Picomolar ‐ Level Melamine Detection via ATP Regulated CeO 2 Nanorods Tunable Peroxidase ‐ Like Nanozyme ‐ Activity ‐ Based Colorimetric Sensor: Logic Gate Implementation and Real Sample Analysis

: The capability of functional logic operations is highly intriguing, but far from being realized owing to limited recognition element (RE) and complex readout signals, which limit their applications. In this contribution, for a visual colorimetric sensor for melamine (MEL) we described the construction of two ‐ and three ‐ input AND logic gate by exploiting the intrinsic peroxidase (POD) ‐ like activity of CeO 2 nanorods (NRs) (~23.04% Ce 3+ fraction and aspect ratio (R TEM ) of 3.85 ± 0.18) as RE at acidic pH (4.5). Further ATP piloted catalytic tuning of POD ‐ like activity in CeO 2 NRs employed for a functional logic gate ‐ controlled MEL sensing at neutral pH (7.4). AND logic circuit operated MEL sensing record colorimetric response time of 15 minutes to produce blue color proportionate to MEL concentration. The fabricated nanozyme (CeO 2 ) ‐ based logic gate sensor probe for MEL at pH 4.5 showed a linear response from 0.004 nM to 1.56 nM with a limit of detection (LOD) of 4 pM; while translation from acidic to neutral pH (at 7.4) sensor exhibited linear response ranging from 0.2 nM to 3.12 nM with a LOD value of 17 pM. Through CeO 2 POD ‐ like nanozyme behavior under acidic and neutral pH, the fabricated logic gate sensor showed high affinity for MEL, generating prominent visual output with picomolar sensitivity, good reproducibility, and stability with relative standard deviation (RSD) <1% and 2%, respectively. A feasibility study in real samples (raw milk and milk powder) showed good recoveries with negligible matrix effect, an anti ‐ interference experiment revealed sensor selectivity, highlighting robust sensor practical utility. With the merits of high sensitivity, specificity, low cost, and simplified sample processing, the developed logic ‐ controlled colorimetric MEL sensing platform with appropriate modifications can be recognized as a potent methodology for on ‐ site analysis of various food adulterants and related applications.


Introduction
The synergy within the mounting field of nanotechnology and material science is revealing innovative feats toward the design and fabrication of nanomaterials exhibiting enzyme-like activity (known as nanozymes) [1]. The accidental discovery of magnetic Fe3O4 nanoparticles (NPs) possessing peroxidase (POD)-like activity reported by Gao et al. [2], thereafter opened doors for a variety of nanomaterials, including noble metals [3], metal oxides [2,4] and carbon-based nanomaterials [5], which have been found mimicking natural horseradish peroxidase (HRP) enzyme for catalysis. By proficiently imitating the catalytic sites of natural enzymes or harboring multi-valent elements for catalytic reactions nanozymes have effectively served as "direct surrogates of traditional enzymes," offering the unflinching biocatalytic potential for imaging, sensing, and theranostics applications [1].
In the recent literature enzymatic reactions have been widely explored in the food sector and play an indispensable role to combat the increasing food safety issues [6]. Nevertheless, enzymes demonstrate high substrate specificity and catalytic activity but present certain drawbacks, such as low operation stability, vulnerability under harsh environmental conditions, complicated recovery and recycling procedures, high cost of synthesis and purification, which greatly curtail their practical applications [7]. As a result of such obstacles causing the ineffectiveness of practical application, "nanozymes" have emerged as a promising candidate for artificial enzymes, owing to small size, high surfaceto-volume ratio, increased reactivity, enhanced stabilities, and tunable catalytic activities that have garnered ever-growing research attention. Among a large community of artificial nanozymes, nanostructured CeO2 offers a potential foundation for practical implementation in the agri-food industry, predominantly as an optical sensor for antioxidants [8], mycotoxins [9], organophosphate pesticides [10], and food adulterants [11,12]. To date, there have been very limited reviews that have discussed nanozymes' primarily POD-like activity-based catalytic potential concentrating on food safety and quality parameters [13,14].
Food "safety and quality" monitoring is of major public health importance because the growing incidences of food poisoning pose a cumulative threat worldwide, such as the outbreaks of melamine (MEL) contamination of milk and dairy products in China (2008) [15]. MEL a triazine heterocyclic organic compound, is being utilized in the manufacture of fertilizer, flame retardant paint, plywood, plastics, adhesives, and cement [16]. With high nitrogen content (66% by mass) MEL has been illegally added to infant formula, human and animal foods to elevate the apparent protein content, which directly or indirectly affects the food quality consumed worldwide. This fraudulent practice of MEL adulteration of different milk products attracted the attention of many organizations and researchers globally. Incidents of illness and the death of hundreds of pets were reported due to MEL-contaminated pet food [17]. The high concentration of MEL in Chinese infant formula causes the illness of >51,900 infants and young children, who were hospitalized due to urinary problems, resulting in six deaths [18]. Although MEL is a lowtoxicity material, co-exposure of MEL with cyanuric acid forms an insoluble MELcyanurate complex, resulting in kidney stones that may cause renal failure, bladder carcinoma, and even death [19]. This calls for an innovative, cost-effective, and reformative analytical methodology able to deliver real-time measurement of crude foodstuffs at the source with high sensitivity, specificity, and reproducible output.
Prevailing conventional methods, including gas chromatography/mass spectrometry (GC/MS) [18], liquid chromatography−mass spectrometry (LC-MS) [20], enzyme-linked immunosorbent assay (ELISA) [21], and surface-enhanced Raman scattering (SERS) [22], are sensitive and selective but make use of cumbersome laboratory equipment with a high operating cost and need skilled operators and time-consuming pre-concentration procedures, reflecting their inaptness, thus creating a developmental bottleneck toward on-site applicability. Alternatively, colorimetric sensing offers low cost, easy fabrication, portability, and concentration-dependent real-time naked eye recognition, which makes it attractive for on-site analysis, Table S1.
Advancing nanostructured-based sensing has routed interest toward real-time pointof-care (POC) sensing devices, while efforts in emulating the Boolean functions on the molecular scale [23], which are capable of performing logic gate operations to produce an output signal in the form of optical, electrical responses, have opened the possibility of design and realization of "stimuli-responsive nanoscale sensing devices" [24]. A variety of metal, metal oxide NPs, and metal ions [12,[25][26][27] have already been reported as colorimetric sensors for MEL. Though some hold high accuracy and sensitivity, it is vital to develop a simplified operational and highly sensitive platform to meet the established safety limits [28,29]. To address the limitation or challenges (Table S1), our method needs no complicated operations or probe modification, making it a simple, feasible visualbased detection for MEL,. Herein we report on the realization of a two-and three-input AND logic gate colorimetric sensor for MEL based on the catalytic tunability offered by CeO2 nanorods (NRs), which remarkably executed POD-like nanozyme activity at pH 4.5 when used as a recognition element (RE). Further, we take advantage of ATP as a catalytic modulator of CeO2 NRs to regulate the POD-like colorimetric reaction at pH 7.4, as detailed in our previous publication [30], which was employed in the fabrication of catalytic logic gate-based colorimetric sensing of MEL at pH 7.4. The successful application of the developed sensor for detection of MEL in real samples (such as raw milk and infant formula) evidences a meaningful achievement, paving the way for innovation in a nanozyme-based logic-gated sensing platform for improving food quality and safety assessment.

Preparation of CeO2 Nanozyme
Following the protocol reported earlier [30], in a bottom-up approach CeO2 NRs were prepared by ammonia-induced co-precipitation method. Having demonstrated previously the POD-like nanozyme activity of CeO2 NRs under acidic and neutral pH [30], they were used in the present study as a RE in the fabrication of a logic gate-based colorimetric sensor for MEL at pH 4.5 and 7.4.

Instrumentation and Characterization
A high-resolution transmission electron microscopy (TEM) image of the synthesized CeO2 nanozyme was obtained using Thermo Scientific high-resolution Cryo-TEM (Talos).
The captured TEM image was analyzed using ImageJ for morphological details [31]. Rietveld refinement study of X-ray diffraction for CeO2 nanozyme was performed using Material Analysis Using Diffraction (MAUD) software (version 2.94) and crystallographic structure is drawn via Visualization for Electronic and Structural Analysis (VESTA) software [32,33]. UV-Visible absorption spectra of CeO2 NRs POD-like activity were viewed and colorimetric detection (at pH 4.5 and 7.4) of MEL was performed in a quartz cuvette (1 cm path length) using a U3900 spectrophotometer (Hitachi). Colorimetric detection of MEL was visually captured from a 12-megapixel camera.

Preparation of Buffer
As a prerequisite for reliable determination, unless otherwise specified, all the experiments in acidic conditions (pH 4.5) were conducted in 0.1 M citrate buffer and 1X PBS at physiological conditions (pH 7.4).

POD-like Activity and Kinetic Measurement of CeO2 NRs
Following our previous work demonstrating CeO2 NRs having low Ce 3+ concentration (~23.04%) and possessing POD-like activity at pH 4.5 and 7.4 [30], we examined the effect of MEL on the kinetics of CeO2 nanozyme catalyzed H2O2-mediated TMB oxidation reactions. Subsequently, kinetic parameters were calculated at a fixed concentration of TMB (0.3 mM) with varying concentrations of H2O2 (1, 1.5, 2, 2.5, 3, 3.5, 4 and 5 mM) for CeO2 NRs + MEL and CeO2 NRs + ATP + MEL at pH 4.5 and 7.4, respectively.

Construction of CeO2 NRs POD-like Nanozyme-Based Logic Operation
We attempted to construct an AND logic gate-configured cascade reaction with two and three input elements under acidic and neutral pH, respectively, thus realizing colorimetric detection of MEL. The absorption intensity of oxidized TMB was monitored at 652 nm and was defined as the output signal with a distinct color change and plotted against the input combinations. The output signal to logic value '1' was optimized experimentally to give an output signal greater than the threshold value. The inclusion conditions for input/output signals are defined in the electronic supplementary sheet (ESS) 1.1.
AND logic operation for sensing MEL at pH 4.5 with two inputs including CeO2 NRs (IN1) and MEL (IN2) was applied to the gate machinery in four possible arrangements (0,0; 0,1; 1,0; 1,1). Citrate buffer was initially added to four vials, then a combination of inputs was executed: To further distinguish the false-positive signals described in ESS 1.3 with the stimulus of different inputs (interfering species), we tested the specificity and selectivity for MEL by the fabricated CeO2 POD-like nanozyme-based two and three AND input logic gate colorimetric sensors, at acidic and neutral pH, respectively.

Experimental Procedure for Visual Colorimetric Detection of MEL
Based on the observed robust POD-like nanozyme activity of CeO2 NRs at room temperature [30], we implemented the AND logic gate operations for the visual colorimetric detection of MEL in acidic (pH 4.5) and neutral (pH 7.4) conditions. Typically

Feasibility Study in Real Samples
To validate the reliability of the present method, real sample analysis was inspected in MEL-challenged raw milk and infant milk powder samples, the detailed protocol is given in the supplementary section (ESS 1.4).

Characterization of CeO2 Nanozyme
We utilized cerium chloride as precursor salt by employing the precipitant into precursor salt (PIS) approach to the CeO2 nanozyme prepared at high pH ⁓10.5 (general procedure schematically illustrated in Figure 1a). As can be seen in Figure 1b the highlighted section of the high-resolution TEM image shows the appearance of spherical particles along the length of the rod, indicating that the growth of CeO2 nanorods possibly happened during the Ostwald ripening process by consuming small spherical particles as "building blocks" [34]. Captured TEM image analyzed for ⁓50 NRs using ImageJ revealed an aspect ratio (R = length to diameter ratio) of 3.85 ± 0.18, as shown in Figure 1b as inset. Rietveld refined structural fit using MAUD software is shown in Figure 1c, affirming the fluorite cubic structure of CeO2 of space group Fm3m. Table S2 presents the structural refinement results. We achieved a good agreement between the experimental and calculated patterns, indicated by the red and black lines in Figure 1c, respectively. Reliability parameters (χ 2 and Rwp %) given in Table S2 show low values, indicating good quality of structural refinement, and agree with Lutterotti criteria [32]. The electronic structural analysis program VESTA was used for the crystal structure representation of CeO2 in Figure 1c as inset. The prepared CeO2 nanorods possessed both the POD-like nanozyme activity reported earlier at acidic and neutral pH [30] and nanoscale properties employed as the RE in the AND logic gate colorimetric sensing of MEL at pH 4.5 and 7.4.

AND Logic Operation for MEL Sensing
Here, the AND logic design exploited CeO2 NRs for its POD mimic activity as the RE on a logic platform of TMB/H2O2 that switched to ON/OFF state upon application of different logic combinations. Figure S1a,b explains the logic operation principle applied for sensing MEL at two different pH (4.5 and 7.4). Reaction cascade operating at pH 4.5 involved the processing of two input signals (IN1:CeO2 NRs; IN2: MEL) to produce a color change from colorless to blue as an output signal. As expected, the UV/visible absorption spectrum in Figure 2a shows the absence of both or either of the inputs (0, 0; 0, 1; 1, 0), reporting no or low absorption response with no significant color, defining an OFF state (output "0"). In the presence of both the input (1,1) results in a high absorption at 652 nm and the reaction solution turning blue, the logic gate switched the output to ON state. Correspondingly, the absorbance intensity (at 652 nm) as output signal plotted against input combinations represented as column bars with logic circuit, given as the inset of Figure 2b. Figure S2a shows the truth table and its corresponding color reactions at pH 4.5.

Analytical Performance of AND Logic Gate-Based Colorimetric Detection of MEL
The  Figure 5b, with the regression equation: ΔA652 = 0.0135 + 0.0712. We investigated the limit of detection (LOD) and limit of quantification (LOQ) values on 20 blank samples at pH 4.5 and 7.4, using the equations LOD = 3 × SD/S and LOQ = 10 × SD/S (where SD: standard deviation of the blank solution, and S: the slope of the calibration plot), detection limit at pH 4.5 and 7.4, estimated to be 4 pM and 17 pM, correspondingly, while LOQ was 12 pM and 51 pM, respectively. The sensitivity of the colorimetric method was far below the prescribed safety limits [25] Analytical performance at both pH 4.5 and 7.4 was comparable and even better than that acquired with a different colorimetric probe, Table S3. Furthermore, the relative standard deviation (RSD) representing precision (for n = 3; where n = number of samples  1,1,0)   (1,1,1)  (1,1,0)  (1,1,1) Absorbance (a.u.)

Wavelength (nm)
(0,0,0) (0,0,1) (1,0,0)  (1,0,1)  (0,1,1)  (0,1,0 at all the concentrations was found to be less than 1% and 0.2%, respectively, implies method reproducibility. Following the logic functions (explained in ESS 1.3) to assess the selectivity of the method and rule out false-positive signals, control experiments were performed in an equimolar ratio of MEL to interfering species, including ascorbic acid (AA), glucose, sucrose, dextrose, and histidine. Results of the selectivity experiment carried out at pH 4.5 and 7.4 indicated that only MEL leads to color reaction (colorless to blue) with a significant increase in the absorbance, Figures 4d and 5d, respectively. Among the various interfering species, it is worth noting that histidine contributed to a very low signal, the presence of AA did not interfere owing to its antioxidant property; thus, this implies an acceptable selectivity and sensitivity for MEL in both acidic and neutral conditions.

Implication of CeO2 NRs POD-Like Activity in MEL Sensing
In the absence (pH 4.5) and presence of ATP (pH 7.4), the reaction of H2O2 and TMB using CeO2 NRs as RE was used to develop a colorimetric detection of MEL (Figures 4 and  5). Reminiscent of what was earlier reported by Jin et al. [12], the addition of MEL probably lowered the colloidal stability and its crosslinking ability resulted in the aggregation of CeO2 NRs, which may initiate decomposition of H2O2-triggered oxidative species generation. ROS trapping experiment ( Figure S3a(v)) reveals the density of •OH species sources TMB oxidation as a function of MEL concentration, as evident from the observable color change, which is also evident in the image shown in Figure 4c. Figure 6 (left) represents the reaction mechanism for the colorimetric sensing of MEL at pH 4.5. In line with the quantification of MEL at pH 4.5, single-step colorimetric detection of MEL at pH 7.4 was attributed to ATP-induced augmented affinity of H2O2 toward CeO2 NRs, Figure S3b. Figure 6 (right) shows the schematic illustration of MEL-induced aggregation of CeO2 NRs and the responsive generation of •OH radical at neutral pH. In addition, Figure S3a,b supports the fact that MEL-induced aggregation of CeO2 NRs was responsible for •OH radical directed oxidation of TMB, thus showing an augmented POD-like activity while the concentration of oxidative species varied at both pH levels. Kinetic observations at both pH levels in the presence of MEL revealed a decline in the Michaelis-Menten constant (Km value) for H2O2, suggesting that the high affinity of CeO2 NRs for H2O2 well corroborates the proposed mechanism ( Figure S4a,b; Table S4).

Real Sample Analysis and Validation of the Method
Briefly, the feasibility study for the use of the logic-operated colorimetric sensor in the quantitative analysis of MEL was carried out on real samples at pH 4.5 and 7.4. Different concentrations of MEL (1.5 nM, 12.5 nM, 50 nM, and 100 nM) were spiked into raw milk and milk powder. The results summarized in Table 1 show the recoveries for MEL in raw milk ranging from 90% to 105%, and 97% to 103% in milk powder at pH 4.5; while those obtained at pH 7.4 were 97% to 103% in raw milk and 100% to 101% in milk powder, reflecting the reliability of the method.
Furthermore, to reveal the influence of the matrix, we compared the signal recorded in the buffer solution and real samples (raw milk and milk powder) at pH 4.5 and 7.4 for the same concentrations of MEL used in recovery studies. As depicted in Figure 5e the percentage of signal suppressed as a function of MEL concentration suggested excellent sensitivity with no harsh matrix influence; the sensor responded to -1.35% to 2.6% signal change, which is within normal sensor-to-sensor variation. However, at both pH level the slight signal gain observed in raw milk was within the error limit of what was seen in the buffer (citrate buffer and PBS) when challenged at 1.5 nM of MEL, Figure S5.

Conclusions
In an attempt to address increasing food safety and quality issues, we proposed an economic visually aided reproducible detection scheme for sensing MEL Overpowering the pH constraint of CeO2 NRs POD-like nanozyme activity as a model reaction, we successfully integrated two and a three-input regulated AND logic circuit for MEL recognition under acidic (4.5) and neutral (7.4) pH, respectively. The sensor was highly specific and outputted by a readable color change generated through the oxidation of TMB in proportion to MEL concentration. The fabricated sensor showed picomolar LOD values, significant recoveries in real samples with negligible matrix effect, and antiinterference studies depicted high selectivity and sensitivity for MEL at both pH levels, outlining the potential practical utility of this robust sensing platform. Outcomes of this study are expected not only to broaden the scope of nanozyme tunable catalysis and Boolean logic operations for sensing analytes relevant to food safety but also to provide a wealth of opportunities for fabricating intelligent biomedical diagnostics deliverable to meet the ASSURED criteria set by the World Health Organization (WHO) [35]. We hope our findings may push forward the mining of novel "next-generation artificial enzymesbased logic gates" in the maturation of practically oriented analytical methods.  Table S1 POD-like Nanozyme activity for MEL Sensing: limitations or challenges and present attainments, Table S2 Reitveld refined structural parameters for CeO2 nanozyme, Table S3 Analytical performance  comparison table between the present and previous reports for MEL sensing, Table S4 Kinetic parameters comparison of CeO2 NRs and horseradish peroxidase (HRP) toward H2O2. Data Availability Statement: The data supporting the findings of the study are accessible from the corresponding author upon reasonable request.