Electrochemical tracing of hypoxia glycolysis by carbon nanotube sensors, a new hallmark for intraoperative detection of suspicious margins to breast neoplasia

Abstract For most people, the first step in treatment is to take out the tumor (surgery), so precise and fast diagnosis of any sign of high‐risk and neoplastic cells, especially in surgical cavity margins, is significant. The frozen pathology method is the conventional standard of intraoperative diagnosis, but the low number of slides prepared from non‐fixed tissues prevents us from achieving a perfect diagnosis. Although many improvements in intraoperative margin detection were achieved, still real‐time detection of neoplastic lesions is crucial to improving diagnostic quality. Functionalized carbon nanotubes grown on the electrode needles lively and selectively determine the H2O2 released from cancer/atypical cells through reverse Warburg effect and hypoxia assisted glycolysis pathways in a quantitative electrochemical manner. The study was carried out on cell lines, 57 in vivo mice models with breast cancer, and 258 fresh in vitro samples of breast cancer tumors. A real‐time electrotechnical system, named cancer diagnostic probe (CDP) (US Patent Pub. No.: US 2018/02991 A1, US 2021/0007638 A1, and US 2021/0022650 A1 [publications], and US 10,786,188 B1 [granted]), has been developed to find pre‐neoplastic/neoplastic cells in vivo in a quantitative electrochemical manner by tracing hypoxia glycolysis byproducts. Matched pathological evaluations with response peaks of CDP were found based on the presence of neoplasia (from atypia to invasive carcinoma) in live breast tissues. The ability of CDP to find neoplastic lesions in mice models in vivo and fresh breast tumors in vitro was verified with sensitivity and specificity of 95% and 97%, respectively. The system may help a surgeon assistant system for usage in the operating room after passing many trials and standard examinations in the future.

2021/0007638 A1, and US 2021/0022650 A1 [publications], and US 10,786,188 B1 [granted]), has been developed to find pre-neoplastic/neoplastic cells in vivo in a quantitative electrochemical manner by tracing hypoxia glycolysis byproducts. Matched pathological evaluations with response peaks of CDP were found based on the presence of neoplasia (from atypia to invasive carcinoma) in live breast tissues. The ability of CDP to find neoplastic lesions in mice models in vivo and fresh breast tumors in vitro was verified with sensitivity and specificity of 95% and 97%, respectively. The system may help a surgeon assistant system for usage in the operating room after passing many trials and standard examinations in the future. laser endomicroscopy for margin detection of brain tumors. 3 Some of the reported advantages and limitations of these techniques are demonstrated in Tables S1 and S2. Although many improvements were achieved, still no intraoperative technique has been reported for the detection of surgical margins with pathological classification in breast cancer (as one of the most important onco-surgeries required to accurate margin detection).
In this paper, a new system based on real-time tracing the hypoxia glycolysis function of cancer cells was introduced as a potential tool for detecting cancer lesions and especially margin detection.
The mechanism of such pathologically classified diagnosis is based on recording the current peaks of H 2 O 2 released during three important pathways activated in epithelial cells during cancerous transformation. First, oncogenic stimulation of normal cells followed by DNA damage and oncogene activation as the signs of tumor initiation. 4 Second, the reverse Warburg effect in which released H 2 O 2 from pre-invasive/neoplastic cells in the microenvironment changes the function of tumor-associated fibroblast (TAF) from aerobic to glycolysis metabolisms. 5,6 Third, hypoxia assisted glycolysis of tumor cells, as their distinct metabolism respect to normal cells. 7,8 It has been shown that H 2 O 2 produced and released by oncogenic stimulated normal cells would result in their transformation to atypical/preinvasive phenotypes. 9 Also, approved reports indicated the strong correlation between activation of hypoxia-assisted glycolysis and neoplastic transformation of breast cells. 7,8 This system, named cancer diagnostic probe (CDP), was investigated on wide ranges of human cell lines followed by freshly dissected breast tumors in vitro and then on mice models with breast cancers in vivo. It is the first time that such an electrical diagnoser would be applied as a real-time cancer detector in live tissues. Here, after recording and calibrating hypoxia based margin diagnosis on 258 fresh breast tumor samples, quantified diagnostic scorings of CDP response peaks were defined in correlation with their permanent histopathology results based on the World Health Organization (WHO) classification of breast tumors. [10][11][12][13] This diagnostic approach showed more than 95% sensitivity in its best calibration, which shed new light for application as a surgeon assistant in the future after passing clinical trials. It is well known that the release of H 2 O 2 molecules is one of the sequential evidence during tumor initiation, reverse Warburg effect, and hypoxia assisted glycolysis of cancer cells (Figure 1b,c). 5-7 CDP's detection mechanism has been based on the real-time breaking of released H 2 O 2 molecules, and selective releasing of two electrons on CNT covered sensing needles, which resulted in peak current recorded by the readout system ( Figure 1b). As a result, a correlation between the cells' pathologic states, the concentration of the generated H 2 O 2 in the tissue microenvironment, and the electrochemical peak current of the CDP would be observed can be analyzed and calibrated as a diagnostic profile. 16 CDP tests on various cell lines (Figure 1f,g and Supplementary Section 1) followed by RT-PCR, FTIR analysis, and lactate-based assays confirmed the specific hypoxia-related responses of CDP, and the investigation of ROS analysis investigated by N-acetyl cysteine (NAC) (see method) ( Figure 2). F I G U R E 1 (a) Image of cancer diagnostic probe (CDP) system with a changeable head probe consists of three needle electrodes coated by multiwalled carbon nanotubes (MWCNTs). (b) Selective electrochemical reactions of released H 2 O 2 on MWCNTs and production of the cathodic ionic peak. The distribution and abundance of nanotubes make a conformal surface for signal extraction, and it is presented in FESEM images. Cancer cells release H 2 O 2 due to hypoxia assisted glycolysis, as their distinct metabolism respect to normal cells.

| RT-PCR analysis
Many reports indicated that pyruvate dehydrogenase inactivation and lactate dehydrogenase activation occurred in hypoxia assisted glycolysis of cancer cells are high in correlation to activation of HIF-1α, and C-Myc activation. 17,18 It is believed that HKII protein mRNA, bound to mitochondria, enhances glucose metabolism through glycolysis in tumors. 19 HIF-1α and C-Myc oncogenes play a crucial role in the upregulation of HKII. [20][21][22] Another mRNA expressed under hypoxia of tumor cells is associated with the PGAM gene. 23,24 Activation of this protein would regulate glycolytic flux and adapt cell hypoxia. 25 Moreover, pyruvate dehydrogenase kinase-1 is another important mRNA activated by HIF-1α during hypoxia-assisted glycolysis. This enzyme induces the inactivation of PDH (the inhibitor of oxidative disposal of pyruvate).
Hence, the diversion of the glycolytic flux to lactate production would be increased. 26  2.2 | FTIR analysis of breast cell lines' secretion FTIR spectroscopy was applied to investigate the lactate-based bonds' presence (including C─H) and oxidative molecules in the media solution of all breast cell lines' phenotypes. Results indicated the increased intensity of C─H, O─H, and molecular bonds due to the invasive progression of cancer cell lines (Figure 1i). The functional group of glycerol, including O─H stretching at 3312 cm À1 , 27 was significantly increased in cancer cells' media solution with malignant grades. Also, the C═O bonds located at 1700 cm À1 could be attributed to the pyruvate produced by lactate de-hydrolysis, 28

| Applying CDP in tumor diagnosis of mice models
The animal model assay is the first step to determine CDP's in vivo efficiency in scoring both EMs and IMs. All mouse studies were per-  Table S3 shows the in vivo scoring results of CDP on 57 tumorized mice verified by H&E/IHC assays to indicate its repeatability.
As it is shown in receiver operating characteristic (ROC) analysis and area under the curve (AUC) table, for CDP the area under the curve is 0.992 (p-value < 0.0001 and CI 99% 0.98-1.00) ( Figure S7 and Table S6), and this value for frozen pathology was 0.982 (p-value < 0.0001 and CI 99% 0.96-1.00) ( Figure S8 and Table S7).
The result of the ROC test shows that the CDP has better results than frozen due to the higher area under the curve of CDP (0.992 > 0.982), and it can be used as a diagnostic test of cancerous specimens. The specificity, sensitivity, specificity, and selectivity of CDP and frozen versus permanent pathology as the gold standard are shown in   This cut-off definition's accuracy and specificity were 92% and 95%, respectively (Table S9)    The length and diameter of nanotubes ranged from 2.5 to 5 μm and from 50 to 70 nm, respectively. Figure 1b shows the FE-SEM image of the CNT biosensor. These CNTs were MWCNTs of high purity, and the presence of nickel on the topside of the CNTs could be related to the tip-growth mechanism. The CNT has been used as the work, counter, and reference electrodes. The CNT grown needles were then attached to an electrical connector with three pins by conductive paste to form the final probe. Just tips of the needle were extended from the connectors up to 1 cm. The probe was reinforced with a user-friendly homemade holder and connected to the readout system by a noiseless cable that handled all three electrodes.

| The CDP electrochemical readout system
The schematic of a CDP integrated portable automatic electrochemical readout board is shown in Figure S10. In this system, we used a low noise, high accuracy, and low power potentiostat. For making this potentiostat to decrease the noise loading effect in the environment caused by the other instruments in the operation room, we used low noise amplifiers. Moreover, to detect the current signal, which flows from the WE, a high-speed potentiostat was required. So, the Current Buffer Amplifier Classes (CBAs) was added to the main board. These two classes of amplifiers produces a creative, effective, and appropriate device for high accuracy tests.
On the other hand, to increase the circuit's accuracy, analog to digital and digital to analog (DAC) converters with 16 bits was used. A processor unit is installed, which receives the converters' data and transmits them via a bluetooth module ( Figure S10a).
Finally, the software was designed to analyze the data, diagnose cancerous or normal, and send the result through an alarm to the surgeon. A 3.3 V and 800 mA lithium-ion cell battery is powering all parts of this circuit, so it does not require to the power supply. The device's ability provides a real-time and accurate diagnostic method to utilize it clinically. The CV studies were done by DC voltage (no AC frequency) for electrochemical measurements. The potential was swept in the range from À800 to +800 m V, using a scan rate of 100 mV s À1 ( Figure S10b). (NCBI) located in the Pasteur Institute. They were kept at 37 C (5% CO 2 , 95% air) in RPMI medium (Gibco) supplemented with 5% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco).

| Cell cultures and reagents
The fresh medium was renewed every other day. All cell lines were examined and found negative for Mycoplasma contamination and counted by neobar lam. Finally, the samples were imaged with a fluorescent microscopy system. 37

| L-Lactate assay kit (colorimetric) procedure
The production of lactate in culture was analyzed to determine the rate of hypoxia-assisted glycolysis. According to the manufacturer's protocol, the intracellular level of lactate was measured by the colorimetric lactate assay kit (Abcam: ab65331, UK). The optical densities were then measured at 450 nm wavelength. The assay was carried on due to the below steps: 1. Reagent preparation: Solubilize lactate substrate mix and lactate enzyme mix, thaw lactate standard, and lactate assay buffer (aliquot if necessary); get the equipment ready. ii. Wash cells with cold PBS. iii.
Resuspend the cell pellet in 500 μl of lactate assay buffer. ii.
Harvest the required quantity of cells for each assay (initial recommendation = 10 mg tissue).
iii. Wash the sample by PBS in low temperature. iv.
Re-suspend cells in 4-6Â volumes assay buffer with the assistance of a homogenizer (kept in ice), with 10-15 passes. To form the standard curve, draw the curve through these points.
Most plate reader software can outline these values and curves.
Based on your standard curve data, calculate the trend line equation (use the equation that presents the most accurate fit).
The concentration of L-lactate in the test samples is determined as: La, amount of lactic acid in the sample well calculated from a standard curve (nmol); Sv, volume of sample added into the well (μl); D, sample dilution factor; lactic acid molecular weight, 90.08 g/mol.

| Tumor formation in mice models
Sixty female inbred BALB/c mice at 6-8 weeks of age were acquired from  Primers and probes (Table S12)