cm−1 known as the G band42 and a peak at ∼1350 cm−1, known as the D band, which are two common bands of graphite43. From the presence of these bands, the defect contained within the crystalline lattice structure of the pure graphite substrate can be deduced. There is a shift for the G band ranging from 1578 to 1588 cm-1 and a D band from 1312 to 1320 cm-1 in the case of the INW platform. The addition of the carbon-oxygen bond and the carbon-carbon bond in the created INW nanostructure could explain this peak shift. Another prominent peak appeared approximately near 2700 cm-1. According to other researchers,44 this peak is designated as G′. The Raman spectra of second order are where the G′band is revealed with regard to the crystalline graphite (without disorder); the overtone of the D band corresponds to this. Based on the known bands (D and G), the ratio of intensity of the SERS-active nanocarbon to the graphite substrate was calculated. For both the Raman shifts, there was a declining trend of these ratios with the increment in the nanocarbon width (Figure 3B). 3.3.1Raman Enhancement factor (EF) calculation Both crystal violet and Rhodamine 6G dyes were utilized to recognize the EF of the SERS of the 3D INW platform (Figure 4). Because of these dyes’ large Raman cross-section, they are common for the analysis of SERS. The details of the procedure utilized for EF calculation of the nanocarbon platform is elaborated in the supporting information (SI) section. According to Figure 4A and 4C, the bulk graphite substrate spectra had no or a minimal dyerelated response when they were covered with either the CV dye or the R6G dye. However, a dye-coated nanocarbon depicted clear and well-defined characteristics of the G band and D band Raman peaks. A significant enhancement in the intensity of these two peaks was observed for all of the nanocarbons of varying C-O concentrations in the presence of these dyes. We observed a



maximum enhancement factor of 3.66×104 and 9.10×103 for both CV and R6G dyes, respectively, at a 10-3 M concentration. Lowering the dye concentration affected the increase in the Raman scattering intensity. With dyes of 10-6 M concentration, the maximum enhancement factor of 3.8×101 and 1.10×101 for both CV and R6G dyes, respectively, was observed.

Figure 4. (A) Raman spectral enhancement with CV dye (B) Raman EF values with CV dye for D and G band (C) Raman spectral enhancement with R6G dye (D) Raman EF values with R6G dye for D and G band at different nanocarbon width.

Two mechanisms, electromagnetic and chemical (EM and CM), are considered for the SERS enhancement phenomena31,45. The local electromagnetic field enhancement, which ultimately creates a substantial increase in the Raman scattering cross-section, is the basis for EM. The incident light that excites the surface plasmons is the main contributor to the electromagnetic


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enhancement46. The enhancement factor (EF) for the EM enhancement can reach above 1010 47. In the CM enhancement48, the charge transfer between the substrate and absorber generates a new resonance state. In this instance, the Raman scattering cross-section increases when more separation occurs between the positive and negative charge inside the molecule. The CM enhancement offers an EF of 10 to 102 36. It is assumed that the CM enhancement might explain the Raman signal enhancement in our study. The achievement of an EF of 103 to 104 is on the higher side in comparison to the EF exhibited by the graphene surface (2 to 17) obtained from other literature36. Therefore, the nanocarbon platform is highly SERS-active and can detect an analyte more readily than the bulk graphite. 3.3.2 SERS efficiency of the 3D INW platform with the CM

Figure 5. Schematic of the charge-transfer mechanism principle.



The SERS efficiency of the 3D INW platform might also be explained with the CM enhancement. The schematics in Figure 5 shows the principle of the charge-transfer mechanism for the CM enhancement. The C-C bond is the core structure of the created INW platform. The CM requires a transfer of charge among the substrate and the molecules at a maximum distance of 0.2 nm between them

49,50

. The core C-C bond of the INW platform might induce a charge

transfer similar to graphene51. When the INW structure surface is neighboring an analyte molecule excited by a photon source, the charge-transfer resonance occurs52. Two energy levels of the INW structure, namely, the valence and conduction band (VB and CB), should be relatively comparable to the lowest unoccupied and the highest occupied molecular orbital (LUMO and HOMO) of the dye molecule, respectively. Electrons can travel along the path shown in the figure thermodynamically from either the INW structure to the molecule or vice versa if these energy levels are within the range of each other and if the incident light has enough energy. When the excitation of an electron (from the VB to the CB) by the photon occurs, the electron is transferred to the LUMO through resonant tunneling because they are neighboring each other and have a comparable energy level. The molecule emits a SERS photon caused by the energy transfer of the electron to the vibrational state of the molecule. Next, the molecule decays back to the CB. Likewise, through resonant tunneling, an electron might be excited by a photon from the HOMO energy level to the LUMO energy level and transferred to the CB of the INW structure. After transferring energy to the vibrational state of the molecule, the electron will decay to the ground state of the molecule, thereby causing emission of a SERS photon. 3.3.3 SERS activity of the 3D INW platform


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Figure 6 depicts the SERS activity of the 3D INW platform. A stability study for SERS activity of the interconnected nanocarbon structure was done with CV dye. The duration of the experiment was 96 hours. Four SERS spectra were observed after 0 hours, 24 hours, 48 hours and 96 hours, respectively. There was a decreasing trend in SERS activity observed within the period of the experiment.

Figure 6. (A) SERS Raman spectra of the INW platform with CV dye within the storage time ranging from 0 to 96 hours. (B) SERS activity showing a declining trend over this period. 3.4 INW cluster uptake The cellular uptake provides necessary local optical fields for ultrasensitive intracellular probing53. Figure 7 portrays the INW cluster uptake process by both fibroblast and HeLa cells during the period of incubation. The results of the SEM, fluorescence and EDX mapping were based on the high C-O concentration INW platform. The results for other two conditions were included in the supporting information section. Both SEM and fluorescence micrographs (Figure 7A) evidenced the presence of INW clusters inside the fibroblast and HeLa cells. When seeded, both fibroblast and HeLa cells had an affinity to attach to the sites that had a greater adsorption of proteins. The biosensitive INW platform, being an area of higher protein adsorption


54,55

,


attracted the cells, and primary attachment took place. It might be hypothesized that the internalization of INW clusters, a constructive component of the interwoven 3D INW platform, occurred through endocytic uptake due to the closeness of the cells to the INW structures. Different parameters of nanoparticles such as surface charge, size and shape along with the type of cells in action determine the endocytic process of taking up INW nanoclusters by the cells56,57.

Figure 7. (A) INW cluster uptake by the fibroblast and HeLa cells adhered to the nanocarbon structure, and (B) The images of EDX mapping of the fibroblasts and HeLa cells adhered to the nanocarbon structure showed internalized INW clusters. The presence of internalized INW clusters inside fibroblast and HeLa cells (figure 7A) evidenced the INW cluster uptake by the endocytosis process58. EDX elemental mapping (Figure 7B)


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further elucidated the presence of INW clusters in the cell cytoskeleton. The spreading pattern of both carbon and oxygen mapping identified the presence of carbon-oxygen and carbon-carbon bond structures and the nanochemistry of the nanocarbon platform and thus demonstrated the uptake of INW clusters in the HeLa and fibroblast cells. 3.5 Live fibroblast and HeLa SERS spectra The live cells (fibroblast, HeLa and breast cancer) SERS spectra seeded on the INW platform were collected during the incubation period of 24 hours at an interval of 6 hours (Figure 8). Figure S6 and S7 (presented in SI) portrayed the SERS spectra of live fibroblast, HeLa and breast cancer cells seeded on different C-O concentrations containing INW platforms for a 24hour incubation period at 6-hour intervals. The cells were seeded covering the entire sample, which constitutes both INW structures containing a graphite substrate and a native graphite substrate. The Raman spectra collected outside the nanostructure zone along with seeded cells were termed native spectra. The Raman spectra after a 24-hour incubation on the high C-O concentration nanocarbon platform were chosen for spectral analysis. During the first 6 hours of incubation, the cells were looking for a suitable site for adhesion. After 6 hours, the live cell response to the Raman signal was significant for fibroblast and HeLa. However, there was a weak intensity of the intracellular components observed for breast cancer cells. There was a variation in peaks over time (24 hours of incubation) as the live cells underwent morphological changes over the period mentioned. The peak variations were dominant for the high C-O concentration INW structure containing the SERS substrate. A Raman spectral peak assignment was done for both live fibroblast and HeLa cells on the high CO concentration nanocarbon platform after 24 hours with reference to the earlier studies (Table S1 and Table S2 in the SI)59,60. The breast cancer Raman spectra also depicted similar Raman



peaks. Different intracellular components, such as DNA/RNA (420 cm-1 - 1000 cm-1), proteins (1100 cm-1 - 1700 cm-1) and lipids (1700 cm-1 - 2900 cm-1), were identified, and the intensity variations at the key wavenumbers were plotted. For HeLa cells, the DNA/RNA peak showed positive intensity variation over the incubation period, and the peaks were prominent and clear. However, in the case of the fibroblast protein peaks, variation was prominent and had the similar positive increments. Breast cancer cells showed positive increments in intensity variation although their peak prominence was lower in comparison to the other two cell lines. 3.5.1 Identification of fibroblast and HeLa cells from live SERS Raman spectra Figure 9 (A-D) depicts the gradual changes in the SERS-based Raman spectra of live HeLa and fibroblast cells for the 24-hour incubation period with 6-hour intervals. Different intracellular components, such as DNA/RNA, protein and lipid peaks, were identified for fibroblasts and HeLa cells over the 24-hour incubation period. Lipid peaks were well defined and had little intensity variation over the 6-hour intervals for both the fibroblast and HeLa cells. For HeLa cells, DNA/RNA peaks established clear prominence after 24 hours and had positive increments in the intensity over the incubation period. However, for fibroblasts, the bundle of protein peaks of fibroblast (1200 cm-1 to 1700 cm-1 range) was dominant and had positive increments in the intensity over the incubation period. Therefore, based on the peak profile and intensity variations, DNA/RNA peaks for HeLa and protein peaks for fibroblasts might be the identifying features for differentiation of these cells.


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Figure 8. SERS Raman spectra of A)-D) live fibroblast, HeLa and breast cancer cells seeded on different C-O concentration INW platforms for the 24-hour incubation period (collected on each



6-hour interval) along with the intensity variation of the intracellular components at key wavenumbers. 3.5.2 SERS effect on live fibroblast and HeLa cells The Raman signal enhancement factor for the intracellular components, such as DNA/RNA, protein and lipids, of HeLa cells and fibroblast was calculated to identify the effect of INW clusters on the Raman spectral quality. The peak intensity of different intracellular components, such as DNA, protein and lipid, of the live fibroblasts and HeLa cells after the 24-hour incubation on high C-O concentration of the INW platform and the corresponding peak intensity of native spectra (the spectra of cells outside the INW platform) were taken for this calculation. The Figure 9 (E, F) showed the overall Raman enhancement factor for DNA, protein and lipid for fibroblast and HeLa cells, respectively. The results are tabulated in the supporting information (Table S3 and Table S4). For HeLa, a six-fold Raman enhancement was produced by DNA. In the case of the fibroblast, the protein showed a four-fold Raman enhancement. Therefore, there was a significant Raman signal enhancement achieved in presence of INW clusters.


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Figure 9. (A-D) Gradual changes in SERS Raman spectra for live HeLa and fibroblast cells on the high C-O concentration INW platform acted as the basis for cell differentiation; (E) SERSbased Raman signal enhancement of intracellular components of fibroblast cells; and (F) SERSbased Raman signal enhancement of intracellular components of HeLa cells.



3.6 SERS Raman spectra of the immobilized fibroblast and HeLa cells The adhesion, SEM and fluorescence micrograph study of the immobilized HeLa and fibroblast cells on the INW platform was performed in depth (Figure 10 A, B). Both fibroblast and HeLa cell-containing samples were fixed after 24 and 48 hours, and Raman spectra of these samples were taken. The native (the area outside the INW platform zone containing cells) Raman spectra after 24 hours showed very weak signals with few peaks for both fibroblast and HeLa cells. The Raman spectra of immobilized fibroblast cells contained peaks of different intracellular components, such as DNA/RNA, protein and lipids, and there was little variation in appearance of these peaks for different C-O concentration nanocarbon platforms. These peaks were more prominent and clear after 48 hours and had positive increments in intensities (Figure 10 and Figure 11 A). The corresponding SEM and fluorescence micrographs of immobilized fibroblast cells adhered on the INW platform might explain the Raman response of the immobilized fibroblast cells. The 24-hour SEM micrograph portrayed the fibroblast cells with an elongated cellular morphology having strong attachment with overextended filopodia and actin filament on the nanostructured surface. Therefore, the Raman responses of these cells contained different intracellular component peaks; the fluorescence micrograph is also in agreement. After 48 hours, these adhered fibroblasts turned to a tissue-like structure because of growth enhancement. As the fibroblast cells turned to an interconnected overlapped tissue-like body, the Raman response in the presence of SERS-active INW clusters was more dominant and was evident from the peaks of DNA/RNA, protein and lipid. Meanwhile, the Raman spectra of the 24-hour immobilized HeLa cells, which had the cells adhered on the INW platform, contained more well-defined, clear peaks of DNA/RNA, protein and lipids in comparison to fibroblast cells (Figure 10B). However, the 48-hour Raman response


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of the immobilized HeLa cells showed a significant difference over the 24-hour response. This difference was more evident with the high carbon-oxygen to carbon-carbon bond ratio INW platform than the low carbon-oxygen to carbon-carbon bond ratio platform. There was a disappearance of some of the DNA/RNA and protein peaks, and there was a lower number of lipid peaks observed for the high carbon-oxygen to carbon-carbon ratio INW platform. After the 48-hour incubation of HeLa on the INW platform, there was a decrease in the Raman peak intensity for the DNA, protein and lipid observed (Figure 11B). These Raman responses might be explained with the analysis of the SEM and fluorescence micrograph of immobilized HeLa cells. The 24-hour SEM and fluorescence micrograph showed strongly attached, evenly spread out and elongated cells with small filopodial extensions. Conversely, the 48-hour SEM and fluorescence micrograph depicted a round-shaped cocoonlike structure. Therefore, the 24-hour spectra contained clear peaks of different intracellular components due to healthy cells, and the 48-hour spectra showed spectral changes due to the morphological change of HeLa cells (apoptotic cells). Subsequently, a comparative Raman shift range-wise analysis of spectral profile of immobilized HeLa and fibroblast cells for 24 and 48 hours was performed to help the detection of the cell health of the fibroblast and HeLa cells. The basis for this analysis was the assigned Raman peaks for immobilized fibroblasts and HeLa cells for 24- and 48-hour samples along with the SEM and fluorescence micrographs. The relative intensity change in the peaks of the intracellular biomolecular components, such as DNA/RNA, protein and lipid, over the 6-hour intervals for the live fibroblast cells might shed light on the probable growth/proliferation behavior of fibroblast cells during the incubation period. The amide III (proteins) peak (Raman shift at 1235 cm-1) was taken as an example for



showing comparison over the 6-hour time intervals of the 24-hour incubation. After first 6 hours of incubation, there was no such peak.

Figure 10. (A)Variation in different intracellular component peaks of immobilized fibroblast cells based on SERS Raman spectra obtained from 24 and 48-hour samples containing different C-O concentration INW platforms along with its SEM and fluorescence micrograph, and (B)


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Variation in different intracellular component peaks of immobilized HeLa cells based on SERS Raman spectra obtained from 24- and 48-hour samples containing different C-O concentration INW platforms along with its SEM and fluorescence micrograph. However, this peak appeared with a degree of variation (1232-1240 cm-1) afterwards and became prominent after 24 hours (Figure 8). The positive change in intensity of the Raman spectral peaks of intracellular components of immobilized fibroblast cells after 24 hours and 48 hours might indicate the presence of healthy cells on the nanostructure and further cell growth (Figure 10 and Figure 11). The SEM and fluorescence micrograph also confirmed this hypothesis.

Figure 11. (A) Positive increase in Raman intensity observed for different intracellular components such as DNA, protein and lipid of fibroblast cells over an incubation of 24 and 48 hours; (B) Decrease in Raman intensity observed for different intracellular components, such as DNA, protein and lipid, of HeLa cells over an incubation period of 24 and 48 hours; (C) Viability of fibroblasts and HeLa cells over an incubation period of 24 and 48 hours.



The Raman spectral profile for both live and immobilized HeLa cells depicted a tendency similar to the fibroblast cells. A 24-hour spectral profile revealed healthy HeLa cells on the nanostructure, and the profile was supported by corresponding SEM and fluorescence micrographs. However, the 48-hour culture depicted a huge difference in peak appearance. Several of the peaks related to different intracellular biochemical components, such as protein, DNA and lipid, that appeared in the low (300-1000 cm-1) and medium (1000-2000 cm-1) Raman shift ranges disappeared in the 48-hour Raman spectral profile (Figure 10 B). Based on such changes, it might be hypothesized that there was a change in growth of the adhered HeLa cell and that they might show a nonproliferating behavior. This hypothesis was supported by the SEM micrograph of HeLa cells after 48 hours of culture (Figure 10). In support of the difference in the Raman spectral changes for fibroblast and HeLa cells, a cell viability measurement was performed (Figure 11C). After the 24-hour incubation, fibroblasts and HeLa showed an 80% viability. After 48 hours, the viability percentage for fibroblast remained unchanged, but there was a sharp decrease, after the 48-hour incubation period, in the viability of the HeLa cells. To support the Raman spectral profile measurement, a numerical study showing the comparison of the divergent forms of HeLa and fibroblast cells on the nanocarbon platform (Figure 12) was conducted. A well-spread, elongated polygonal cell might be indicative of healthy cells, whereas a circular cell revealed apoptotic/nonproliferative cells61. Both the elongated and circular cells that attached on the nanocarbon structure of varying C-O concentrations were counted to identify the cell health in the presence of the nanocarbon structure. According to the graph (Figure 12), the elongated fibroblasts (the indicator of healthy cells on the platform) showed a gradual increment in number for different carbon-oxygen concentrations (from low to high). Further


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growth of fibroblast cells and the formation of a tissue-like structure after 48 hours limited the possibility of the individual counting of fibroblasts. However, although there was no trend with regard to the increasing increments of carbon-oxygen concentrations, the number of HeLa cells within the 24-hour incubation period displayed more elongated shaped cells than circular cells.

Figure 12. Quantitative analysis of different types of fibroblast and HeLa cells on nanocarbon platform with varying C-O concentrations. The standard error of the mean is shown by the error



bars. Triplicate experiments were carried out (n = 3). The *p and **p values were less than 0.05 and 0.01, respectively, which suggested a significant difference. After 48 hours of culture, a huge increment of round nonproliferating cells in comparison to a low number of elongated cells indicated a nonproliferating scenario of the attached HeLa cells. These results are commensurate with the Raman spectra profile obtained for both fibroblast and HeLa cells. 3.7 SERS Raman spectra of the single fibroblast and HeLa cell (immobilized) Identification of the effect of the INW clusters in achieving SERS Raman spectra on an individual cell level was achieved through further investigation of the SERS spectra of individual fibroblast and HeLa cells (Figure 13). The Raman SERS spectra for the solitary fibroblast and HeLa cell that adhered on the nanostructure or on the plain graphite substrate outside the nanostructure zone (termed as native) was collected through another Raman microscopy system (Renishaw confocal), which permitted high-speed, spatially resolved spectroscopy to be accomplished in a confocal manner. In comparison to the native cell spectra, the spectral profile obtained from high carbon-oxygen concentration nanocarbons was prominent. The HeLa cells showed more prominence for different DNA/RNA, protein and lipid peaks in comparison to fibroblast cells. The INW platform characteristic peaks, i.e., D, G and G’ bands, showed the highest intensity in the single cell Raman spectra. The peaks that appeared here for the single cell level for both the cells were in the same region as those peaks for live and immobilized cells that appeared earlier; on this basis, the tentative Raman peak assignment was made in Table S1 and Table S2 for fibroblast and HeLa cells. These peaks also appeared due to the presence of intracellular biochemical components, such as protein, DNA/RNA, and lipid.


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Figure 13. (A) SERS Raman spectra of the immobilized single fibroblast cell along with the confocal image for the 24-hour sample containing different C-O concentrations nanocarbon platforms; and (B) SERS Raman spectra of an immobilized single HeLa cell along with confocal image for the 24-hour sample containing different C-O concentration nanocarbon platforms.



4. Discussion 4.1 3D interconnected nanocarbon web formation The Raman spectroscopic approach along with a nanostructure/nanoparticle-based SERS platform allows the study of the chemical composition of a complex bio-sample, such as cells, and sheds light on their molecular makeup in health and disease53. Fine tuning of the morphology, as well as the nanochemistry of a nanofabricated SERS-active substrate, enables sensitive detection of cells62. In this study, the biosensitive INW platform on the graphite substrate was attained through a photon energy-induced ionization induced by the femtosecond laser under atmospheric conditions. When the surface of the graphite plate was irradiated using femtosecond laser pulses, surface ablation was achieved, and the surface was mechanically fragmented, nucleated homogeneously and vaporized63. The multiphoton ionization creates a plasma plume that is reactive and has a high temperature. The plasma plume contains different forms of carbon, namely, ions (C+, C2+), radicals (C2, C3) and neutral (C) carbons37. The neutral and radical carbons, which are heavy in comparison to the ions, exist at the bottom of the expanding plasma plume. However, carbon ions lie on the upper part of the plume. Variation in the mixture of the different forms of carbon occurs when energy of ionization changes, and it creates an impact on the morphology variation and the change in nanochemistry of the created 3D INW platform. When these carbon nanospecies of the plume condensed and aggregated, they shaped themselves into an interwoven nanocarbon structure. The nanocarbon width varies with the change in ionization energy from low to high and the average width decreases with the increase in ionization energy. The oxygen atom, which is one of the components of the ambient atmospheric gas, comes across the boundary of the growing plume and into contact with C+ and C2+ chemically, thereby creating carbon-oxygen molecular bonds. Therefore, both carbon-carbon


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and carbon-oxygen comprised the chemical structure of created nanocarbons, as they deposited at the time of the condensation process. The nanochemistry in terms of carbon-oxygen concentration increases when the energy of ionization also increases. The change in morphology and chemistry of different INW platforms was displayed from the analysis of FE-SEM (Figure 2C) and XPS (Figure 2(D-E). 4.2 SERS active nanocarbon-based detection of fibroblast and HeLa cells The SERS-based Raman sensing of fibroblast and HeLa cells might be explained by the interactions between the fibroblast and HeLa cells with the INW clusters. When the cells were adhered on the nanocarbon, the uptake mechanism of fibroblast and HeLa cells did not encapsulate the INW clusters. Rather, they merged with the cell membrane and then entered the bodies of the cells. According to Roiter et al.64, the smooth nanoparticles having a diameter ranging from 22 to 200 nm did not encourage the rupture of the membrane because there was a drop of the lipid bilayer curvature onto these nanoparticles. The nanocarbon width distribution in this study varied between a 5.27 and 6.67 nm (Figure 3). The sub-10-nanometer structure of the INW platform helped smooth the uptake of the INW clusters by the cells. Again, researchers showed that an active endocytosis process could take up nanoparticles of sizes up to 100 nm65,66. Upon internalization, the INW clusters were neighboring intracellular components of the cells. The C-C bond of the INW clusters induced charge transfer. The interaction for the transfer of charge among the INW clusters and intracellular biomolecular components, such as proteins, nucleic acids (DNA/RNA) and lipids, might increase the probability of electron transition. Therefore, the Raman response increases. The presence of SERS-active INW clusters inside the cells therefore helped the Raman spectroscopic signal enhancement of the intracellular biochemical components of the cells and thus the detection of HeLa and fibroblast cells.



4.3 Cell health Both the Raman spectral profile and numerical analysis of the different shaped cells that had adhered on the INW platform of varying C-O concentrations suggested two different natures of cell health – a tissue-like structure for the fibroblast and an apoptotic round structure for HeLa. For the fibroblast, the tissue-like structure is an indication of either positive or no impact of the INW cluster on the fibroblast over time. Conversely, the INW cluster had a negative effect on the HeLa cells with the passage of time and resulted in the nonproliferating round-shaped apoptotic cells. Our previous study67 supports these results. The carbon-oxygen chemical bond presence in the nanocarbon clusters that was internalized into the cell body through endocytic uptake might contribute in the positive response of both HeLa and fibroblast cells after 24 hours of culture and helped in cell survival, growth and proliferation. These facts were supported by recent research on cell survival and proliferation in an oxygenated environment68. Again, the nanocarbon clusters present inside the cytoplasm might deregulate both the genes and corresponding proteins that are associated with adhesion and affect the energy metabolism of the HeLa cells over the passage of time67. With elapsing time, the nanocarbon clusters could become cytotoxic and might create stress to the HeLa cells69. All of these phenomena result in cell rounding, i.e., apoptotic cell structure. 5. Conclusions The development of noninvasive label-free analytical techniques for in vitro cell study is advancing substantially. The SERS technique identifies components in cells through an intrinsic contrast mechanism. Therefore, the SERS technique has attracted the attention of researchers. To date, pure noble metal (gold, silver- based SERS substrates) or noble metal-assisted carbon nanoplatforms have been used for limited scale biosensing of cells and detection of biomolecules


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only. Carbon nanomaterials/nanostructures were conjugated with noble particles due to their low/no SERS ability and toxicity. However, the carbon nanomaterials were not explored individually to the same extent as a nonplasmonic SERS-active platform for in vitro cancer/normal cell detection. In this paper, we have reported the creation of a novel SERS-active nonplasmonic biosensing platform based on a self-functionalized biocompatible 3D interconnected nanocarbon web structure for effective detection of HeLa and fibroblast cells along with a guidance regarding their cell health. The sub-10-nanometer physical morphology of the INW helps the endocytic uptake of INW clusters to cells, and its SERS functionality introduces live cell Raman sensing. The INW platform has achieved an enhancement factor (EF) of 3.66×104 and 9.10×103 with crystal violet and Rhodamine 6G dyes, respectively, which is significant in comparison to the EF exhibited by the graphene surface (2 to 17). The SERS-based Raman spectra of the time-based Raman spectroscopic monitoring of both live HeLa and fibroblast cells revealed chemical fingerprints of intracellular components, such as DNA/RNA, protein and lipids. A guideline has been introduced to specify each cell based on the spectroscopic differences of DNA/RNA and protein peaks. The highest Raman enhancement achieved for fibroblasts (protein) was four-fold, and that for HeLa cells (DNA) was six-fold. Additionally, the SERS spectra along with the SEM and FM analysis of the immobilized cells after 24 and 48 hours shed light on fibroblast and HeLa cell health. A photon energy-induced ionization brought with the femtosecond laser fabricated a biocompatible INW platform with the said unique attributes. With the ionization energy alteration, there was a variation in the physicochemical properties and the morphology of the created 3D INW structure. This in vitro study recognized the distinct functionality of the self-functionalized 3D INW platform as a nonplasmonic SERS-active biosensing nanoprobe for the identification of both HeLa and



fibroblast cells through the chemical fingerprints of intracellular components, such as DNA/RNA, protein and lipids, without the need of conjugation of any noble metals. Overall, the 3D INW structure can be employed as a feasible and adaptable SERS-active sensing platform for probing intracellular compounds of HeLa and fibroblast cells and thus detection of each cell separately while elucidating the health of cells. Associated content Supporting information Raman enhancement factor calculation, Raman spectral enhancement with CV dye, R6G dye and mixture of CV and R6G dyes on INW platform, nanocarbon uptake for medium carbon-oxygen to carbon-carbon ratio INW platform, nanocarbon uptake for low carbon-oxygen to carboncarbon ratio INW platform, SERS Raman spectra of live fibroblast and HeLa cells seeded on different carbon-oxygen concentration INW platform for a 24-hour incubation period with 6hour intervals, SERS Raman spectra of live breast cancer cells seeded on different C-O concentration INW platform for 24-hour incubation period with 6-hour intervals, Table S1 Raman spectra peak assignments for fibroblast cells, Table S2 Raman spectra peak assignments for HeLa cells, Table S3 Raman signal enhancement for DNA, protein and lipid peaks for live fibroblast cells, Table S4 Raman signal enhancement for DNA, protein and lipid peaks for live HeLa cells. Authors’ Information Corresponding Author * Email: [email protected] (K.V.), Phone: 416-979-5000 Ext: 4984. ORCID A. K. M. Rezaul Haque Chowdhury: 0000-0001-9514-3589 Krishnan Venkatakrishnan: 0000-0002-6491-3971 Authors’ Contributions


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A. K. M. R. C. accomplished fabrication of samples with laser, characterization of the created nanocarbon and the manuscript drafting. K.V. and B.T. assisted and coordinated in its design. Competing interests No competing interests as per author’s declaration. Acknowledgments The NSERC (Natural Science and Engineering Research Council of Canada) has contributed funds to this research (NSERC Discovery Grants 126042 and 119087). References (1)

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Graphical Abstract


SERS-Active 3D Interconnected Nanocarbon Web ... - ACS Publications

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