Rapid, Objective Intraoperative Mapping of Hirschsprung Disease Using a Portable Electrochemical Acetylcholine Sensor

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Ashis Mathur, Souradeep Roy, Suchanda Sahu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6288646/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: Surgical procedures for Hirschsprung Disease (HD) require accurate identification of enteric ganglion cells and cholinergic hypertrophic nerve bundles. Current intraoperative mapping through frozen section histopathology-histochemistry is time-consuming and demands skilled interpretation. This study explores an electrochemical sensor for objective, rapid intraoperative mapping of aganglionic bowel segments via tissue acetylcholine (ACh) detection. Methods: An electrochemical biosensor was developed using anodized laser-induced graphene electrodes functionalized with acetylcholinesterase enzyme (AChE). Electrochemical analyses were conducted on homogenized intestinal biopsies obtained intraoperatively from ten patients, including eight diagnosed with Hirschsprung Disease (HD) and two with total colonic aganglionosis (TCA). Biopsy samples representing ganglionic, transitional, and aganglionic bowel segments were evaluated. The sensor quantified tissue acetylcholine (ACh) levels, which serve as markers of cholinergic neuronal hypertrophy, by measuring the generated electrical current. Results: The electrochemical analysis demonstrated significantly higher current levels in the aganglionic segments compared to ganglionic segments in 87.5% (7 out of 8) of classical Hirschsprung disease (HD) patients. The mean peak currents observed in HD cases were 2.62 µA in aganglionic segments, 3.66 µA in transition segments, and 2.04 µA in ganglionic segments. In contrast, electrochemical patterns in patients diagnosed with total colonic aganglionosis (TCA) were atypical; ileal tissue samples from these patients generally yielded higher current measurements than colonic samples across all zones examined. Additionally, there was a progressive increase in tissue current values correlating positively with patient age. Conclusion: The electrochemical sensor effectively differentiated aganglionic from ganglionic zones in HD, suggesting potential as a quick, objective tool for intraoperative bowel leveling. Further validation in larger cohorts is necessary to confirm clinical utility. Biotechnology and Bioengineering Nanoscience Hirschsprung Disease Electrochemical Sensor Acetylcholine Ganglion cells Biosensor Figures Figure 1 Figure 2 Figure 3 Introduction Hirschsprung disease (HD) is a common cause of large bowel obstruction in children with a global incidence of 1 in 4000–7000 live births, probably higher in India. [ 1 ] The disease is characterized by a congenital absence of neural crest derived ganglion cells in the colon, starting from the rectum and extending proximally to varying lengths. This deficiency leads to neural hypertrophy in the aganglionic segment, causing excessive acetylcholine (ACh) release, leading to spasticity of the smooth muscles and intestinal obstruction. Histopathology remains the gold standard for HD diagnosis and management. Diagnosis is typically confirmed through a rectal biopsy demonstrating aganglionosis, followed by serial biopsies of the proximal colon during surgery to accurately map the transition from ganglionic to aganglionic zones. To make an intraoperative diagnosis, a skilled pathologist employs routine frozen section analysis with hematoxylin-eosin (H&E) staining and specific enzyme histochemical staining. Acetylcholinesterase (AChE) staining enhances the accuracy of frozen section diagnoses by identifying cholinergic neural hypertrophy. The traditional biopsy and histology process, including AChE histochemistry, requires 30–40 minutes, occasionally requiring repeat biopsies due to technical difficulties. This stepwise intraoperative process prior to a definitive surgical procedure extends anaesthesia duration, posing increased risks and requiring expert pathological support, which is available only in specialized centers. [ 2 ] The prolonged intraoperative duration and subjective nature inherent to frozen-section histopathology call for a rapid, objective, and reproducible diagnostic alternative to accurately map bowel innervation during Hirschsprung’s disease surgery. There is an increased acetylcholine level in the aganglionic segments of the intestine compared to normal, as detected by Ikawa et al. [ 3 ]. AChE catalyzes the hydrolysis of ACh into acetate and choline (reaction a), while choline oxidase further oxidizes the electroactive later to betaine (reaction b), producing two protons and electrons, that correlate with acetylcholine concentrations. These reactions yield electrons detectable via electrochemical means using a redox-active probe, with the resulting electrical current reflecting the concentration of tissue acetylcholine and the extent of cholinergic neural hypertrophy (Fig. 1 ). Such a quantitative, electrochemical approach could precisely distinguish aganglionic segments from normal intestinal tissue. The aim of this study is therefore to develop and validate an electrochemical sensor capable of accurately detecting acetylcholine-derived electric currents in intestinal tissue homogenates, thereby enabling objective identification of aganglionic bowel segments in patients with Hirschsprung’s disease. Materials and Methods Biosensor development and characterization: An electrochemical enzymatic biosensor was developed by immobilizing acetylcholinesterase (AChE) and choline oxidase (ChOx) enzymes onto anodized laser-induced graphene (aLIG) electrodes, forming a nanocomposite-modified screen-printed electrode (SPE) as previously described [ 4 ]. This biosensor utilized a voltammetric detection mechanism for acetylcholine (ACh), a recognized biomarker of Hirschsprung's disease (HD), in tissue homogenates. The electrochemical behavior and performance characteristics of the biosensor were initially assessed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Analytical performance was further validated through cyclic voltammetry using serum samples spiked with known concentrations of acetylcholine. For sensor testing, a potentiostat setup was custom-designed to quantify the electrical current resulting from enzymatic conversion of ACh by AChE and ChOx, which releases electrons correlating with acetylcholine concentration (Fig. 2 a). The potentiostat incorporated a dedicated electrode slot along with a built-in well on the electrode surface designed specifically to accommodate 40 µL of tissue homogenate supernatant (Fig. 2 b). Preclinical Validation: The electrochemical biosensor was evaluated using residual biopsy samples from patients clinically diagnosed with Hirschsprung’s disease. Approval from the Institutional Ethics Committee (IEC reference no.: T/EMF/Ped Surg/21/20) was obtained before initiation of the study. All patients underwent surgery following standard HD treatment protocols. Biopsies were taken intraoperatively under general anesthesia, with access to the colon obtained either via laparotomy or a transanal approach. Seromuscular biopsies (approximately 5 × 5 mm in size) were systematically collected, beginning from the clinically identified narrow zone (aganglionic segment) and progressing proximally into the dilated zone (ganglionic segment) at intervals of approximately 6–8 cm. These biopsies were obtained from residual tissue samples originally intended for routine frozen-section histopathological analysis. Immediately after harvesting, biopsy specimens were gently washed in phosphate-buffered saline (PBS) to remove residual blood, carefully blotted dry on filter paper to eliminate excess fluid, and subsequently weighed. A precisely weighed tissue sample (25 mg) was minced and suspended in 10 mL of phosphate buffer solution. Each intestinal tissue sample was homogenized thoroughly in 0.1 M phosphate buffer (pH 8.0) using a Potter-Elvehjem homogenizer operated at 8000 rpm until a uniform homogenate without visible tissue remnants was achieved. The homogenate was centrifuged at 1500 g for 5 minutes, after which the clear supernatant was carefully collected and immediately subjected to electrochemical analysis. A precise volume of 40 µL supernatant from each sample was loaded onto the biosensor electrode surface (Fig. 2 b), and cyclic voltammetry was conducted by inserting the sensor electrode into the potentiostat apparatus (Fig. 2 a). A potential range of -700 mV to + 700 mV was applied at a scan rate of 100 mV/s. For each sample, cyclic scans were repeated five times, and the corresponding peak current values were recorded. To assure measurement accuracy and reproducibility and minimize variability, each tissue sample was analyzed in triplicate, using separate biosensor electrodes each time. Mean peak current values for each sample were then calculated. Finally, the electrical current values generated by tissue samples were compared between histologically confirmed aganglionic and ganglionic segments, as identified by conventional frozen-section histopathology, the established gold-standard diagnostic technique. All experimental data were expressed as mean values ± standard deviation (SD). To assess the diagnostic efficacy of the electrochemical sensor relative to conventional histopathological analysis (gold standard), measures of diagnostic accuracy—including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy—were calculated. The sensor-generated current values across different intestinal zones (aganglionic, transition, and ganglionic) were summarized as mean ± standard deviation and compared using non-parametric tests (e.g., Mann–Whitney U test), due to the relatively small sample size and distribution characteristics. The correlation between sensor-generated current values and patient age was analyzed using Spearman's rank correlation coefficient. Linear regression analysis was performed to assess the linearity of sensor response to increasing acetylcholine concentration, and the correlation coefficient (R²) was computed to evaluate goodness-of-fit. A p-value less than 0.05 was considered statistically significant. All statistical analyses were performed using standard statistical software. Results Biosensor analytical performance validation results: The performance of the electrochemical sensor in detecting acetylcholine was evaluated using Differential Pulse Voltammetry (DPV) across a potential window ranging from − 0.4 to + 0.4 V at a scan rate of 100 mV/s. The sensor demonstrated a low detection limit of 0.26 µM and exhibited robust stability, maintaining reliable performance for up to one month (at 4 0 C) under storage conditions, even in the presence of common biochemical interferents. The sensor’s linear response was tested using human serum samples spiked with acetylcholine concentrations ranging from 1 µM to 100 µM alongside the negative controls to verify its analytical accuracy. DPV voltammograms clearly illustrated a concentration-dependent increase in peak current amplitude, with current intensities approximately doubling when acetylcholine concentration increased from 1 µM (24.7 µA) to 100 µM (50 µA). A calibration curve was generated by plotting sensor current responses against the logarithm of acetylcholine concentrations, revealing a strong linear correlation (R² = 0.99), indicative of excellent sensor performance and reliability (Fig. 3 ). This clear linear relationship confirms the sensor's ability to differentiate between normal acetylcholine levels (lower currents typical of ganglionic segments) and elevated acetylcholine concentrations associated with aganglionic segments characteristic of Hirschsprung’s disease. Preclinical evaluation results: In this study, a total of 39 intestinal biopsy samples were obtained from ten patients, each analyzed using both the electrochemical biosensor and conventional histopathological methods (frozen section and paraffin-embedded H&E staining). Based on the histopathological results, eight patients were diagnosed with Hirschsprung's disease (HD), while two patients had total colonic aganglionosis (TCA). Electrochemical analysis was performed on residual tissue obtained from frozen section biopsies, and the generated electrical current values were recorded. Sensor current amplitude distinctly differentiated aganglionic from ganglionic segments in 7 out of 10 cases (70%), with aganglionic segments generating higher currents. In patients with HD, the mean peak current recorded by the sensor was highest in the transition zone (mean: 3.66 µA, range: 1.1–8.78 µA), followed by the aganglionic zone (mean: 2.62 µA, range: 0.95–5.77 µA), and lowest in the ganglionic zone (mean: 2.04 µA, range: 0.92–4.16 µA). Notably, the aganglionic zone demonstrated higher current values compared to the ganglionic zone in 7 out of 8 patients (87.5%). Additionally, a distinct trend emerged in HD patients: sensor current progressively increased from the aganglionic segment, peaked in the transition zone, and subsequently decreased as it approached the ganglionic region. In contrast, biopsies were analyzed for the two patients diagnosed with TCA from both colonic and ileal segments, covering aganglionic, transition, and ganglionic zones. Interestingly, ileal samples consistently exhibited higher current values compared to colonic samples across all intestinal segments. Specifically, while colonic biopsies yielded current values of 1.28 µA and 1.40 µA, ileal biopsies produced markedly higher average currents of 5.68 µA, 4.77 µA, and 6.66 µA in the aganglionic, transition, and ganglionic zones, respectively. This pattern was opposite to that observed in HD patients; in TCA samples, the lowest currents were observed in the transition zone, increasing again towards the ganglionic region. The overall variation in current between ganglionic and aganglionic segments ranged from 0.92 µA to 8.78 µA. Notably, in 4 patients (40%), the difference between these zones was less than 1 µA, indicating minimal contrast. Additionally, a consistent and progressive increase in sensor current values was observed with increasing patient age, a phenomenon noted across all three intestinal zones evaluated. The electrochemical biosensor findings were directly compared to conventional histopathological analysis (frozen-section biopsy and H&E staining), which established the gold standard for diagnosing and mapping Hirschsprung’s disease. In instances where multiple biopsies were obtained from the same intestinal zone, the mean current value was calculated to standardize results (Table 1 ). The biosensor successfully detected higher acetylcholine-derived current levels in aganglionic segments in 7 out of 8 patients with classical HD, corresponding to a sensitivity of 87.5%. However, one HD patient (a neonate) demonstrated lower current levels in the aganglionic region, resulting in a false-negative rate of 12.5%. Both total colonic aganglionosis (TCA) cases showed atypical electrochemical patterns, highlighting potential diagnostic limitations in these distinct disease variants. Given the absence of false-positive results in identifying aganglionic segments within classical HD cases, the calculated specificity was 100%. Considering the entire patient cohort (HD and TCA, n = 10), the electrochemical sensor showed a sensitivity of 70%, specificity of 100% overall diagnostic accuracy of 70% for distinguishing aganglionic from ganglionic bowel segments intraoperatively. Table 1 Patient-wise Electrochemical Current Values in Ganglionic, Transition, and Aganglionic Zones of the Bowel Each row represents an individual patient and provides their age, sex, and diagnosis [Hirschsprung disease (HD) or total colonic aganglionosis (TCA)], along with the recorded electrochemical current values in the ganglionic, transitional, and aganglionic segments of the bowel. Sl.no. Age/Sex Electrochemical current (µA ) Remarks Ganglionic zone (± SD) Transition zone (± SD) Aganglionic zone (± SD) 1. 1m/M 1.11 (0.07) 1.20 (0.05) 0.98 (0.02) HD.Only instance with a lower level in the aganglionic zone than the ganglionic zone 2. 3m/M 0.92 (0.04) 1.10 (0.03) 0.95 (0.05) HD -Rectosigmoid TZ 3. 3m/M 0.93 (0.1) 1.16 (0.04) 0.95 (0.03) HD -Rectosigmoid TZ 4. 1y/F 1.91 (0.1) 4.46 (0.2) 2.84 (0.09) HD- Long segment, splenic flexure TZ 5. 2y/M 2.46 (0..9) 4.60 (0.4) 3.12 (0.09) HD Long segment, descending colon TZ 6. 2y/F 2.12 (0.07) 2.90 (0.08) 2.37 (0.06) HD -Rectosigmoid TZ 7. 3y/M 4.16 (0.09) 8.78 (0.2) 5.77 (0.5) HD-Rectosigmoid TZ 8. 5 y/M 2.77 (0.07) 5.08 (0.05) 3.95 (0.05) HD Rectosigmoid TZ 9. 5m/F 6.2 (1.2) 4.0 (1.1) 4.8 (0.7) TCA. Ileal TZ. Colonic sample − 1.4 10. 2y/F 7.12 (0.9) 5.55 (1.4) 6.56 (1.2) TCA. Ileal TZ. Colonic sample 1.28 SD - Standard Deviation. m-month, y-year, M-male ,F-female, HD - Hirschsprung Disease, TCA- Total colonic aganglionosis, TZ- Transition Zone The Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of the electrochemical sensor were estimated based on comparison with histopathological findings. Given the sensor correctly identified 7 out of 8 confirmed aganglionic segments, the positive predictive value approached 100%, assuming no false-positive cases were recorded. However, the presence of a single false-negative case yielded a negative predictive value slightly below 100%. Further analysis with a larger sample size is required to establish these diagnostic parameters precisely. Discussion The biosensor could successfully detect the aganglionic segment of the colon in 7 out of 8 (87.5%) patients with Hirschsprung Disease, and both the TCA patients showed atypical electrochemical patterns. Following a diagnosis of aganglionosis by rectal biopsy, the traditional histological and histochemical leveling extends operative and anesthesia time by approximately an hour and relies on considerable diagnostic expertise. Often, surgeons experience downtime during this period as they await the pathologist’s report. Such delays present logistical challenges, especially in developing nations already burdened with heavy patient loads. In addition, there is an increased risk from prolonged general anesthesia. Leveling inaccuracies can lead to either the removal of functional ganglionic bowel or the retention of aganglionic segments, both of which are detrimental. Residual aganglionosis is a known factor contributing to morbidity post-pull-through surgery, necessitating further surgical intervention, and significantly impacting the quality of life [ 1 ]. In fact, a meta-analysis suggests that up to one-third of reoperations for HD are due to residual aganglionosis [ 5 ]. Various experimental methods have been explored in pursuit of more efficient intraoperative techniques. Frykman et al. reported that spectral imaging could differentiate between ganglionic and aganglionic bowel tissues in real time with high sensitivity and specificity in a mouse model of HD [ 6 ]. They demonstrated a statistical difference between the spectral curves for ganglionic and aganglionic bowel at around 610nm wavelength. Similarly, Soares de Oliveira et al. demonstrated the use of optical modalities for near real-time intraoperative detection of pathological segments in murine models [ 7 ]. Confocal laser endomicroscopy (CLE) appears to be a promising diagnostic alternative to conventional histopathology. Harada et al. employed CLE to detect aganglionosis in resected colonic specimens from nine cases of HD [ 8 ]. This method successfully differentiated the appearance of the enteric nervous system in ganglionic versus aganglionic segments; it correlated with the histopathological reference standards in 88% of the samples. CLE allows visualization of the enteric nervous system within the submucosal layer. However, the technique faces practical challenges - creating a submucosal space from the serosal side is technically demanding, and creating this space can adversely impact postoperative colon function. Furthermore, a significant limitation remains the absence of a human-approved neural-affinity dye that can highlight the myenteric plexus during microendoscopy. Thus, practical limitations include the difficulty of imaging the entire bowel and creating a submucosal space without affecting postoperatively colon function. Besides aganglionosis, cholinergic neural hypertrophy in the aganglionic zone has traditionally served as a foundation for leveling of Hirschsprung Disease (HD) before bowel resection. There is a substantial need for a practical, swift, and dependable sensor technology that is cost-effective, compact, and user-friendly to facilitate intraoperative management of HD. Our work introduces a portable electrochemical biosensor that correlates the generated current with tissue acetylcholine (ACh) levels, potentially aiding in the intraoperative leveling of HD. Existing acetylcholine biosensors are mostly sizable benchtop units and fall short in sensitivity for intraoperative use on tissue fluids [ 9 – 12 ]. In contrast, our prototype sensor is a portable unit akin to a Universal Serial Bus (USB) device that is designed to interface with a notebook laptop and enable the generation of colon mapping data through specialized software (Fig. 2 b). The hallmark of HD is the absence of ganglion cells within the intestinal myenteric and submucosal plexuses. The rectosigmoid region is implicated in approximately 85% of cases, where a transition zone of several centimeters separates the distal aganglionic and proximal ganglionic regions. Notably, this aganglionic zone is characterized by a significant increase in the size of preganglionic parasympathetic cholinergic nerve fibers [ 13 ], which correspond to a surge in AChE enzyme concentration—a phenomenon readily detected through histochemical staining [ 14 ]. The aganglionic zone is known to exhibit heightened levels of acetylcholine release, both at rest and under stimulation, relative to the proximal ganglionic zone [ 15 ], with the distal aganglionic rectum demonstrating the peak AChE activity, diminishing in intensity towards the normoganglionic bowel [ 16 ]. Several studies report elevated acetylcholine concentrations in the aganglionic zones when compared to ganglionic ones [ 13 – 16 ]. Ikawa et al. noted concentrations of 23.79 nmol/g in aganglionic zones versus 8.51 nmol/g in ganglionic zones of tissue [ 3 ]. Additionally, the concentration of cholinergic vesicles is denser at the nerve endings within the aganglionic zones [ 15 ]. Our projections of increased current values in aganglionic zones were confirmed, although the electrical activity displayed by the transition zone—peaking in patients with HD and dipping in patients with TCA— remains unexplained. This phenomenon could be attributed to atypical structural and secretory characteristics of ganglion cells in these transitional areas. [ 17 ]. Also, though both HD and TCA are aganglionic states, TCA is gradually being recognized as not merely a longer version of HD but a biologically distinct form with its own neuronal characteristics. [ 18 ] The detected current levels are dependent on the tissue ACh levels, which are, in turn, a consequence of the degree of nerve hypertrophy. Kapur et al. have observed that such hypertrophy tends to be focal or multifocal, with about half of the cases demonstrating diffuse hypertrophy. [ 19 ] In our methods, we halved the harvested biopsy samples, performing frozen biopsies on one part and electrochemical analysis on the other. Thus it is probable that hypertrophic nerves present in one half of the sample would also be in the other, thereby reducing the likelihood of sampling errors due to focal hypertrophy. The interpretation of acetylcholinesterase (AChE) enzyme histochemistry requires a level of technical skill and experience that is not always readily available, and inconclusive results can occur in a significant portion of biopsies [ 20 ]. While alternative staining methods have been explored to conclusively map ganglion cells and hypertrophic nerves, recent findings by Kapur et al. indicate atypical choline transporter innervation in both aganglionic segments and transition zones [ 21 ]. Such irregularities in choline transport could potentially lead to an accumulation of tissue ACh, a factor our electrochemical method can quantify. In this series, an elevated current in the aganglionic segment was absent in one neonate with classic HD, aligning with observations from Garrett et al., who reported an absence of heightened AChE activity in younger patients [ 22 ]. Furthermore, Ambartsumyan et al. have suggested that nerve caliber—and consequently, acetylcholine release—increases with patient age, accounting for the higher levels of acetylcholine observed in older individuals [ 23 ]. In total colonic aganglionosis (TCA) cases, the proliferation patterns and proximal extensions of AChE-positive nerve fibers are less understood. Some reports indicate a lack of the typical positive AChE reaction seen in classical HD, raising questions about the reliability of histochemical diagnosis in TCA [ 18 ]. Meier-Ruge et al. report a significant reduction in AChE-positive nerve fibers in TCA patients as one moves orally from the rectum towards the splenic flexure [ 14 ], a finding that correlates with the minimal nerve hypertrophy and, thus, reduced acetylcholine levels in the colon. Our findings corroborate this, with TCA patients demonstrating low acetylcholine levels in colonic tissues, distinct from those observed in classical HD. Compared to previously reported experimental and clinical methods aimed at identifying aganglionic bowel segments in Hirschsprung’s disease (HD), the electrochemical biosensor described in our study offers distinct advantages in terms of simplicity, portability, and cost-effectiveness. While existing methodologies such as spectral imaging, confocal laser endomicroscopy, and conventional histopathological analysis require sophisticated equipment, specialized infrastructure, and expert interpretation, our proposed electrochemical biosensor provides a portable, user-friendly platform capable of real-time intraoperative analysis. This approach significantly reduces reliance on specialized pathology support, minimizes operative downtime, and potentially shortens anesthesia duration, thus offering substantial logistical and clinical benefits, especially in resource-constrained healthcare settings. Potential limitations of this method include variability introduced during sample preparation and analysis. Specifically, errors may arise due to the uneven distribution of hypertrophic nerve fibers within biopsy samples, potentially affecting acetylcholine levels and resultant electrochemical currents. Additionally, variability in tissue homogenization, efficiency of acetylcholine extraction, and sample processing may also influence the accuracy and reproducibility of sensor measurements. To mitigate these issues, careful standardization of biopsy collection techniques, rigorous homogenization protocols, and optimization of acetylcholine extraction procedures are recommended for future studies. In addition to the above complexities and technical limitations, we note that the current generated varied less than 1 µA between normal and pathological bowel in 40% of the patients. The sensitivity of the electrode, calibrated using a stock solution of ACh, was 0.42µA/µM/mm 2 , which translates to a minimal difference in acetylcholine concentrations between the aganglionic and ganglionic zones—less than 2.38 µM. To enhance the sensitivity and reliability of our measurements, we plan to expand the electrode's reactive surface area and refine our sample processing technique, such as extracting ACh from the tissue prior to electrochemical analysis. We acknowledge the potential for other artifacts within the homogenized samples that may generate free electrons and impact the potentiostat's readings. Further optimization to enhance sensor sensitivity and specificity, possibly through advanced nanomaterials or refined sample preparation techniques, will be necessary to increase diagnostic accuracy, especially in samples showing minimal contrast. Our prototype sensor continues to undergo meticulous evaluation in terms of sample processing and clinicohistopathological correlation. We intend to further refine our electrodes and methods based on current data and engage in a multicenter pivotal study with a larger sample size to validate our findings. Conclusion The electrochemical sensor developed for detecting ACh in homogenized tissue samples has been validated using stock solutions and spiked serum. In a preclinical test, the sensor demonstrated a higher level of ACh in the aganglionic zone compared to the normal ganglionic zone in 87.5% of patients with classical HD. The promising preliminary outcome requires further testing on a broader pediatric cohort to establish the clinical utility of the sensor. The quantitative nature of the assessment reduces the risk of observer bias and could be a revolutionary device for intraoperative mapping/leveling of the ganglionic -aganglionic zones in the management of Hirschsprung Disease and allied disorders. This study demonstrates the feasibility of an electrochemical biosensor for objectively and rapidly differentiating ganglionic from aganglionic segments intraoperatively in patients with Hirschsprung's disease. While initial results are promising, broader multicenter validation studies are essential to establish its clinical reliability and utility before adoption into routine practice. Declarations Acknowledgement: This research was supported by funding from the Biotechnology Industry Research Assistance Council (BIRAC), Department of Biotechnology, Government of India, under the Biotechnology Ignition Grant (BIG) scheme. The authors gratefully acknowledge BIRAC for the financial support provided to carry out this work. 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Am J Surg Pathol. ;40(12):1637–1646. 10.1097/PAS.0000000000000711 . PMID: 27526297 de Haro Jorge I, Palazón Bellver P, Julia Masip V, Saura García L, Ribalta Farres T, Cuadras Pallejà D, Tarrado Castellarnau X (2016) Effectiveness of calretinin and role of age in the diagnosis of Hirschsprung disease. Pediatr Surg Int 32(8):723–727. 10.1007/s00383-016-3912-3 Epub 2016 Jul 1. PMID: 27369965 Kapur RP, Raess PW, Hwang S, Winter C (2017 Jul-Aug) Choline Transporter Immunohistochemistry: An Effective Substitute for Acetylcholinesterase Histochemistry to Diagnose Hirschsprung Disease With Formalin-fixed Paraffin-embedded Rectal Biopsies. Pediatr Dev Pathol 20(4):308–320 Epub 2017 Mar 23. PMID: 28649946 Garrett JR, Howard ER, Nixon HH (1969) Autonomic nerves in rectum and colon in Hirschsprung's disease. A cholinesterase and catecholamine histochemical study. Arch Dis Child 44(235):406–417. 10.1136/adc.44.235.406 PMID: 5785192; PMCID: PMC2020300 Ambartsumyan L, Smith C, Kapur RP (2020) Jan-Feb;23(1):8–22 Diagnosis of Hirschsprung Disease. Pediatr Dev Pathol. doi: 10.1177/1093526619892351. Epub 2019 Dec 2. PMID: 31791203 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6288646","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":432713218,"identity":"8f1dcfbe-3fe3-45d9-8911-44d126216e72","order_by":0,"name":"AKASH BIHARI PATI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYJCCA0DIYABifQBiNnaCGpgRWhhngLQwE6GFAaaFmQcmgA+Ys58/eODHGTt7cwbmZ49tfm2T52NmYPzwMQe3FsueZIaDPTeSE3c2sJkb5/bdNmxjZmCWnLkNtxaDA8kMB3g+MCcYHGAwk87tuc0I1MLGzItPy/nHDAf/fKi3NzjA/k3asue2PWEtN5IZDvPcOMy44QCPmTTDj9uJBLVYznhscFjmzPHEDYd5yg17G24ntzEzNuP1izl/4uOPb45V2xscb9/24Mef27bz25sPfviIz2FwFtA9DIxtIBZjA271KFqA8c7A8Aev4lEwCkbBKBihAADDkVSk8vGhnwAAAABJRU5ErkJggg==","orcid":"","institution":"All India Institute of Medical Sciences, Bhubaneswar","correspondingAuthor":true,"prefix":"","firstName":"AKASH","middleName":"BIHARI","lastName":"PATI","suffix":""},{"id":432713219,"identity":"dc46ee3e-ff9a-4d0f-82d5-d6133b0f19b8","order_by":1,"name":". Ashis Mathur","email":"data:image/png;base64,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","orcid":"","institution":"University of Petroleum and Energy Studies, Dehradun, Uttarakhand, India","correspondingAuthor":true,"prefix":"","firstName":".","middleName":"Ashis","lastName":"Mathur","suffix":""},{"id":432713220,"identity":"a083c09d-f27a-42ac-93c4-072d2110bc39","order_by":2,"name":"Souradeep Roy","email":"","orcid":"","institution":"University of Petroleum and Energy Studies, Dehradun, Uttarakhand, India","correspondingAuthor":false,"prefix":"","firstName":"Souradeep","middleName":"","lastName":"Roy","suffix":""},{"id":432713221,"identity":"da1819fa-bddd-47f0-a24f-9be15866bc83","order_by":3,"name":"Suchanda Sahu","email":"","orcid":"","institution":"All India Institute of Medical Sciences, Bhubaneswar","correspondingAuthor":false,"prefix":"","firstName":"Suchanda","middleName":"","lastName":"Sahu","suffix":""},{"id":432713222,"identity":"a524f6b4-dd9b-4ae3-8c00-2d8fb342fff0","order_by":4,"name":"Pritinanada Mishra","email":"","orcid":"","institution":"All India Institute of Medical Sciences, Bhubaneswar","correspondingAuthor":false,"prefix":"","firstName":"Pritinanada","middleName":"","lastName":"Mishra","suffix":""},{"id":432713223,"identity":"dc709426-56c4-4cd9-87bc-29465038a931","order_by":5,"name":"Santosh Kumar Mahalik","email":"","orcid":"","institution":"All India Institute of Medical Sciences, Bhubaneswar","correspondingAuthor":false,"prefix":"","firstName":"Santosh","middleName":"Kumar","lastName":"Mahalik","suffix":""},{"id":432713224,"identity":"420313ed-0aec-46c7-ad5b-b61fedd8393e","order_by":6,"name":"Antony Tsai","email":"","orcid":"","institution":"Penn State Health Milton S. Hershey Medical Center, Pennsylvania, USA","correspondingAuthor":false,"prefix":"","firstName":"Antony","middleName":"","lastName":"Tsai","suffix":""},{"id":432713225,"identity":"8b5247cf-9aed-4892-a3c4-198a469cf498","order_by":7,"name":"Kanishka Das","email":"","orcid":"","institution":"All India Institute of Medical Sciences, Bhubaneswar","correspondingAuthor":false,"prefix":"","firstName":"Kanishka","middleName":"","lastName":"Das","suffix":""}],"badges":[],"createdAt":"2025-03-23 13:57:13","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6288646/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6288646/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79260548,"identity":"5b5266bf-e8c6-4bb4-9595-f0159c335076","added_by":"auto","created_at":"2025-03-26 09:23:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the electrochemical biosensor mechanism for acetylcholine (ACh) detection. Acetylcholine esterase (AChE) catalyzes the hydrolysis of acetylcholine into choline, subsequently oxidized by choline oxidase (ChOx), producing electrons measurable as electrical current. The magnitude of this current correlates directly with acetylcholine concentration, facilitating the identification of aganglionic bowel segments in Hirschsprung Disease.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"ACh1.png","url":"https://assets-eu.researchsquare.com/files/rs-6288646/v1/85bf9073251edf51e6517ef3.png"},{"id":79260575,"identity":"cd6f84e7-b7c5-4407-a7af-04994e91e30b","added_by":"auto","created_at":"2025-03-26 09:23:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":798360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectrochemical sensor device and operational setup. (a) Portable potentiostat device (USB interface) showing the sensor electrode insertion slot; (b) Sensor electrode positioned within the device connected to a laptop for real-time electrochemical data acquisition and intraoperative bowel segment mapping\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"ACh2.png","url":"https://assets-eu.researchsquare.com/files/rs-6288646/v1/92492c28be1c037c797d53bf.png"},{"id":79260552,"identity":"e9cf0340-6fd4-452a-873c-6ec2ee2b1a11","added_by":"auto","created_at":"2025-03-26 09:23:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCalibration curve demonstrating the linear relationship between sensor-generated current and acetylcholine concentrations (1 µM to 100 µM) in spiked serum samples.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eThe high correlation coefficient (R² = 0.99) confirms the reliability and sensitivity of the electrochemical biosensor for detecting acetylcholine.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"ACh3.png","url":"https://assets-eu.researchsquare.com/files/rs-6288646/v1/536a7e8e4ddf005e2eb5c1ef.png"},{"id":79261154,"identity":"8e62b05c-b940-48e7-861a-a6d702458139","added_by":"auto","created_at":"2025-03-26 09:31:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2085513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6288646/v1/d24c49ad-6e45-4c0c-8db7-6fec7196dd6d.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRapid, Objective Intraoperative Mapping of Hirschsprung Disease Using a Portable Electrochemical Acetylcholine Sensor\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHirschsprung disease (HD) is a common cause of large bowel obstruction in children with a global incidence of 1 in 4000\u0026ndash;7000 live births, probably higher in India. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] The disease is characterized by a congenital absence of neural crest derived ganglion cells in the colon, starting from the rectum and extending proximally to varying lengths. This deficiency leads to neural hypertrophy in the aganglionic segment, causing excessive acetylcholine (ACh) release, leading to spasticity of the smooth muscles and intestinal obstruction.\u003c/p\u003e \u003cp\u003eHistopathology remains the gold standard for HD diagnosis and management. Diagnosis is typically confirmed through a rectal biopsy demonstrating aganglionosis, followed by serial biopsies of the proximal colon during surgery to accurately map the transition from ganglionic to aganglionic zones. To make an intraoperative diagnosis, a skilled pathologist employs routine frozen section analysis with hematoxylin-eosin (H\u0026amp;E) staining and specific enzyme histochemical staining. Acetylcholinesterase (AChE) staining enhances the accuracy of frozen section diagnoses by identifying cholinergic neural hypertrophy. The traditional biopsy and histology process, including AChE histochemistry, requires 30\u0026ndash;40 minutes, occasionally requiring repeat biopsies due to technical difficulties. This stepwise intraoperative process prior to a definitive surgical procedure extends anaesthesia duration, posing increased risks and requiring expert pathological support, which is available only in specialized centers. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] The prolonged intraoperative duration and subjective nature inherent to frozen-section histopathology call for a rapid, objective, and reproducible diagnostic alternative to accurately map bowel innervation during Hirschsprung\u0026rsquo;s disease surgery.\u003c/p\u003e \u003cp\u003eThere is an increased acetylcholine level in the aganglionic segments of the intestine compared to normal, as detected by Ikawa et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. AChE catalyzes the hydrolysis of ACh into acetate and choline (reaction a), while choline oxidase further oxidizes the electroactive later to betaine (reaction b), producing two protons and electrons, that correlate with acetylcholine concentrations. These reactions yield electrons detectable via electrochemical means using a redox-active probe, with the resulting electrical current reflecting the concentration of tissue acetylcholine and the extent of cholinergic neural hypertrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Such a quantitative, electrochemical approach could precisely distinguish aganglionic segments from normal intestinal tissue. The aim of this study is therefore to develop and validate an electrochemical sensor capable of accurately detecting acetylcholine-derived electric currents in intestinal tissue homogenates, thereby enabling objective identification of aganglionic bowel segments in patients with Hirschsprung\u0026rsquo;s disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBiosensor development and characterization:\u003c/h2\u003e \u003cp\u003eAn electrochemical enzymatic biosensor was developed by immobilizing acetylcholinesterase (AChE) and choline oxidase (ChOx) enzymes onto anodized laser-induced graphene (aLIG) electrodes, forming a nanocomposite-modified screen-printed electrode (SPE) as previously described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This biosensor utilized a voltammetric detection mechanism for acetylcholine (ACh), a recognized biomarker of Hirschsprung's disease (HD), in tissue homogenates. The electrochemical behavior and performance characteristics of the biosensor were initially assessed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Analytical performance was further validated through cyclic voltammetry using serum samples spiked with known concentrations of acetylcholine.\u003c/p\u003e \u003cp\u003eFor sensor testing, a potentiostat setup was custom-designed to quantify the electrical current resulting from enzymatic conversion of ACh by AChE and ChOx, which releases electrons correlating with acetylcholine concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The potentiostat incorporated a dedicated electrode slot along with a built-in well on the electrode surface designed specifically to accommodate 40 \u0026micro;L of tissue homogenate supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreclinical Validation:\u003c/h3\u003e\n\u003cp\u003eThe electrochemical biosensor was evaluated using residual biopsy samples from patients clinically diagnosed with Hirschsprung\u0026rsquo;s disease. Approval from the Institutional Ethics Committee (IEC reference no.: T/EMF/Ped Surg/21/20) was obtained before initiation of the study. All patients underwent surgery following standard HD treatment protocols. Biopsies were taken intraoperatively under general anesthesia, with access to the colon obtained either via laparotomy or a transanal approach. Seromuscular biopsies (approximately 5 \u0026times; 5 mm in size) were systematically collected, beginning from the clinically identified narrow zone (aganglionic segment) and progressing proximally into the dilated zone (ganglionic segment) at intervals of approximately 6\u0026ndash;8 cm. These biopsies were obtained from residual tissue samples originally intended for routine frozen-section histopathological analysis.\u003c/p\u003e \u003cp\u003eImmediately after harvesting, biopsy specimens were gently washed in phosphate-buffered saline (PBS) to remove residual blood, carefully blotted dry on filter paper to eliminate excess fluid, and subsequently weighed. A precisely weighed tissue sample (25 mg) was minced and suspended in 10 mL of phosphate buffer solution. Each intestinal tissue sample was homogenized thoroughly in 0.1 M phosphate buffer (pH 8.0) using a Potter-Elvehjem homogenizer operated at 8000 rpm until a uniform homogenate without visible tissue remnants was achieved. The homogenate was centrifuged at 1500 g for 5 minutes, after which the clear supernatant was carefully collected and immediately subjected to electrochemical analysis.\u003c/p\u003e \u003cp\u003eA precise volume of 40 \u0026micro;L supernatant from each sample was loaded onto the biosensor electrode surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and cyclic voltammetry was conducted by inserting the sensor electrode into the potentiostat apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). A potential range of -700 mV to +\u0026thinsp;700 mV was applied at a scan rate of 100 mV/s. For each sample, cyclic scans were repeated five times, and the corresponding peak current values were recorded. To assure measurement accuracy and reproducibility and minimize variability, each tissue sample was analyzed in triplicate, using separate biosensor electrodes each time. Mean peak current values for each sample were then calculated.\u003c/p\u003e \u003cp\u003eFinally, the electrical current values generated by tissue samples were compared between histologically confirmed aganglionic and ganglionic segments, as identified by conventional frozen-section histopathology, the established gold-standard diagnostic technique.\u003c/p\u003e \u003cp\u003eAll experimental data were expressed as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). To assess the diagnostic efficacy of the electrochemical sensor relative to conventional histopathological analysis (gold standard), measures of diagnostic accuracy\u0026mdash;including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy\u0026mdash;were calculated. The sensor-generated current values across different intestinal zones (aganglionic, transition, and ganglionic) were summarized as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and compared using non-parametric tests (e.g., Mann\u0026ndash;Whitney U test), due to the relatively small sample size and distribution characteristics.\u003c/p\u003e \u003cp\u003eThe correlation between sensor-generated current values and patient age was analyzed using Spearman's rank correlation coefficient. Linear regression analysis was performed to assess the linearity of sensor response to increasing acetylcholine concentration, and the correlation coefficient (R\u0026sup2;) was computed to evaluate goodness-of-fit. A p-value less than 0.05 was considered statistically significant. All statistical analyses were performed using standard statistical software.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiosensor analytical performance validation results:\u003c/h2\u003e \u003cp\u003eThe performance of the electrochemical sensor in detecting acetylcholine was evaluated using Differential Pulse Voltammetry (DPV) across a potential window ranging from \u0026minus;\u0026thinsp;0.4 to +\u0026thinsp;0.4 V at a scan rate of 100 mV/s. The sensor demonstrated a low detection limit of 0.26 \u0026micro;M and exhibited robust stability, maintaining reliable performance for up to one month (at 4\u003csup\u003e0\u003c/sup\u003eC) under storage conditions, even in the presence of common biochemical interferents.\u003c/p\u003e \u003cp\u003eThe sensor\u0026rsquo;s linear response was tested using human serum samples spiked with acetylcholine concentrations ranging from 1 \u0026micro;M to 100 \u0026micro;M alongside the negative controls to verify its analytical accuracy. DPV voltammograms clearly illustrated a concentration-dependent increase in peak current amplitude, with current intensities approximately doubling when acetylcholine concentration increased from 1 \u0026micro;M (24.7 \u0026micro;A) to 100 \u0026micro;M (50 \u0026micro;A).\u003c/p\u003e \u003cp\u003eA calibration curve was generated by plotting sensor current responses against the logarithm of acetylcholine concentrations, revealing a strong linear correlation (R\u0026sup2; = 0.99), indicative of excellent sensor performance and reliability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This clear linear relationship confirms the sensor's ability to differentiate between normal acetylcholine levels (lower currents typical of ganglionic segments) and elevated acetylcholine concentrations associated with aganglionic segments characteristic of Hirschsprung\u0026rsquo;s disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreclinical evaluation results:\u003c/h3\u003e\n\u003cp\u003eIn this study, a total of 39 intestinal biopsy samples were obtained from ten patients, each analyzed using both the electrochemical biosensor and conventional histopathological methods (frozen section and paraffin-embedded H\u0026amp;E staining). Based on the histopathological results, eight patients were diagnosed with Hirschsprung's disease (HD), while two patients had total colonic aganglionosis (TCA). Electrochemical analysis was performed on residual tissue obtained from frozen section biopsies, and the generated electrical current values were recorded. Sensor current amplitude distinctly differentiated aganglionic from ganglionic segments in 7 out of 10 cases (70%), with aganglionic segments generating higher currents.\u003c/p\u003e \u003cp\u003eIn patients with HD, the mean peak current recorded by the sensor was highest in the transition zone (mean: 3.66 \u0026micro;A, range: 1.1\u0026ndash;8.78 \u0026micro;A), followed by the aganglionic zone (mean: 2.62 \u0026micro;A, range: 0.95\u0026ndash;5.77 \u0026micro;A), and lowest in the ganglionic zone (mean: 2.04 \u0026micro;A, range: 0.92\u0026ndash;4.16 \u0026micro;A). Notably, the aganglionic zone demonstrated higher current values compared to the ganglionic zone in 7 out of 8 patients (87.5%). Additionally, a distinct trend emerged in HD patients: sensor current progressively increased from the aganglionic segment, peaked in the transition zone, and subsequently decreased as it approached the ganglionic region.\u003c/p\u003e \u003cp\u003eIn contrast, biopsies were analyzed for the two patients diagnosed with TCA from both colonic and ileal segments, covering aganglionic, transition, and ganglionic zones. Interestingly, ileal samples consistently exhibited higher current values compared to colonic samples across all intestinal segments. Specifically, while colonic biopsies yielded current values of 1.28 \u0026micro;A and 1.40 \u0026micro;A, ileal biopsies produced markedly higher average currents of 5.68 \u0026micro;A, 4.77 \u0026micro;A, and 6.66 \u0026micro;A in the aganglionic, transition, and ganglionic zones, respectively. This pattern was opposite to that observed in HD patients; in TCA samples, the lowest currents were observed in the transition zone, increasing again towards the ganglionic region.\u003c/p\u003e \u003cp\u003eThe overall variation in current between ganglionic and aganglionic segments ranged from 0.92 \u0026micro;A to 8.78 \u0026micro;A. Notably, in 4 patients (40%), the difference between these zones was less than 1 \u0026micro;A, indicating minimal contrast. Additionally, a consistent and progressive increase in sensor current values was observed with increasing patient age, a phenomenon noted across all three intestinal zones evaluated.\u003c/p\u003e \u003cp\u003eThe electrochemical biosensor findings were directly compared to conventional histopathological analysis (frozen-section biopsy and H\u0026amp;E staining), which established the gold standard for diagnosing and mapping Hirschsprung\u0026rsquo;s disease. In instances where multiple biopsies were obtained from the same intestinal zone, the mean current value was calculated to standardize results (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The biosensor successfully detected higher acetylcholine-derived current levels in aganglionic segments in 7 out of 8 patients with classical HD, corresponding to a sensitivity of 87.5%. However, one HD patient (a neonate) demonstrated lower current levels in the aganglionic region, resulting in a false-negative rate of 12.5%. Both total colonic aganglionosis (TCA) cases showed atypical electrochemical patterns, highlighting potential diagnostic limitations in these distinct disease variants. Given the absence of false-positive results in identifying aganglionic segments within classical HD cases, the calculated specificity was 100%. Considering the entire patient cohort (HD and TCA, n\u0026thinsp;=\u0026thinsp;10), the electrochemical sensor showed a sensitivity of 70%, specificity of 100% overall diagnostic accuracy of 70% for distinguishing aganglionic from ganglionic bowel segments intraoperatively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient-wise Electrochemical Current Values in Ganglionic, Transition, and Aganglionic Zones of the Bowel Each row represents an individual patient and provides their age, sex, and diagnosis [Hirschsprung disease (HD) or total colonic aganglionosis (TCA)], along with the recorded electrochemical current values in the ganglionic, transitional, and aganglionic segments of the bowel.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSl.no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAge/Sex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eElectrochemical current (\u0026micro;A )\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRemarks\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGanglionic zone (\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransition zone (\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAganglionic zone (\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1m/M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.11 (0.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.20 (0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.98 (0.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHD.Only instance with a lower level in the aganglionic zone than the ganglionic zone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3m/M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.92 (0.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.10 (0.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.95 (0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHD -Rectosigmoid TZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3m/M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.93 (0.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.16 (0.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.95 (0.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHD -Rectosigmoid TZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1y/F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.91 (0.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.46 (0.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.84 (0.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHD- Long segment, splenic flexure TZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2y/M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.46 (0..9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.60 (0.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.12 (0.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHD Long segment, descending colon TZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2y/F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.12 (0.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.90 (0.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.37 (0.06)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHD -Rectosigmoid TZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3y/M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.16 (0.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.78 (0.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.77 (0.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHD-Rectosigmoid TZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 y/M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.77 (0.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.08 (0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.95 (0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHD Rectosigmoid TZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5m/F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.2 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.0 (1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.8 (0.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTCA. Ileal TZ. Colonic sample \u0026minus;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2y/F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.12 (0.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.55 (1.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.56 (1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTCA. Ileal TZ. Colonic sample 1.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSD - Standard Deviation. m-month, y-year, M-male ,F-female, HD - Hirschsprung Disease, TCA- Total colonic aganglionosis, TZ- Transition Zone\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of the electrochemical sensor were estimated based on comparison with histopathological findings. Given the sensor correctly identified 7 out of 8 confirmed aganglionic segments, the positive predictive value approached 100%, assuming no false-positive cases were recorded. However, the presence of a single false-negative case yielded a negative predictive value slightly below 100%. Further analysis with a larger sample size is required to establish these diagnostic parameters precisely.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe biosensor could successfully detect the aganglionic segment of the colon in 7 out of 8 (87.5%) patients with Hirschsprung Disease, and both the TCA patients showed atypical electrochemical patterns.\u003c/p\u003e \u003cp\u003eFollowing a diagnosis of aganglionosis by rectal biopsy, the traditional histological and histochemical leveling extends operative and anesthesia time by approximately an hour and relies on considerable diagnostic expertise. Often, surgeons experience downtime during this period as they await the pathologist\u0026rsquo;s report. Such delays present logistical challenges, especially in developing nations already burdened with heavy patient loads. In addition, there is an increased risk from prolonged general anesthesia. Leveling inaccuracies can lead to either the removal of functional ganglionic bowel or the retention of aganglionic segments, both of which are detrimental. Residual aganglionosis is a known factor contributing to morbidity post-pull-through surgery, necessitating further surgical intervention, and significantly impacting the quality of life [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In fact, a meta-analysis suggests that up to one-third of reoperations for HD are due to residual aganglionosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVarious experimental methods have been explored in pursuit of more efficient intraoperative techniques. Frykman et al. reported that spectral imaging could differentiate between ganglionic and aganglionic bowel tissues in real time with high sensitivity and specificity in a mouse model of HD [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. They demonstrated a statistical difference between the spectral curves for ganglionic and aganglionic bowel at around 610nm wavelength. Similarly, Soares de Oliveira et al. demonstrated the use of optical modalities for near real-time intraoperative detection of pathological segments in murine models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConfocal laser endomicroscopy (CLE) appears to be a promising diagnostic alternative to conventional histopathology. Harada et al. employed CLE to detect aganglionosis in resected colonic specimens from nine cases of HD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This method successfully differentiated the appearance of the enteric nervous system in ganglionic versus aganglionic segments; it correlated with the histopathological reference standards in 88% of the samples. CLE allows visualization of the enteric nervous system within the submucosal layer. However, the technique faces practical challenges - creating a submucosal space from the serosal side is technically demanding, and creating this space can adversely impact postoperative colon function. Furthermore, a significant limitation remains the absence of a human-approved neural-affinity dye that can highlight the myenteric plexus during microendoscopy. Thus, practical limitations include the difficulty of imaging the entire bowel and creating a submucosal space without affecting postoperatively colon function.\u003c/p\u003e \u003cp\u003eBesides aganglionosis, cholinergic neural hypertrophy in the aganglionic zone has traditionally served as a foundation for leveling of Hirschsprung Disease (HD) before bowel resection. There is a substantial need for a practical, swift, and dependable sensor technology that is cost-effective, compact, and user-friendly to facilitate intraoperative management of HD. Our work introduces a portable electrochemical biosensor that correlates the generated current with tissue acetylcholine (ACh) levels, potentially aiding in the intraoperative leveling of HD. Existing acetylcholine biosensors are mostly sizable benchtop units and fall short in sensitivity for intraoperative use on tissue fluids [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In contrast, our prototype sensor is a portable unit akin to a Universal Serial Bus (USB) device that is designed to interface with a notebook laptop and enable the generation of colon mapping data through specialized software (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe hallmark of HD is the absence of ganglion cells within the intestinal myenteric and submucosal plexuses. The rectosigmoid region is implicated in approximately 85% of cases, where a transition zone of several centimeters separates the distal aganglionic and proximal ganglionic regions. Notably, this aganglionic zone is characterized by a significant increase in the size of preganglionic parasympathetic cholinergic nerve fibers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which correspond to a surge in AChE enzyme concentration\u0026mdash;a phenomenon readily detected through histochemical staining [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The aganglionic zone is known to exhibit heightened levels of acetylcholine release, both at rest and under stimulation, relative to the proximal ganglionic zone [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], with the distal aganglionic rectum demonstrating the peak AChE activity, diminishing in intensity towards the normoganglionic bowel [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral studies report elevated acetylcholine concentrations in the aganglionic zones when compared to ganglionic ones [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Ikawa et al. noted concentrations of 23.79 nmol/g in aganglionic zones versus 8.51 nmol/g in ganglionic zones of tissue [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, the concentration of cholinergic vesicles is denser at the nerve endings within the aganglionic zones [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Our projections of increased current values in aganglionic zones were confirmed, although the electrical activity displayed by the transition zone\u0026mdash;peaking in patients with HD and dipping in patients with TCA\u0026mdash; remains unexplained. This phenomenon could be attributed to atypical structural and secretory characteristics of ganglion cells in these transitional areas. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Also, though both HD and TCA are aganglionic states, TCA is gradually being recognized as not merely a longer version of HD but a biologically distinct form with its own neuronal characteristics. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe detected current levels are dependent on the tissue ACh levels, which are, in turn, a consequence of the degree of nerve hypertrophy. Kapur et al. have observed that such hypertrophy tends to be focal or multifocal, with about half of the cases demonstrating diffuse hypertrophy. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] In our methods, we halved the harvested biopsy samples, performing frozen biopsies on one part and electrochemical analysis on the other. Thus it is probable that hypertrophic nerves present in one half of the sample would also be in the other, thereby reducing the likelihood of sampling errors due to focal hypertrophy.\u003c/p\u003e \u003cp\u003eThe interpretation of acetylcholinesterase (AChE) enzyme histochemistry requires a level of technical skill and experience that is not always readily available, and inconclusive results can occur in a significant portion of biopsies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. While alternative staining methods have been explored to conclusively map ganglion cells and hypertrophic nerves, recent findings by Kapur et al. indicate atypical choline transporter innervation in both aganglionic segments and transition zones [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Such irregularities in choline transport could potentially lead to an accumulation of tissue ACh, a factor our electrochemical method can quantify.\u003c/p\u003e \u003cp\u003eIn this series, an elevated current in the aganglionic segment was absent in one neonate with classic HD, aligning with observations from Garrett et al., who reported an absence of heightened AChE activity in younger patients [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, Ambartsumyan et al. have suggested that nerve caliber\u0026mdash;and consequently, acetylcholine release\u0026mdash;increases with patient age, accounting for the higher levels of acetylcholine observed in older individuals [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn total colonic aganglionosis (TCA) cases, the proliferation patterns and proximal extensions of AChE-positive nerve fibers are less understood. Some reports indicate a lack of the typical positive AChE reaction seen in classical HD, raising questions about the reliability of histochemical diagnosis in TCA [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Meier-Ruge et al. report a significant reduction in AChE-positive nerve fibers in TCA patients as one moves orally from the rectum towards the splenic flexure [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], a finding that correlates with the minimal nerve hypertrophy and, thus, reduced acetylcholine levels in the colon. Our findings corroborate this, with TCA patients demonstrating low acetylcholine levels in colonic tissues, distinct from those observed in classical HD.\u003c/p\u003e \u003cp\u003eCompared to previously reported experimental and clinical methods aimed at identifying aganglionic bowel segments in Hirschsprung\u0026rsquo;s disease (HD), the electrochemical biosensor described in our study offers distinct advantages in terms of simplicity, portability, and cost-effectiveness. While existing methodologies such as spectral imaging, confocal laser endomicroscopy, and conventional histopathological analysis require sophisticated equipment, specialized infrastructure, and expert interpretation, our proposed electrochemical biosensor provides a portable, user-friendly platform capable of real-time intraoperative analysis. This approach significantly reduces reliance on specialized pathology support, minimizes operative downtime, and potentially shortens anesthesia duration, thus offering substantial logistical and clinical benefits, especially in resource-constrained healthcare settings.\u003c/p\u003e \u003cp\u003ePotential limitations of this method include variability introduced during sample preparation and analysis. Specifically, errors may arise due to the uneven distribution of hypertrophic nerve fibers within biopsy samples, potentially affecting acetylcholine levels and resultant electrochemical currents. Additionally, variability in tissue homogenization, efficiency of acetylcholine extraction, and sample processing may also influence the accuracy and reproducibility of sensor measurements. To mitigate these issues, careful standardization of biopsy collection techniques, rigorous homogenization protocols, and optimization of acetylcholine extraction procedures are recommended for future studies.\u003c/p\u003e \u003cp\u003eIn addition to the above complexities and technical limitations, we note that the current generated varied less than 1 \u0026micro;A between normal and pathological bowel in 40% of the patients. The sensitivity of the electrode, calibrated using a stock solution of ACh, was 0.42\u0026micro;A/\u0026micro;M/mm\u003csup\u003e2\u003c/sup\u003e, which translates to a minimal difference in acetylcholine concentrations between the aganglionic and ganglionic zones\u0026mdash;less than 2.38 \u0026micro;M. To enhance the sensitivity and reliability of our measurements, we plan to expand the electrode's reactive surface area and refine our sample processing technique, such as extracting ACh from the tissue prior to electrochemical analysis. We acknowledge the potential for other artifacts within the homogenized samples that may generate free electrons and impact the potentiostat's readings. Further optimization to enhance sensor sensitivity and specificity, possibly through advanced nanomaterials or refined sample preparation techniques, will be necessary to increase diagnostic accuracy, especially in samples showing minimal contrast.\u003c/p\u003e \u003cp\u003eOur prototype sensor continues to undergo meticulous evaluation in terms of sample processing and clinicohistopathological correlation. We intend to further refine our electrodes and methods based on current data and engage in a multicenter pivotal study with a larger sample size to validate our findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe electrochemical sensor developed for detecting ACh in homogenized tissue samples has been validated using stock solutions and spiked serum. In a preclinical test, the sensor demonstrated a higher level of ACh in the aganglionic zone compared to the normal ganglionic zone in 87.5% of patients with classical HD. The promising preliminary outcome requires further testing on a broader pediatric cohort to establish the clinical utility of the sensor. The quantitative nature of the assessment reduces the risk of observer bias and could be a revolutionary device for intraoperative mapping/leveling of the ganglionic -aganglionic zones in the management of Hirschsprung Disease and allied disorders. This study demonstrates the feasibility of an electrochemical biosensor for objectively and rapidly differentiating ganglionic from aganglionic segments intraoperatively in patients with Hirschsprung's disease. While initial results are promising, broader multicenter validation studies are essential to establish its clinical reliability and utility before adoption into routine practice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement:\u003c/h2\u003e \u003cp\u003e \u003cem\u003eThis research was supported by funding from the Biotechnology Industry Research Assistance Council (BIRAC), Department of Biotechnology, Government of India, under the Biotechnology Ignition Grant (BIG) scheme. The authors gratefully acknowledge BIRAC for the financial support provided to carry out this work.\u003c/em\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDas K, Kini U, Babu MK (2010) The distal level of normally innervated bowel in long segment colonic Hirschsprung\u0026rsquo;s disease. 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PMID: 31791203\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"33b6afec-532e-4605-9abe-889118445508","identifier":"10.13039/501100014825","name":"Biotechnology Industry Research Assistance Council","awardNumber":"BIRAC/KIIT01161/BIG-17/20","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"All India Institute of Medical Sciences Bhubaneswar","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hirschsprung Disease, Electrochemical Sensor, Acetylcholine, Ganglion cells, Biosensor","lastPublishedDoi":"10.21203/rs.3.rs-6288646/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6288646/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePurpose: Surgical procedures for Hirschsprung Disease (HD) require accurate identification of enteric ganglion cells and cholinergic hypertrophic nerve bundles. Current intraoperative mapping through frozen section histopathology-histochemistry is time-consuming and demands skilled interpretation. This study explores an electrochemical sensor for objective, rapid intraoperative mapping of aganglionic bowel segments via tissue acetylcholine (ACh) detection.\u003c/p\u003e\n\u003cp\u003eMethods: An electrochemical biosensor was developed using anodized laser-induced graphene electrodes functionalized with acetylcholinesterase enzyme (AChE). Electrochemical analyses were conducted on homogenized intestinal biopsies obtained intraoperatively from ten patients, including eight diagnosed with Hirschsprung Disease (HD) and two with total colonic aganglionosis (TCA). Biopsy samples representing ganglionic, transitional, and aganglionic bowel segments were evaluated. The sensor quantified tissue acetylcholine (ACh) levels, which serve as markers of cholinergic neuronal hypertrophy, by measuring the generated electrical current.\u003c/p\u003e\n\u003cp\u003eResults: The electrochemical analysis demonstrated significantly higher current levels in the aganglionic segments compared to ganglionic segments in 87.5% (7 out of 8) of classical Hirschsprung disease (HD) patients. The mean peak currents observed in HD cases were 2.62 µA in aganglionic segments, 3.66 µA in transition segments, and 2.04 µA in ganglionic segments. In contrast, electrochemical patterns in patients diagnosed with total colonic aganglionosis (TCA) were atypical; ileal tissue samples from these patients generally yielded higher current measurements than colonic samples across all zones examined. Additionally, there was a progressive increase in tissue current values correlating positively with patient age.\u003c/p\u003e\n\u003cp\u003eConclusion: The electrochemical sensor effectively differentiated aganglionic from ganglionic zones in HD, suggesting potential as a quick, objective tool for intraoperative bowel leveling. Further validation in larger cohorts is necessary to confirm clinical utility.\u003c/p\u003e","manuscriptTitle":"Rapid, Objective Intraoperative Mapping of Hirschsprung Disease Using a Portable Electrochemical Acetylcholine Sensor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 09:23:36","doi":"10.21203/rs.3.rs-6288646/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f5517ec-7eab-44ab-b523-56e0e4597d4c","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46082810,"name":"Biotechnology and Bioengineering"},{"id":46082811,"name":"Nanoscience"}],"tags":[],"updatedAt":"2025-03-26T09:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-26 09:23:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6288646","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6288646","identity":"rs-6288646","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}