Intelligent SERS Navigation System Guiding Brain Tumor Surgery by Intraoperatively Delineating the Metabolic Acidosis

Abstract Surgeons face challenges in intraoperatively defining margin of brain tumors due to its infiltrative nature. Extracellular acidosis caused by metabolic reprogramming of cancer cells is a reliable marker for tumor infiltrative regions. Although the acidic margin‐guided surgery shows promise in improving surgical prognosis, its clinical transition is delayed by having the exogenous probes approved by the drug supervision authority. Here, an intelligent surface‐enhanced Raman scattering (SERS) navigation system delineating glioma acidic margins without administration of exogenous probes is reported. With assistance of this system, the metabolites at the tumor cutting edges can be nondestructively transferred within a water droplet to a SERS chip with pH sensitivity. Homemade deep learning model automatically processes the Raman spectra collected from the SERS chip and delineates the pH map of tumor resection bed with increased speed. Acidity correlated cancer cell density and proliferation level are demonstrated in tumor cutting edges of animal models and excised tissues from glioma patients. The overall survival of animal models post the SERS system guided surgery is significantly increased in comparison to the conventional strategy used in clinical practice. This SERS system holds the promise in accelerating clinical transition of acidic margin‐guided surgery for solid tumors with infiltrative nature.


Synthesis of Raman reporter molecules.
The detailed synthesis of IR7p can be found in our previous report [5] . In brief (Fig S1), compound a (100 mg, 0.58 mmol) and compound b (365 mg, 1.13 mmol) were dissolved in mixed solvent (C 4  SEM studies. SEM images were captured by ZEISS Gemini 500. The chips were pasted on the nail table by carbon conductive tapes. EHT=3.00 or 6.00 kV, WD=3.7 or 4.0 mm, Mag=15.00/30.00/50.00/200.00 K X. Signal A= Inlens, Aperture Size=20.00 μm. The gold nanostars distributed on the silicon wafer uniformly. There were about 1.3610 10 nanoparticles per square centimeter ( Figure S2). The gold nanoparticles in the SERS chips growing for 20 min had some protuberances with height of 2-6 nm. Compared with that before growth, the diameter of nanoparticles also increased 10-15 nm.
Extinction spectra studies. The silicon wafers in SERS chips were replaced by the glass coverslips (24 mm50 mm) in this study for the optical transparency of glass. The other fabrication parameters were the same. The extinction spectra were obtained by fixing the chips in the sample pool location in the spectrophotometer (UV2550, Shimadzu Co.). The clean coverslips were used as the control. The scanning wavelength range was 400-900 nm.
The maximal extinction wavelength of the SERS chips of gold nanospheres was 529 nm, shorter than that of gold nanostars chips, whose maximal extinction wavelength was 608 nm.
The increased of extinction in the range of 638 nm-900 nm and 475 nm-571 nm suggested that the molecule IR7p were successfully modified on the surface of gold nanostars (Fig. S3).

Raman spectroscopy studies.
No new peaks were observed by comparing the SERS spectra of IR7p to that of IR7p-pre. The main difference between the spectra of IR7p and IR7p-pre is that the SERS peak at 1097 cm -1 and 1199 cm -1 of IR7p is relatively stronger than that of IR7p-pre ( Fig S5). Therefore, we attributed all the SERS peaks to the bond in the compound IR7p-pre.
The SERS substrate post different nanoparticle growth time were treated with the methanol solution of IR7p for 12 h. The SERS signal intensities of chips after IR7p modification were significantly stronger than that of chips without IR7p fabrication. The fitting curves of I 558/ I 311 and pH value showed that the SERS chips with longer growth time have steeper trend, which is helpful to calculate pH more accurately ( Figure S9C and S9D).
The results of limit of detection (LOD) and peak assignment were obtained by using Ocean Optics QE65 Pro handheld Raman scanner. The power of 785 nm laser focused on samples are 350 mW and the acquisition time was 1 s. The LOD experiment was performed by dropping the methanol solution of IR7p onto the SERS chips (growth time: 90 min). The spectra were recorded by focusing the laser in the center of the droplet area. The signal to noise ratio (SNR) of LOD was measured as 3. The spectra of IR7p with the concentration higher than 10 -5 M can be used to calculated pH accurately because the noise in this condition is weak enough to be ignored. We choose the IR7p concentration of 2 10 -5 M to carry out the follow-up pH measuring experiment ( Figure S10).
The SERS signal homogeneity was investigated by dropping the buffer solution (pH 6.0, 2.0 μL) onto 10 spots across the SERS chip. Two similar experiments were performed by replacing the buffer solution with pH 6.5 and 7.0. Five spectra were acquired for each spot (excitation laser wavelength: 785 nm, excitation laser intensity: 350 mW, grating: 600 gr/mm, acquisition time: 500 ms). The experimental results were showed in Figure S4. Even though the Raman intensities collected at different locations of the SERS chip were diverse, the Raman intensity ratios between the Peak at 558 cm -1 and Peak at 311 cm -1 kept constant for all the spots. The standard deviations of the pH values were measured as 4.1%, 3.5% and 4.8% respectively for buffer solutions with pH 6.0, 6.5 and 7.0. Above experimental data indicated the satisfactory homogeneity of the SERS chip.
In order to study the effect of contact area on the pH measurement accuracy, four different pipette tips with diameters ranged from 469 μm to 956 μm were applied to extract samples from the mimetic tissue (agarose gel, pH 7.5). The mimetic tissue was fabricated as following. The buffer solution of about 48.0 mEq/L/pH buffer capacity was prepared by mixing the solution of disodium hydrogen phosphate (0.2 M) and citric acid (0.1 M). 100 mg of agarose powder was added into 10 mL of buffer solution. The mixture changed to gel after boiling for 10 seconds and cooling to room temperature. The diameter of tips was measured in microscope image (Olympus BX53, DP27) by the software Cellsens. The cross section of the tips is considered to be an ideal circle, and the contact area is calculated by the circle area formula. The tips containing 0.4 μL ultrapure water were contact with the agarose gel for 1 s or 2 s. Then the water was added to the SERS chip for Raman spectra acquisition. Sample extraction and pH detection procedure were carried out ten times for each kind of tips and 5 spectra were acquired for every Raman detection. When the contact time was 1.0 s, the average pH value measured by pipette tip with diameter of 469 μm was remarkably lower than the tip with contacting area diameter above 622 μm. Notably, tip area showed negligible effect to pH measurement if the contact time was longer than 2 s ( Figure S6).
In order to study the effect of blood contamination on the pH measurement by the SERS system. Three pieces of mimetic tissue made of agarose gel (pH 6.0 and pH 6.5) were prepared ( Figure S11). The fresh blood from mice was dropped on the centre of agarose gel and cleaned with cotton swabs 5 s later. The pH was measured at the same location 20 s later (ultrapure water volume:0.4 μL, contact time: 2 s). The measured pH for agarose gel of pH 6.0 ,6.5 and 7.0 was 5.98 (SD: 0.146), 6.45 (SD: 0.150) and 7.13 (SD: 0.170) respectively. So we believe the blood contamination has little effect on the measured pH by SERS system. simulation software package [1][2][3] in 3D mode. The simulation region is inside a cuboid with dimension of 500nm ×500nm ×100nm, and a 50-nm-thick perfect matched layer is added at each face boundary. The resolution of the simulation in space region is 2Å and the corresponding Courant factor is 0.95. For the calculation electric field distribution and charge distribution, a monochromatic light source (781nm) was used. The primitivity of Au reported by Johnson and Christy in 1972 [4] was used to describe optical properties of all the Au nano-structures. The FDTD results showed that the |E/E 0 | in the gap of two nanostars (maximum: 178.9) is much higher than that on the surface of nanospheres (maximum: 4.5)  Female 28 Ⅲ Anaplastic Astrocytoma          The diagram illustrating the experimental procedure. 10 μL fresh mouse blood was dropped on the agarose gel prepared with buffered solutions with pH 6.0, 6.5 and 7.0 respectively. The pH was measured before and 20 s after wiping the blood. (B) The pH values measured at the locations after wiping the blood were close to the original pH of agarose gel.