Category: 3. Business

Introduction

Acetylcholinesterase (AChE), an essential enzyme in the cholinergic nervous system, is responsible for breaking down the neurotransmitter acetylcholine (ACh) into choline and acetate, thereby modulating the levels of ACh at synapses in a dynamic manner.^1,2 Dysregulation of AChE activity—whether excessive or deficient—is closely associated with severe neurological disorders. Notably, enhanced AChE activity leads to the depletion of ACh, a hallmark of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease.³ Conversely, elevated ACh accumulation due to suppressed AChE activity can disrupt neurotransmission, potentially resulting in fatal outcomes.⁴ Given its central role in neuroregulation, AChE has emerged as a key biomarker for neurodegenerative conditions,⁵ particularly AD, where its activity is significantly elevated in patients. Consequently, AChE inhibitors have garnered substantial attention as potential therapeutic agents for AD treatment. The development of highly sensitive and efficient biosensors to detect AChE activity and evaluate inhibitor efficacy is thus imperative—not only for early diagnosis of neurodegenerative diseases but also for accelerating drug discovery and personalized therapeutic strategies.

Various sensing strategies have been developed for detecting AChE activity or screening its inhibitors, including colorimetric assays,⁶ fluorescent assays,⁷ chromatography-mass spectrometry,⁸ electrochemical sensors,⁸ and others. Among these, colorimetric analysis has gained significant attention in AChE activity assays due to its convenience, low cost, fast readout, ease of visual detection, and strong potential for point-of-care or ready-to-use applications.^9,10 Nowadays, colorimetric detections of acetylcholinesterase and its drug inhibitors have been developed using molecularly imprinted polymers,¹¹ nanomaterials,¹² antibodies,¹³ and natural enzymes¹⁴ as recognition units. Nanoenzymes are a series of biological nanomaterials with enzyme-like catalytic capabilities, which have many advantages such as high catalytic activity, low cost, and ease of large-scale preparation.^15,16 In addition, nanoenzymes also overcome some limitations of natural enzymes, such as low tolerance to pH, temperature, and organic solvents, impaired activity during long-term use, and difficult purification.¹⁷ For example, Lin’s research group prepared manganese dioxide (MnO₂) nanosheets as an oxidase-mimicking nanomaterial, which could directly oxidize TMB into oxTMB without the need for horseradish peroxidase (HRP) and H₂O₂,¹⁸ supporting the colorimetric detection of acetylcholinesterase activity and its inhibitor. Other biomimetic enzyme-like nanomaterials, such as 2D Zn-TCPP(Fe) nanosheets,¹ α-FeOOH nanorods,¹⁹ and FeMn DSAs/N-CNTs nanozymes,²⁰ are also employed to develop colorimetric biosensors for the detection of AChE and its inhibitor. Although great progress in nanozyme-based colorimetric sensors has been made, it is necessary to develop a real-time, on-site, and portable approach for AChE detection and inhibitor screening. Amazingly, the development of microneedle-mediated POCT strategies hinges on the synergistic integration of biosensing and biofluid sampling. On one hand, the emergence of highly sensitive microneedle-mediated biosensors allows for the direct transduction of biochemical signals in the dermal layers, paving the way for novel closed-loop diagnostic systems.²¹ For instance, Ruan et al constructed a dual-continuous microneedle patch integrating transdermal delivery of pH-sensitive licorzinc MOFs and Zn²⁺ hydrogel sensors for managing alopecia areata.²² Meanwhile, these microneedle-based biosensors are powerfully complemented by substantial progress in using microneedles to efficiently extract interstitial fluid,^23–25 which establishes a reliable, minimally invasive method to obtain a rich source of biomarkers, making subsequent laboratory-grade analysis possible at the point-of-care. Therefore, the integration of microneedle-mediated POCT strategies into nanozyme-based colorimetric sensors probably offer a potential approach for on-site monitoring of AChE in interstitial fluid, heralding a new era in point-of-care diagnostics of neurodegenerative diseases. A key advantage of this POCT platform is its ability to provide rapid, real-time feedback on AChE levels through minimally invasive sample analysis, enabling immediate clinical or therapeutic decision-making at the point of sampling.²⁶ This capability empowers healthcare providers to optimize treatment protocols and monitor disease progression dynamically. Moreover, the developing smartphone-assisted POCT colorimetric system streamlines traditional AChE detection workflows, effectively shifting the diagnostic paradigm from centralized laboratories to decentralized settings such as clinics or even home care conditions. Importantly, this microneedle-based user-friendly smart POCT colorimetric sensor not only enhances patient engagement in biomarker monitoring but also supports data-driven choices for personalized healthcare interventions.^27,28

To achieve this aim, we ingeniously designed and constructed a portable Colorisensor coupling with microneedle and metal-phenol nanozyme for smartphone-assisted point-of-care testings of acetylcholinesterase activity and its drug inhibitor (Scheme 1). When pressing the microneedle array for contacting with the skin layer, the analytes in ISF could respond to Fe-PD nanozymes. In the presence of AChE, it catalytically hydrolyzes ATCh into TCh. With its high reducing ability, TCh induces the decomposition of Fe-PD nanozymes, causing the inhibition of their POD-like activity. Hence, with the increasing concentration of AChE, there is an apparently fading change in the color of the oxTMB solution oxidized by H₂O₂ under the catalysis of Fe-PD nanozymes. Furthermore, to make the detection more smart, convenient, and minimally invasive, we have combined colorimetric methods with microneedle technology and an RGB identification strategy to create a colorimetric microneedle-mediated biosensing array for intelligent detection of AChE activity and its inhibitors. The developed Colorisensor exhibits outstanding sensitivity, selectivity, repeatability, and long-term stability, which hold promising prospects in the early diagnosis and screening of therapeutic drugs for neurodegenerative diseases.

Materials and Methods

Reagents and Materials

Dopamine hydrochloride (DA·HCl), iron(III) chloride hexahydrate (FeCl₃·6H₂O), hydrogen peroxide (H₂O₂), 3,3′,5,5′-tetramethylbenzidine (TMB), acetylcholinesterase (AChE), acetylthiocholine iodide (ATCh), berberine hydrochloride, potassium chloride (KCl), glucose (Glu), L-cysteine (L-Cys), glutathione (GSH), glucoseoxidase (GOx), lysozyme (Lyz), tyrosinase (Tyr), N-vinylpyrrolidone (NVP), ethoxylated trimethylolpropane triacrylate (ETPTA), 2-hydroxy-2-methylpropiophenone (HMPP), ethylene glycol dimethacrylate (EGDMA), 1×PBS buffer (pH=7.4), acetic acid (HAc), and sodium acetate (NaAc) were purchased from NanJing WanQing Chemical Glassware Instrument Co., Ltd. All chemicals were of analytical grade and used without further purification. All porcine ear skin samples were collected from healthy male domestic pigs, aged 8 months and weighing ~100 kg, obtained from a local market. The sacrifice of the pigs were not involved in this study. These specific sources and application basis of porcine skins were stated in Source Declaration of Porcine Ear Skin.

Preparation and Characterization of Fe-PD Nanozymes

To synthesize Fe-PD nanozymes, NH₃·H₂O was initially added to a conical flask containing a mixture of ethanol and deionized water (2/8, v/v), followed by stirring for 1 hour until the solution became homogeneous at a pH of 10. Subsequently, solutions of DA (15 mL, 50 mg/mL) and FeCl₃·6H₂O (5 mL, 50 mg/mL) were sequentially introduced into the aforementioned flask. The reaction solution was maintained at room temperature and stirred for 7 hours. Thereafter, the resulting Fe-PD product was subjected to centrifugation (13500 rpm, 30 min) and washed three times with deionized water and ethanol, respectively. Ultimately, the Fe-PD nanozymes were dried at 45 °C.

The morphology and chemical composition of the synthesized Fe-PD nanozymes were thoroughly characterized using various analytical techniques. Scanning electron microscopy (SEM, Hitachi S4800) was employed to investigate the nanozymes’ structural features at an accelerating voltage of 5.0 kV, a beam current of 10 μA, and a working distance of 13.5 mm, with micrographs captured at a magnification of 30.0k. The detailed morphology and elemental composition of the nanozymes were analyzed using high-resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray spectroscopy (EDX) on an FEI Talos 200x instrument. The analysis was conducted in STEM mode with a high tension of 200 kV. Key parameters for the EDX spectrum imaging included a beam convergence of 10.5 rmad, a camera length of 98 mm, a spot size of 5, and a dwell time of 10.0 μm. The spectrum image was acquired at a magnification of 261kx with an image size of 1024×1024 pixels over 24 frames. The UV-Vis absorption spectrum of the Fe-PD nanozyme-based colorimetric system was recorded using an ultraviolet-visible spectrophotometer (UV-Vis, Shanghai MAPADA). The UV-Vis absorption spectra within the range of 550–750 nm were recorded at 25 °C. Quantitative analysis was performed based on the maximum absorption peak observed at 652 nm. Additionally, the zeta potential was measured with a laser particle size analyzer (Brookhaven, ZetaPALS) to evaluate the surface charge of the nanozymes.

Enzyme-Like Activity and Steady-State Kinetics of Fe-PD Nanozymes

The Enzyme-like activity of Fe-PD nanozymes was evaluated by the system, including Fe-PD (50 µL, 50 µg/mL), TMB (100 µL,2 mM), and H₂O₂ (100 µL, 10 mM) in the NaAC-HAC buffer (0.1 M, pH 4.0). After incubation for 6 minutes at 37 °C, UV-Vis absorption spectra were recorded at 652 nm, and corresponding photographs were taken to visually assess the reaction process. Steady-state kinetic analysis of Fe-PD nanozymes was conducted by varying the substrate concentrations of TMB (0.05 mM to 8.0 mM) and H₂O₂ (0.125 mM to 32.0 mM), while keeping the concentration of Fe-PD nanozymes constant. Absorbance spectra of the solutions were recorded at 652 nm. The Michaelis-Menten constant (K_m) and the maximum reaction velocity (V_max) were then calculated using the Michaelis-Menten equation, based on the relationship between substrate concentrations and reaction velocity.^29,30

Colorimetric Detection of AChE Activity and Its Inhibitor

According to previous studies,^31,32 the enzymatic activity of AChE and its inhibition by berberine hydrochloride were evaluated using a colorimetric assay of an aqueous TMB + H₂O₂ system. The assay was based on the oxidation of TMB catalyzed by Fe-PD nanozymes in the presence of H₂O₂, with the enzymatic hydrolysis product thiocholine (TCh) serving as an inhibitor of the oxidation reaction. Then, Fe-PD suspension (0.05 mg/mL in acetate buffer, pH 4.0), TMB solution (2 mM in ethanol), H₂O₂ solution (10 mM in deionized water), AChE solution (0.1–1000 mU/mL in PBS, pH 7.4), ATCh solution (30 mM in PBS), NaAc-HAc buffer (0.1 M, pH 4.0) were prepared. To validate the sensing mechanism, five control experiments were conducted: Group a (Control): NaAc-HAc buffer only. Group b (Fe-PD + H₂O₂ + TMB): To confirm TMB oxidation by Fe-PD/H₂O₂. Group c (Fe-PD + H₂O₂ + TMB + AChE): To assess AChE’s direct effect. Group d (Fe-PD + H₂O₂ + TMB + ATCh): To examine ATCh interference. Group e (Fe-PD + H₂O₂ + TMB + AChE + ATCh): To verify TCh-mediated inhibition of TMB oxidation. Then, different concentrations of AChE (0.01–1000.0 mU/mL) were incubated with ATCh (5 mM, 20 min, 37°C) to generate TCh. The reaction mixture was then added to a solution containing Fe-PD, H₂O₂, and TMB. The absorbance at 652 nm (oxTMB) was recorded using a UV-Vis spectrophotometer. To evaluate inhibitory effects, varying concentrations of berberine hydrochloride (0.1–150 μM) were pre-incubated with AChE (50 mU/mL) for 15 min at 37°C before adding ATCh. The residual AChE activity was determined by measuring the UV-Vis absorbance peak intensity at 652 nm from the suppression of TMB oxidation.

Construction of Microneedle-Based Colorimetric Sensing Array and Characterizations of the Micronnedle Patch

First, 23.75 mg of 3A-PBA, 750 μL of NVP, 100 μL of ETPTA, and 19 μL of EGDMA were added to a centrifuge tube, followed by shaking and ultrasonic treatment until the solution was thoroughly dissolved. Then, 9 μL of HMPP was added as a photoinitiator, and the mixture was shaken to ensure complete blending. The resulting solution was carefully injected into a designed polydimethylsiloxane (PDMS) microneedle mold (depth: 800 μm, base width: 400 μm, center space: 900 μm and placed in a vacuum chamber for 5 minutes. Subsequently, photo-crosslinking was performed under UV light (360 nm, 5 W) to form the microneedle array. The microneedle array was then carefully removed from the PDMS mold and stored in a desiccator for future use. Microneedle arrays were first incubated in Fe-PD nanozyme solution (0.05 mg/mL) for 1 h to allow boronate ester bond formation, then rinsed with PBS to remove unbound nanozymes. For ATCh loading, the above pre-functionalized microneedle arrays were incubated in the 30 mM ATCh solution for 30 min and dried under nitrogen.

For the microneedle characterization, SEM imaging was performed under an accelerating voltage of 5.0 kV and a beam current of 10 μA, with a working distance of 18.5 mm, a stage tilt of 30°, and a magnification of 50. The demolding rate was calculated following the defined equation 1:

Where No. _{Demolded microneedles} represents the number of needles in the demolded microneedle patch, and No. _{Designed microneedles} represents the number of needles in the designed microneedle mold.

For the mechanical hardness of the microneedles, we positioned the microneedle patch on the sample stage of a single-column material testing machine (Instron 5940). Relevant test parameters were configured prior to the experiment, and the compression table was carefully adjusted to achieve proper clamping of the microneedle patch. Subsequently, a compression test was performed at a constant speed of 1.0 mm/min to determine the corresponding compressive mechanical curve.

The microneedle array was immersed in 1× PBS solution (pH 7.4), and its weight was recorded at various soaking time points (0 min, 1 min, 3 min, 5 min, 7 min, 10 min, 20 min, 30 min, 60 min). According to the previous reference,³³ the water absorption expansion rate was calculated following the equation 2:

Where Ws is the weight of the microneedle patch after swelling, and W₀ is the initial weight of the microneedle patch.

POCT Analysis of AChE and Its Drug Inhibitor in Simulated Samples

Before detection, fresh porcine ear skin purchased from the local market were used as model samples simulating human skin and processed through the following steps: (1) The pig skin was disinfected with 75% ethanol, followed by cleaning with phosphate-buffered saline (PBS, 10 mM, pH 7.4) to remove surface contaminants; (2) The clean skin was cut into uniform small pieces and incubated overnight at 4°C in an artificial interstitial fluid (AISF) containing different concentrations of AChE (1, 10, 100, 1000 mU/mL) and varying concentrations of berberine (0.1, 10, 20, 40, 80, 120, 160 μM); (3) After incubation, the excess liquid on the surface of the skin sample, containing the AChE or its inhibitor-AISF complex, was gently blotted with a lint-free cloth. This pre-treatment ensured the uniform distribution of AChE or its inhibitor in the dermal matrix, thereby ensuring the accuracy of the subsequent detection results. Next, the engineered nanozyme-based microneedle Colorisensor patches were pressed onto the surface of the pre-treated porcine skin. After 20 minutes of contact, the patches were immersed in TMB (2 mM, 1000 μL) and H₂O₂ (10 mM, 1000 μL) for 3 minutes. The patches were then removed, and the color change of the patches was observed. The RGB values of the patches were measured using a color detection app on a smartphone.

Statistical Analysis

Statistical analysis of the data was performed using GraphPad Prism 10 Software. All data were presented as the mean ± the standard deviation (SD). Specifically, Tukey’s multiple comparison test was employed to further elaborate on significant differences among various groups. The data were marked as (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****) P < 0.0001. The P-value above 0.05 was considered non-significant (^ns).

Results and Discussions

Preparation and Characterization of Fe-PD Nanorods

To our knowledge, dopamine tends to form the 0D sphere or 2D film when metal and ligands undergo cross-linking.^34,35 In this study, we employed a novel approach through Fe (III)-catechol coordination interaction, producing Fe-PD nanorods that served as catalytic components of the POCT colorimetric sensor. Our template-free strategy has overcome key issues in the fabrication process of nanorod-structured synthesized enzymes, including complicated experimental routines, strict conditions, long time-consuming, difficult removal, and residual side effects of the template.³⁶ To prepare Fe-PD nanorods, dopamine and Fe³⁺ were successively added for chelation in an alkaline ethanol/water reaction system (Figure 1A). During the reaction stage, the catechol groups of dopamine can strongly chelate and cross-link with Fe to form Fe-PD precursor.^37,38 These chemical chelates were further aggregated and polymerized by radical polymerization, resulting in Fe-PD nanorods for the next step of ATCh sensing. The SEM images in Figure 1B and C exhibited the change of the resulting DA-Fe chelates from nanospheres to nanorods. The Fe-PD nanorods were synthesized with a length of about 346.0 nm and a diameter of about 88.2 nm in a mold alkaline environment, as shown in the high-angle angular dark field-scanning transmission electron microscope (HAADF-STEM) image of Figure 1D. The EDS spectroscopy characterization (Figure 1E and Table S1 in Supporting Information) indicated that ≈6.0 wt% Fe element is uniformly distributed in the Fe-PD nanorods, which means effective Fe (III) chelate with dopamine molecules. Meanwhile, the zeta potential of the Fe-PD⁺ nanorods was measured at −17.2 mV, representing a notable positive shift compared to pristine PDA (−38.7 mV) (Figure S1). These findings collectively confirm the successful formation of Fe-PD nanorods.

Figure 1 (A) The synthesis procedure of Fe-PD nanozymes. (B) The SEM image of the initial coordinated Fe-PD nanospheres; (C) The SEM image of the finally prepared Fe-PD nanorods. (D) HAADF-STEM image of Fe-PD nanorods. (E) EDS mapping of the Fe-PD nanorods. (F) Schematic illustration of the POD-like catalytic process of Fe-PD nanozymes. (G) Kinetics for POD-like activity of Fe-PD nanozymes with different concentrations of TMB (0.05–8 mM). Inset: Corresponding Lineweaver-Burk plot with a resulting linear equation of y = 0.055x + 0.065 (R2 = 0.9992). (H) Kinetics for POD-like activity of Fe-PD nanozymes with different concentrations of H2O2 (0.125–32.0 mM). Inset: Corresponding Lineweaver-Burk plot with a resulting linear equation of y = 0.0262x + 0.1092 (R2 = 0.9974).

Afterwards, potential possibilities of Fe-PD nanorods were investigated for the role of nanozymes in POCT colorimetric sensors. Herein, to determine the enzymatic-like activity of Fe-PD nanorods, we further studied their enzymatic catalytic behavior using the TMB-H₂O₂ reaction system. Generally, peroxidase can catalyze colorless TMB to produce blue oxTMB by generating active hydroxyl radicals in the presence of H₂O₂,³⁹ accompanied by the appearance of a characteristic absorption peak at 652 nm.⁴⁰ To systematically evaluate the catalytic efficiency of Fe-PD nanorods as nanoenzymes (Figure 1F), we conducted steady-state kinetic parameter analysis, including the Michaelis constant (K_m) and maximum reaction rate (V_max). By adjusting the concentrations of TMB and H₂O₂, the steady-state kinetics were investigated to better understand the enzyme-like activity of the prepared Fe-PD nanorods. In Figure 1G and H, Fe-PD nanorods exhibit typical Michaelis-Menten models at different concentrations of TMB and H₂O₂, respectively. Moreover, when changing the concentration of the other substrate, Lineweaver-Burk curves can be obtained (Insets of Figure 1G and H). The resulting K_m value and V_max of Fe-PD nanorods were evaluated by fitting the Lineweaver-Burk equation in the double reciprocal plot. The K_m of Fe-PD nanorods for TMB and H₂O₂ are calculated as 0.85 and 0.24 mM, respectively. Its K_m (TMB) value is lower compared to that of the HRP [K_m (TMB): 0.43 mM]; meanwhile, its K_m (H₂O₂) value is smaller than that of the HRP [K_m (H₂O₂): 3.7 mM].⁴¹ Additionally, when using the TMB as the substrate, Fe-PD nanorods achieved a stronger affinity with higher catalytic efficiency (V_max = 15.38×10⁻⁸ MS⁻¹) compared to that of previously reported other nanozymes using TMB as substrate (Table S2). Further, this nanorod-structured Fe-PD enzyme exhibits outstanding affinity with a higher maximum reaction rate (9.16×10⁻⁸ MS⁻¹) when using H₂O₂ as the substrate, which exceeds most of the counterpart nanozymes (Table S3). The outstanding POD-like activity of the Fe-PD nanozyme is attributed to its three-dimensional rod-shaped structure (as illustrated in Figure 1C) and numerous catalytic sites, significantly promoting electron transfer.⁴² The inclusion of Fe³⁺ serves to reconfigure the electronic distribution,⁴³ further enhancing its POD-like activity. More importantly, Fe-PD nanorods, as metal-catechol ligand cross-linking nanomaterials, could maintain their enzymatic-like catalytic activity over a long time with a low RSD value of 1.06% and attenuation rate less than 3.0% (until 85 days, Figure S2). These exciting results indicate the stronger affinity and catalytic activity of Fe-PD nanozymes than natural HRP and other reported POD-like nanozymes, with excellent practical stability. This further proves that Fe-PD nanozymes have reliable POD-like behavior, and this preparation approach could be advantageous for practical applications.

Analytical Performance of This Colorisensor for AChE

Taking advantage of excellent POD-like activity, Fe-PD nanozymes could effectively catalyze the oxidation of colorless TMB (Curve a in Figure 2A) to produce the blue oxTMB (Curve b in Figure 2A) in the presence of hydrogen peroxide, resulting in a marked increase in absorbance at 652 nm. Experimental results showed that the only addition of AChE (Curve c in Figure 2A) or ATCh (Curve d in Figure 2A) had no significant impact on the absorbance of the system, indicating that these components did not interfere with the colorimetric assay. As shown in Figure 2A-Curve e and Figure 2B, when both AChE and ATCh were present in the system (with their pre-reaction producing TCh), the absorbance significantly decreased, suggesting that TCh generated by AChE-catalyzed hydrolysis of ATCh could effectively inhibit the catalytic activity of Fe-PD nanozymes and prevent the oxidation of TMB. These findings demonstrate that the Colorisensor, based on the specific inhibition effect of TCh on Fe-PD nanozyme activity, can achieve AChE detection. This proves the AChE analytical feasibility of the colorimetric assay.

Figure 2 (A) UV-vis spectra of the colorimetric biosensing system in the absence and in the presence of AA. The colorimetric biosensing system includes a: TMB + Fe-PD, b: TMB + Fe-PD + H2O2, c: TMB + Fe-PD + H2O2 + AChE, d: TMB + Fe-PD + H2O2 + ATCh, and e: TMB + Fe-PD + H2O2 + AChE + ATCh. Inset: corresponding solution color photographs. (B) Comparison of the above corresponding absorbance peak intensity. (C) Effects of Different incubation time (3 min, 5 min, 10 min, 20 min, and 60 min), (D) Different incubation temperature (4 °C, 25 °C, 37 °C, 45 °C, and 60 °C), and (E) added volume ratio of ATCh and AChE on the absorbance peak intensity of the Colorisensor system. VATCh and VAChE represent the added volumes of ATCh and AChE, respectively. (F) The relationship calibration curve between the absorbance of the Colorisensor system and the logarithm of the AChE activities (CAChE: from 0.01 to 1000.0 mU/mL) obtained by the Colorisensor. Inset: corresponding solution color photographs. Lg(CAChE) represents the logarithm values of AChE concentration to the base 10. R2 represents fitting coefficient of the linear relationship curve. Data were expressed as mean ± SD, n = 4, nsP> 0.05, *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.

To obtain better analytical performance of this Colorisensor, the experimental conditions, including incubation time, incubation temperature, and content ratio of ATCh and AChE, were optimized. As shown in Figure 2C, the relationship between incubation time and the absorbance peak intensity of oxTMB at 652 nm decreased with increasing incubation time, ranging from 3 to 60 min. When the reaction time was 20 min, the peak intensity almost reached the plateau. Thus, 20 min was chosen as the optimal reaction time. Similarly, reaction temperature and content ratio of ATCh and AChE were also optimized as 37 °C (Figure 2D) and 1:1 (Figure 2E), respectively. Under the optimal experimental conditions, we conducted a detailed analysis of the detection performance of the Colorisensor. Plotting the longitudinal absorbance values versus the logarithm of the AChE activities within the range of 0.01–1000.0 mU/mL (Figure 2F), exhibits a good linear relationship. According to the linear regression equation of y = −0.0954x + 0.2907 (R²=0.9990), the detection limit of this Colorisensor was estimated as 0.007 mU/mL. Correspondingly, the solution color changed from blue to colorless with the increase of AChE (Inset in Figure 2F), further validating the excellent detection ability of the Colorisensor for AChE activities.

Repeatability, Stability, and Selectivity of This Colorisensor

Before practical analysis, other key detection parameters (including repeatability, stability, and selectivity) of this Colorisensor were tested. As shown in Figure 3A, the relative standard deviation (RSD) of eight independent sensors for 50 mU/mL of AChE detection is about 1.29%, suggesting that the Colorisensor holds outstanding repeatability. To investigate the selectivity of our developed colorimetric biosensing system, several potential interfering substances, including KCl, Glu, L-Cys, GSH, GOx, Lyz, and Tyr, were selected. In Figure 3B, the detection of AChE could not be interfered by these interfering substances, proving that this Colorisensor has excellent selectivity. Prominently, the colorimetric sensor also has long-term storage stability. The RSD of this Colorisensor for 50 mU/mL of AChE detection over 30 days is only 1.83% (Figure 3C). The above results demonstrate the great potential analytical performance of the Colorisensor for the diagnosis of degenerative diseases.

Figure 3 (A) Repeatability of eight independent Colorisensors used to detect 50 mU/mL of AChE in AISF. (B) Effect of several interferences on the absorbance of the developed Colorisensor at 652 nm in the presence of interferences [KCl (0.15 mM); Glu (4 mM); L-cys and GSH (0.18 mM); GOx, Lyz, and Tyr (50 mU/mL)]; and AChE (50 mU/mL). (C) Storage stability of our Colorisensors for 30 days.

Detection of the AChE Inhibitor

Because of the great performance of the colorimetric sensing platform, it was further expanded by exploring its potential application for the determination of AChE inhibitor. Berberine is a promising effective inhibitor of the activity of AChE owing to its neuroprotective effects and treatment of Alzheimer’s Disease.⁴⁴ As shown in Figure 4A, the absorption intensity gradually increased with increasing berberine concentrations. Figure 4B displayed the trend of inhibition efficiency with berberine concentration ranging from 0.1 to 40.0 μM, which can be determined by the equation: y = 0.0089x + 0.0418 (R²=0.9920). The corresponding solution gradually returned to its blue color of oxTMB. The LOD was 0.034 μM by using the 3σ/slope method, suggesting that the proposed Colorisensor platform was able to achieve berberine detection visually.

Figure 4 (A) Absorbance of the colorimetric system at 652 nm with increasing berberine concentration from 0.1 to 160 μM. (B) Corresponding relationship between absorption peak intensity and the berberine concentration (0.1–40 μM). The linear equation: y=0.0089x + 0.0418 (R2=0.9920).

POCT Analysis in Simulated Samples by the Microneedle-Based Color-Sensing Array

To evaluate the POCT application in the condition surrounding simulated complex components in vivo, we first constructed the microneedle-based colorisensing array to carry out the actual detection of the activity of target AChE or the inhibiting effects of drug berberine on enzyme activity. The microscopic images of the microneedle array in Figure 5A displayed the well-arranged microneedle structures with uniform morphology, which is the basis for sampling and analyzing biofluid in the sensing platform. To ensure optimal penetration into the skin while minimizing tissue damage, the microneedle arrays were designed with a specific geometry. As shown in Figure 5B, dimensional information of the microneedle array could be provided with their height (H) of 775.4 μm, width (W) of 393.8 μm at the base, and center space (S) of 984.6 μm, ensuring an appropriate size to penetrate the epidermis layer and perform sensing functions. The Colorisensor microneedle array was fabricated via a photopolymerization-micromold method, as Figure 5C exhibited its SEM image for revealing the uniformity and sharpness of the microneedle tips, which are crucial for effective contact with tissue fluid. As shown in Figure S3, the microneedle patch was fabricated with a calculated demolding rate of 99.73%. To test the mechanical hardness of the microneedles, we conducted compression mechanical experiments at the speed of 1.0 mm/min to obtaining a force of approximately 0.18 N per needle (Figure S4), which exceeds the transdermal threshold ~0.058 N per needle.⁴⁵ The resulting penetration depth of microneedles to pig skin was about 716 μm (Figure S5), indicating their ability to penetrate the stratum corneum and access the dermis layer for analyte detections. To better exhibit the sample ability, we investigated the swelling behavior of the microneedle patch, and defined the water absorption expansion rate as the evaluation index of its swelling performance. As a result, the water absorption expansion rate was calculated as about 156% (Figure S6).

Figure 5 (A) microscopic image of part regions, (B) Cross-section image of the microneedle sensing array, and (C) SEM image of the microneedle sensing array. (D) Schematic illustration of Fe-PD nanozyme-mediated colorisensing visual evaluation of AChE activity and its drug inhibitors by the easy-to-use smartphone-assisted microneedle array platform. (E) Change of visible light G·B/R values of microneedle arrays with AChE concentrations of 1, 10, 100, and 1000 mU/mL, along with 30 mM of ATCh in porcine ear skin. Inset: Corresponding color photographs of the microneedle arrays. (F) Corresponding relationship between Visible light G·B/R values of microneedle arrays and Lg(AChE concentration), with the linear equation of y = −35.1x + 228.4 and R2= 0.9873. (G) Change of visible light G·B/R values of microneedle arrays with berberine concentrations (0.1, 1, 10, 20, 40 μM) in porcine skin. Inset: Corresponding color photographs of the microneedle arrays. (H) Corresponding relationship between visible light G·B/R values of microneedle arrays and berberine concentration, with the linear equation of y = 3.789x + 140.5 and R2= 0.9827.

Afterwards, the sampling duration for the microneedle patch was determined through a combination of quantitative in vitro kinetics and ex vivo visual confirmation. We immersed the microneedle patches in artificial interstitial fluid (AISF) and quantitatively monitored the water absorption expansion rate over time. The data indicated that the water absorption expansion rate reached a plateau (the equilibrium stage) after approximately 20 min (Figure S7), suggesting that the patch’s fluid uptake capacity was nearly saturated. To corroborate the in vitro findings and visually demonstrate the sampling process, we applied the patches to pig skin. The microneedle patch contains a colorimetric indicator (CoCl₂) whose color changes from blue to pink upon interaction with the extracted interstitial fluid. We observed that the color change progressed gradually and then stabilized, reaching a consistent and unchanging state after a period of 20 minutes (Figure S8). This visual endpoint provided direct evidence that the active sampling process was complete. The strong agreement between the quantitative swelling equilibrium and the qualitative color stabilization gave us high confidence that a 20-min sampling duration is sufficient to ensure the patch operates at its full capacity, guaranteeing complete and efficient sample collection for reliable analysis. The covalent linkage between 3A-PBA and Fe-PD nanozymes minimized the nanozyme detachment, while the low RSD values (2.80%, Figure S9) verified the consistent loading efficiency across batches. The characterization of nanozyme coating stability via continuous washing tests (RSD=3.12%, Figure S10) to further validate the robustness of the modification on the microneedle arrays. To evaluate the actual POCT performance of the Colorisensor platform, we conducted the detection experiment of AChE and its inhibitor on a fresh porcine ear skin as the simulated real in vivo condition, which is reported as a typical in vitro model due to its content of ISF (about 70% of human skin).^46,47 The RGB recognition strategy was employed to quantify the color changes induced by the enzymatic activity of AChE. As depicted in Figure 5D, the microneedle array was applied to the porcine skin spiked with different activities of AChE, and the resulting color changes were captured using a smartphone app. The RGB values were then analyzed to determine the AChE activity. The visible light intensity was defined as (G·B)/R values for quantitative analysis of enzyme activity. As shown in Figure 5E, a clear activity-dependent response to different concentrations of AChE was exhibited. The linear relationship between the logarithm of AChE concentration and the visible light (G·B)/R value is illustrated in Figure 5F, with a correlation coefficient (R²) of 0.9973. This indicates that the Colorisensor platform can accurately detect AChE activity over a broad range from 0.01 mU/mL to 1000 mU/mL at point-of-care (POC), with a limit of detection (LOD) as low as 0.049 mU/mL. Meanwhile, the developed Colorisensor demonstrates comparable accuracy to the standard Ellman’s method under spiked conditions (Figure S11), while exhibiting a wider detection range and a lower detection limit compared to both traditional assays and emerging nanomaterial-based colorimetric methods (Table S4).

To further validate the inhibitor detection utility of the Colorisensor platform, we chose berberine hydrochloride as an inhibitor candidate of AChE. The results presented in Figure 5G and H demonstrate a significant decrease in the visible light intensity (G·B)/R value with increasing concentrations of berberine. The linear regression analysis showed a strong correlation (R² = 0.9827) between the berberine concentration and the observed color changes from 0.1 μM to 40 μM, with a low LOD of 0.098 μM indicating the high sensitivity and accuracy of the Colorisensor for POC detecting AChE inhibitors.

Potential Applications and Future Perspectives

This study primarily demonstrates the application of our microneedle and nanozyme-based Colorisensor for AChE detection, focusing on its potential in the early diagnosis and drug screening of neurodegenerative diseases. The minimally invasive nature, high sensitivity, and portability of our platform address a critical need for point-of-care monitoring of chronic biochemical changes, such as the subtle fluctuations in AChE activity associated with the onset and progression of conditions like Alzheimer’s disease.^48,49

Beyond this primary focus, we recognize that the significant potential of our platform in other AChE-related testing scenarios. A prominent example is the rapid screening of organophosphate and carbamate pesticide poisoning, where AChE inhibition is a well-established clinical biomarker.^50,51 While current field tests for poisoning are often qualitative, our Colorisensor offers a quantitative, highly sensitive, and user-friendly alternative. It could not only confirm exposure but also help stratify poisoning severity and objectively monitor the efficacy of antidote administration at the point of care, which is crucial in agricultural and low-resource settings. We envision that our Colorisensor will evolve into a multi-scenario POCT tool, capable of addressing diverse needs in both chronic disease management and emergency medicine.

Conclusion

In summary, we develop a highly sensitive acetylcholinesterase assay based on a microneedle-based colorimetric nanosensing platform. Integrating metal-phenol nanozymes with peroxidase-like activity that responds to target-induced changes, and combining smartphone-based RGB color recognition, the platform achieves rapid and easy-to-operate AChE activity detection with a broad linear range of 0.01–1000 mU/mL and excellent selectivity. It successfully identifies berberine as a candidate AChE inhibitor, demonstrating promising potential for applications in drug inhibitor screening fields. Notably, the microneedle-based sensing concept presented herein is a proof-of-concept. To realize point-of-care testing, further engineering optimizations, such as miniaturization of supporting detection devices, improvement of on-site readout convenience, and enhancement of practical applicability in complex biological scenarios are required. Furthermore, the versatility of this sensing strategy suggests promising potential for expansion into other application fields, such as rapid on-site screening for pesticide exposure, highlighting its broad impact in both clinical and public health settings.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This research was supported by Science and Technology Program of Suzhou (SYW2025037) and Science and Technology Program of Taicang (TC2024JCYL23).

Disclosure

The authors declare no conflict of interest.

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The German bank will take about twice as much space in the YY building on South Colonnade as Revolut, the report said, citing people familiar with the matter.

Sign up here.

Deutsche Bank declined to comment on the report. Canary Wharf Group referred Reuters to asset manager Oaktree Capital Management, which owns the building, when asked for a comment. Oaktree declined to comment.

Oaktree bought the building in a joint venture with real estate firm Quadrant Estates in 2019, according to Quadrant’s website. Quadrant could not be reached for comment.

($1 = 0.7502 pounds)

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Story Continues

Head to the Valuation section of our Company Report for more details on how we arrive at this Fair Value for First Quantum Minerals.

For cyclical and capital intensive businesses like miners, price to sales is often a more reliable yardstick than earnings based multiples, because profits can swing wildly with commodity prices while revenue tends to be more stable and reflective of underlying scale.

In general, faster expected growth and lower perceived risk justify a higher price to sales multiple, while slower growth or elevated risk warrant a discount, so there is no single “right” number that fits every company or cycle.

First Quantum Minerals currently trades at about 3.98x sales, a shade below the Metals and Mining industry average of roughly 6.41x and slightly under the 4.12x peer group average. This suggests the market is still assigning it a modest discount despite the strong share price performance.

Simply Wall St’s proprietary Fair Ratio framework estimates what a reasonable price to sales multiple should be for First Quantum, given its growth outlook, margins, industry, size and risk profile. This approach is more tailored than simple peer comparisons because it adjusts for company specific strengths and weaknesses.

On this basis, First Quantum’s Fair Ratio comes out at about 4.42x, which sits above the current 3.98x sales. This indicates the shares still screen as undervalued on a sales multiple basis.

Result: UNDERVALUED

PS ratios tell one story, but what if the real opportunity lies elsewhere? Discover 1442 companies where insiders are betting big on explosive growth.

Earlier we mentioned that there is an even better way to understand valuation, so let us introduce you to Narratives, a simple way to connect your view of First Quantum Minerals with the numbers behind it. A Narrative is your story for the company, where you spell out how you think its revenue, earnings and margins will develop, and link that story directly to a financial forecast and then to a fair value estimate. On Simply Wall St’s Community page, used by millions of investors, Narratives are an easy to use tool that help you assess opportunities by constantly comparing your fair value view to the current share price. They update dynamically as new information such as news or earnings is released, so your investment thesis does not sit still while the world moves on. For example, one Narrative for First Quantum Minerals might assume strong copper demand and place fair value far above today’s price, while another builds in higher political and project risk and concludes that the stock is already fairly valued.

Do you think there’s more to the story for First Quantum Minerals? Head over to our Community to see what others are saying!

This article by Simply Wall St is general in nature. We provide commentary based on historical data and analyst forecasts only using an unbiased methodology and our articles are not intended to be financial advice. It does not constitute a recommendation to buy or sell any stock, and does not take account of your objectives, or your financial situation. We aim to bring you long-term focused analysis driven by fundamental data. Note that our analysis may not factor in the latest price-sensitive company announcements or qualitative material. Simply Wall St has no position in any stocks mentioned.

Companies discussed in this article include FM.TO.

Have feedback on this article? Concerned about the content? Get in touch with us directly. Alternatively, email editorial-team@simplywallst.com

Accenture (ACN) has been grinding higher recently, with the stock up about 7% over the past month despite a rough year for shareholders. That move has investors rechecking whether today’s price still lines up with fundamentals.

See our latest analysis for Accenture.

The recent rebound follows a tough stretch, with a negative year to date share price return and a roughly 12 month total shareholder return still in the red. This hints that sentiment is improving but not fully repaired.

If Accenture has you rethinking your tech exposure, this could be a good moment to explore high growth tech and AI stocks for other potential opportunities riding similar digital transformation themes.

With earnings still growing and the share price lagging its recent peak, investors now face a key question: is Accenture quietly offering value at today’s levels, or is the market already pricing in its next leg of growth?

According to FCruz, the narrative implies a fair value well below Accenture’s last close of $266.59, setting up a tension between quality and price.

Bottom line (fundamental stance) I’m moderately constructive over 12 to 18 months. Accenture combines (i) scaled exposure to GenAI-led reinvention with tangible bookings, (ii) high-quality margins, returns, and FCF, and (iii) a reset valuation near historical norms. The near-term swing factor is bookings momentum; if that stabilizes or improves, upside to the Street’s mid-30s EPS multiple case becomes more plausible.

Read the complete narrative.

Want to see how modest revenue growth, steady margins and a premium future earnings multiple still argue for a much lower fair value than today? The full narrative walks through those moving parts step by step, but keeps one core valuation lever front and center. Curious which assumption does most of the heavy lifting, and how sensitive the outcome is if it shifts?

Result: Fair Value of $202.38 (OVERVALUED)

Have a read of the narrative in full and understand what’s behind the forecasts.

However, persistent weakness in bookings or a sharper slowdown in consulting spend could quickly challenge the case for Accenture’s current premium valuation.

Find out about the key risks to this Accenture narrative.

While the most popular narrative sees Accenture as roughly 31.7% overvalued, our valuation work using a simple earnings multiple lands in a different place. At 21.5 times earnings, the stock trades well below the US IT industry average of 30.3 times and peers at 25.3 times, and also below a fair ratio of 36.7 times that the market could drift toward over time.

That gap suggests investors are paying a noticeable discount for a business with high quality earnings and strong returns on equity, which could limit downside if growth stays steady. However, it also raises a tougher question: what if the market never fully closes that valuation gap?

See what the numbers say about this price — find out in our valuation breakdown.

If you see the story differently or prefer your own due diligence, you can build a personalized view in just minutes with Do it your way.

A good starting point is our analysis highlighting 5 key rewards investors are optimistic about regarding Accenture.

Before you move on, lock in an edge by scanning fresh opportunities with the Simply Wall Street Screener so your next decision is intentional and not reactive.

This article by Simply Wall St is general in nature. We provide commentary based on historical data and analyst forecasts only using an unbiased methodology and our articles are not intended to be financial advice. It does not constitute a recommendation to buy or sell any stock, and does not take account of your objectives, or your financial situation. We aim to bring you long-term focused analysis driven by fundamental data. Note that our analysis may not factor in the latest price-sensitive company announcements or qualitative material. Simply Wall St has no position in any stocks mentioned.

Companies discussed in this article include ACN.

Have feedback on this article? Concerned about the content? Get in touch with us directly. Alternatively, email editorial-team@simplywallst.com

• Institutions’ substantial holdings in Ameriprise Financial implies that they have significant influence over the company’s share price

• A total of 17 investors have a majority stake in the company with 50% ownership

• Recent sales by insiders

Trump has pledged to “unleash” American oil and gas and these 15 US stocks have developments that are poised to benefit.

A look at the shareholders of Ameriprise Financial, Inc. (NYSE:AMP) can tell us which group is most powerful. And the group that holds the biggest piece of the pie are institutions with 87% ownership. In other words, the group stands to gain the most (or lose the most) from their investment into the company.

Institutional investors would appreciate the 4.7% increase in share price last week, given their one-year losses have totalled a disappointing 14%.

Let’s delve deeper into each type of owner of Ameriprise Financial, beginning with the chart below.

See our latest analysis for Ameriprise Financial

Many institutions measure their performance against an index that approximates the local market. So they usually pay more attention to companies that are included in major indices.

We can see that Ameriprise Financial does have institutional investors; and they hold a good portion of the company’s stock. This can indicate that the company has a certain degree of credibility in the investment community. However, it is best to be wary of relying on the supposed validation that comes with institutional investors. They too, get it wrong sometimes. If multiple institutions change their view on a stock at the same time, you could see the share price drop fast. It’s therefore worth looking at Ameriprise Financial’s earnings history below. Of course, the future is what really matters.

Investors should note that institutions actually own more than half the company, so they can collectively wield significant power. Hedge funds don’t have many shares in Ameriprise Financial. The Vanguard Group, Inc. is currently the largest shareholder, with 13% of shares outstanding. Meanwhile, the second and third largest shareholders, hold 9.7% and 4.8%, of the shares outstanding, respectively.

Looking at the shareholder registry, we can see that 50% of the ownership is controlled by the top 17 shareholders, meaning that no single shareholder has a majority interest in the ownership.

While it makes sense to study institutional ownership data for a company, it also makes sense to study analyst sentiments to know which way the wind is blowing. There are plenty of analysts covering the stock, so it might be worth seeing what they are forecasting, too.

Story Continues

By Charles Passy

America’s retirement system desperately needs reform

President Trump suggested the U.S. should consider the Australian retirement-savings model.

Could America’s signature retirement-savings program look a lot like Australia’s one day?

That’s the idea President Trump hinted at earlier this week, saying the Australian model is a “good plan” that has “worked out very well.”

“We’re looking at it very seriously,” Trump said.

In a nutshell, the Australian plan takes some of what’s already baked into the American 401(k) retirement-savings model, but expands upon it in significant ways that assure more people have more savings by the time they reach retirement age. Called the “superannuation” (or “super”) model, the program requires employers to make a 12% contribution to a retirement fund on behalf of the employee. The employee can also contribute an amount beyond that.

‘We’re looking at it very seriously.’President Donald Trump, on the U.S. possibly adopting the Australian retirement-savings model

In the U.S., the 401(k) model works by giving employees the chance to participate in a retirement-savings program through their employer, with tax benefits to employees for doing so. But the employer is not obligated to offer a 401(k) plan – and even if they do, there’s no requirement they make any kind of contribution to it.

Indeed, research has shown that 56 million private-sector workers in the U.S. lack access to a retirement-savings plan. And even among employees who have a 401(k), the employer contribution is typically in the form of a match, which often equates to 4% to 6% of an employee’s salary – far below that 12% Australian figure.

Australia’s program, with the mandatory employer-contribution aspect, has been in place since 1992, but it didn’t start at 12%. In fact, it began with just 3%, but over time the figure grew incrementally to the current 12%. Still, it has resulted in Australian workers, on average, accumulating the equivalent of around $115,000 (that’s roughly $173,000 in Australian currency (AUDUSD)).

Those enrolled in U.S. 401(k) plans actually have a bit more than that; the average 401(k) balance is $148,153, though it should be noted that wages and the cost of living are lower in Australia.

Perhaps the more relevant data point, however, is the fact that 78% of Australians participate in the “super” program. By contrast, just 59% of Americans have a retirement-savings plan, be it a 401(k), 403(b) or an individual retirement account (IRA), according to a Gallup survey.

But some financial experts say it might be politically tough to push through an Australian-style program in the U.S., especially given the financial burden it places on companies – and small ones in particular.

A plan that mandates that businesses contribute to employee retirement plans at such a high level “will never happen,” said Teresa Ghilarducci, a noted retirement authority who’s an economics professor at the New School in New York City.

Plus, even those who give the Australian system high marks point to issues within it. A key one: Even though the system helps ensure that workers save a significant sum for retirement, it doesn’t necessarily guide them on how to tap that money once they retire – by turning it into, say, a monthly income stream they can parse out carefully over time as they deal with any number of medical or other issues they may face as they age.

“The system still struggles to help retirees navigate longevity risk, inflation and cognitive decline,” said Tomas A. Geoghegan, founder of Beacon Hill Private Wealth in New Jersey.

That said, the American 401(k) model doesn’t offer any systemized way of parsing out, or annuitizing, one’s retirement savings, either.

In any case, there’s little question that the current retirement-savings system in the U.S. needs to be revamped. Without an improved safety net, Americans will be relying more heavily on Social Security than ever, experts note. And as Americans are constantly reminded, Social Security is under threat as it is.

“We absolutely have to do something,” said Holly Verdeyen, a partner at Mercer, a consulting firm that focuses heavily on retirement planning.

Mercer rates retirement systems throughout the world, and gives the Australian model a solid B+. By contrast, the U.S. gets a C+.

The U.S. has already been looking at ways to revamp its retirement-savings model, regardless of whether or not it considers the Australian one.

For starters, under what’s commonly referred to as the Secure 2.0 Act, Congress authorized such changes as letting employers automatically enroll employees into 401(k) plans and allowing employees between the ages of 60 and 63 to increase their maximum retirement contributions.

On top of that, a number of states are looking at ways for employees to access retirement-savings programs.

But more sweeping national reform is still needed, many argue. And some say it could come in the form of the Retirement Savings for Americans Act (RSAA), which is currently making its way through Congress. It calls for a program that would broaden accessibility to tax-advantaged retirement-savings accounts and would have the federal government match contributions for workers below certain income levels.

In the meantime, the Australian model is still out there.

White House spokesman Kush Desai wouldn’t get into specifics about how the model could work in the U.S., but told MarketWatch: “The administration is closely examining all options to help Americans build wealth and achieve prosperity.”

-Charles Passy

This content was created by MarketWatch, which is operated by Dow Jones & Co. MarketWatch is published independently from Dow Jones Newswires and The Wall Street Journal.

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  12-06-25 0800ET
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Good afternoon and thank you for the flexibility in allowing me to deliver my remarks towards the end of the day due to scheduling issues. Earlier, the Committee engaged in discussions on corporate governance and tokenization and will later discuss artificial intelligence disclosures.

Today, also, is the final Committee Meeting for our Investor Advocate. I want to extend my appreciation to Cristina [Martin Firvida] for her dedication and professionalism. Cristina is the second Investor Advocate in the Commission’s history. When she was selected, I was not quite sure what to expect from her. However, Cristina won me over by finding areas of mutual agreement and then executing upon them. One of Cristina’s important contributions is reforming the selection process for Committee members. As an SEC staff member, I was here at the very beginning of the Committee. It became clear that the departure of a significant majority of the Committee members every four years was not conducive to longer-term thinking. Through Cristina’s efforts, we now have a plan where approximately a quarter of the Committee will be replaced annually, thereby obtaining a smoother transition of new members into the Committee. Thank you, Cristina, for your outstanding service and I wish you the best in your future endeavors!

Corporate governance plays a role in investor confidence and market integrity. It encompasses the systems, principles, and processes by which companies are directed and controlled. At its core, effective governance ensures that companies are accountable to shareholders and resilient in the face of evolving risks.

Over time, the role of governance has expanded beyond traditional oversight. Companies are increasingly expected to navigate complex issues such as cybersecurity and emerging technologies. A well-functioning board must not only monitor management but also anticipate and adapt to systemic shifts that could affect long-term value creation.

However, Congress specifically left the states in charge of governance under state corporate law. The federal securities laws and regulations play an important complimentary role in providing disclosure, describing the governance structure, the rights of shareholders, and potential risks associated with a company’s particular structure as well as for providing a regulatory framework for proxy solicitation.

Yet, as we consider reforms, we must be mindful of the temptation to use the Commission’s disclosure authority for registration statements and proxy materials, as well as oversight of exchange listing requirements, to impose prescriptive governance mandates. It is inappropriate to mistake the “investor protection” and “public interest” standards contained in the federal securities laws as Congressional authority to set national corporate governance standards. Congress does not “hide elephants in mouse holes.”

Corporate governance is best left to the market to decide. If a potential shareholder does not like a particular governance framework — such as whether it is board composition, independence, expertise, then there is a simple solution: do not invest. Companies with corporate governance structures that are not well received by investors will have a higher cost of capital and a depressed stock price.

Similarly, another area of recent discussion is the Commission’s clarification regarding mandatory arbitration provisions in registration statements and how the presence of such provisions will not be an impediment to acceleration of effectiveness. Some panelists might have argued earlier today that this represents a reversal of prior Commission policy or weakening of investor protections. In fact, the Commission has never had a policy prohibiting such provisions. Our recent Policy Statement simply articulates that, absent a clear congressional directive, the applicability of the Federal Arbitration Act was not overruled by the federal securities laws, and the existence of an arbitration provision is not grounds for denying effectiveness under Section 8(a) of the Securities Act.

This approach is consistent with judicial precedent and ensures that our rulebook is clear, transparent, objective, and predictable. To suggest otherwise is to mischaracterize both the law and the absence of any prior Commission’s policy.

During consideration of this Policy Statement at the open meeting, I specifically asked whether the Commission could adopt the opposite position. In other words, assume that the parade of horrible described by critics of mandatory arbitration provisions were to occur. Would the Commission have the legal authority to issue a policy statement specifically denying the acceleration of effectiveness of any registration statement if it disclosed the preference of a mandatory arbitration provision? The answer was essentially no — if the disclosure was adequate, then that would be essentially merit regulation, which was not authorized by the federal securities laws.

Finally, let me express my appreciation for the Committee’s recent work on artificial intelligence disclosures. This is a rapidly evolving area of corporate activity. As AI becomes more deeply integrated into business operations, the need for material information by investors may grow. The Committee has proposed that issuers:

1. Define what they mean by “artificial intelligence” in their disclosures;
2. Disclose board oversight mechanisms, if any, for AI deployment; and
3. Report separately on the material effects of AI on internal operations and consumer-facing matters.

I recognize that these recommendations are grounded in a materiality-based approach and are designed to fit within the existing disclosure framework under Regulation S-K. Furthermore, the Committee’s decision to build on existing disclosure items—rather than propose a standalone AI regime—is a pragmatic one that avoids unnecessary structural complexity.

That said, it is important to approach this area with caution. There are practical and conceptual challenges that merit further consideration. For example, the lack of a universally accepted definition of “artificial intelligence” could create interpretative and disclosure issues. Boards, management, and their outside counsel may struggle to determine what qualifies as AI, particularly when distinguishing between traditional automation and more advanced machine learning systems.

Similarly, while board oversight is a critical governance function, mandating disclosures in this area may not always yield meaningful insight for investors—especially if oversight responsibilities are diffuse or still evolving. And while reporting on the material effects of AI is a reasonable goal, it may be difficult in practice to isolate those effects separately from regular business operations.

Moreover, we must be mindful of the potential for regulatory overreach. Prematurely codifying rigid disclosure mandates could stifle innovation, particularly for smaller issuers that may lack the resources to implement complex compliance systems. A one-size-fits-all approach may not be appropriate in a space where use cases, risks, and maturity levels vary widely across industries.

As we consider the Committee’s recommendation, our goal should not be to use the federal securities laws as a backdoor attempt to regulate AI. Rather, we must ensure that investors are not left in the dark about material risks and opportunities that may arise from the use of AI in business operations and strategy and to do so in a manner without being encumbered by prescriptive or duplicative requirements.

The Committee provides a forum for this ongoing dialogue, and I look forward to continued engagement on this issue.

Total spending, updated annually as part of Volkswagen’s rolling five-year investment plan, compares with 165 billion euros for the 2025-2029 period and 180 billion for 2024-2028, with 2024 marking a peak.

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Blume told the weekly Frankfurter Allgemeine Sonntagszeitung that the focus in the latest spending plan was “on Germany and Europe,” including in products, technology and infrastructure. He said talks about an extended savings programme at Porsche would run into 2026.

PORSCHE NOT EXPECTED TO GROW IN CHINA, BLUME SAYS

“But it is true, of course, that shareholders have suffered losses since Porsche went public three years ago. I, too, must face up to this criticism.”

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– Data from pivotal studies of CASGEVY in children ages 5-11 years with severe sickle cell disease or transfusion-dependent beta thalassemia demonstrates the transformative potential of the therapy in younger patients –

– Efficacy and safety data in children 5-11 years are consistent with the durable and positive benefit/risk profile established from clinical studies in patients 12 years of age and older –

– Vertex expects to initiate global regulatory submissions for CASGEVY in children 5-11 years in 1H 2026 –

BOSTON–(BUSINESS WIRE)–Dec. 6, 2025–
Vertex Pharmaceuticals Incorporated (Nasdaq: VRTX) today announced data from multiple studies demonstrating the clinical benefits of CASGEVY^® (exagamglogene autotemcel) in people ages 5 years and older living with severe sickle cell disease (SCD) or transfusion-dependent beta thalassemia (TDT). The results, including the first presentation of clinical data from pivotal studies in children ages 5-11 years, and longer-term data from the pivotal studies of people with severe SCD and TDT ages 12 years and older, will be presented at the American Society of Hematology (ASH) Annual Meeting. CASGEVY is currently approved for eligible people ages 12 years and older with SCD or TDT in the United States, Great Britain, the European Union, the Kingdom of Saudi Arabia, the Kingdom of Bahrain, Kuwait, Qatar, Canada, Switzerland and the United Arab Emirates.

“These results — the first clinical data ever presented on any genetic therapy for children ages 5-11 years with SCD — again demonstrate the transformative potential of CASGEVY,” said Carmen Bozic, M.D., Executive Vice President, Global Medicines Development and Medical Affairs, and Chief Medical Officer at Vertex. “With dosing completed in the 5-11 age group and the Commissioner’s National Priority Voucher for CASGEVY in this population in hand, we are excited to begin global regulatory filings in the first half of next year and bring this potentially transformative therapy to eligible children as soon as possible.”

“As an investigator in the clinical program for patients 12 years and older and after having real-world experience with CASGEVY as an early commercial treatment center, I have seen firsthand the transformative impact this therapy has had on older patients with SCD or TDT. I am excited to hopefully be able to offer this option to my younger patients soon, early in life, before some of the most devastating impacts of these diseases begin,” said Haydar Frangoul, M.D., M.S., Medical Director of Pediatric Hematology and Oncology at Sarah Cannon Research Institute and HCA Healthcare’s TriStar Centennial Children’s Hospital, Member of Vertex’s SCD Program Steering Committee, and presenting author of the 5-11 years old CASGEVY data at ASH.

First presentation of data in children ages 5-11 years treated with CASGEVY

• In children with SCD, 11 patients have been dosed with CASGEVY in the Phase 3 CLIMB-151 clinical study, and all (4/4) patients with sufficient follow-up achieved the primary endpoint of being free from vaso-occlusive crises (VOCs) for at least 12 consecutive months (VF12).
  • No patient experienced a VOC following infusion with CASGEVY, with the longest duration of VOC-free of approximately two years (range 3.2–24.1 months).
• In children with TDT, 13 patients have been dosed with CASGEVY in the Phase 3 CLIMB-141 clinical study, and all (6/6) patients with sufficient follow-up achieved the primary endpoint of transfusion independence for at least 12 consecutive months while maintaining a weighted average hemoglobin (Hb) of at least 9 g/dL (TI12).
  • Following CASGEVY infusion, 12/13 are transfusion free, with the longest duration of transfusion free just under two years (range 2.3–22.5 months).
  • One patient died from pneumonia in the setting of multi-organ failure due to severe veno-occlusive disease related to the busulfan conditioning.
• The safety profile of CASGEVY in younger patients is consistent with myeloablative conditioning and autologous transplant in both SCD and TDT, as established in clinical studies in older patients.
• Consistent with studies in older patients, children treated with CASGEVY have durable increases in fetal hemoglobin (HbF) and stable allelic editing.

Longer-term data for people with SCD and TDT ages 12 years and older treated with CASGEVY

New longer-term data from the pivotal clinical studies of CASGEVY in people 12 years and older will also be presented at ASH. These data, as of April 2025, continue to demonstrate the transformative, durable clinical benefits that CASGEVY provides to people living with SCD or TDT. In SCD, 100% of patients (45/45) achieved VF12 in either CLIMB-121 or the long-term follow-up study CLIMB-131, with a mean duration of VOC-free for 35.3 months (range 12.9–67.7 months). In TDT, 98.2% (55/56) achieved TI12 in either CLIMB-111 or CLIMB-131 with a mean duration of transfusion independence of 41.4 months (range 13–72.3 months). The safety profile remained consistent with myeloablative conditioning and autologous transplant in both SCD and TDT.

About Sickle Cell Disease (SCD)

SCD is a debilitating, progressive and life-shortening disease. It is an inherited blood disorder that affects the red blood cells, which are essential for carrying oxygen to all organs and tissues of the body. SCD causes severe pain, organ damage and shortened life span due to misshapen or “sickled” red blood cells. The clinical hallmark of SCD is vaso-occlusive crises (VOCs), which are caused by blockages of blood vessels by sickled red blood cells and result in severe and debilitating pain that can happen anywhere in the body at any time. SCD requires a lifetime of treatment and results in a reduced life expectancy. In the U.S., the median age of death for patients living with SCD is approximately 45 years. SCD patients report health-related quality of life scores well below the general population, and the lifetime health care costs in the U.S. of managing SCD for patients with recurrent VOCs is estimated between $4 and $6 million.

About Transfusion-Dependent Beta Thalassemia (TDT)

TDT is a serious, life-threatening genetic disease. It requires frequent blood transfusions and iron chelation therapy throughout a person’s life. Due to anemia, patients living with TDT may experience fatigue and shortness of breath, and infants may develop failure to thrive, jaundice and feeding problems. Complications of TDT can also include an enlarged spleen, liver and/or heart, misshapen bones and delayed puberty. TDT requires lifelong treatment and significant use of health care resources, and ultimately results in reduced life expectancy, decreased quality of life and reduced lifetime earnings and productivity. In the U.S., the median age of death for patients living with TDT is 37 years. TDT patients report health-related quality of life scores below the general population and the lifetime health care costs in the U.S. of managing TDT are estimated between $5 and $5.7 million.

About CASGEVY^® (exagamglogene autotemcel)

CASGEVY is a non-viral, ex vivo CRISPR/Cas9 gene-edited cell therapy for eligible patients with SCD or TDT, in which a patient’s own hematopoietic stem and progenitor cells are edited at the erythroid specific enhancer region of the BCL11A gene through a precise double-strand break. This edit results in the production of high levels of fetal hemoglobin (HbF; hemoglobin F) in red blood cells. HbF is the form of the oxygen-carrying hemoglobin that is naturally present during fetal development, which then switches to the adult form of hemoglobin after birth. CASGEVY has been shown to reduce or eliminate VOCs for patients with SCD and transfusion requirements for patients with TDT.

The use of CASGEVY in children ages 5-11 years is investigational.

About the CLIMB Studies

The Phase 1/2/3 open-label studies, CLIMB-111 and CLIMB-121, are designed to assess the safety and efficacy of a single dose of CASGEVY in patients ages 12-35 years with TDT or with SCD and recurrent VOCs. Patients will be followed for approximately two years after CASGEVY infusion in these studies. CLIMB-141 and CLIMB-151 are ongoing Phase 3 open-label studies, designed to assess the safety and efficacy of a single dose of exagamglogene autotemcel in patients ages 2-11 years with TDT or with SCD and recurrent VOCs. Enrollment and dosing are complete for the 5-11-years-old cohort in both studies with the plan to extend to ages 2-4 years.

Each patient will be asked to participate in the ongoing long-term, open-label study, CLIMB-131. CLIMB-131 is designed to evaluate the long-term safety and efficacy of CASGEVY in patients with up to 15 years of follow up after CASGEVY infusion.

Next steps for CASGEVY in children ages 5-11 years

Enrollment and dosing are complete for the 5-11 years cohort in both studies. Vertex expects to initiate global regulatory filings for this age group, including a supplemental Biologics License Application (sBLA) in the U.S., in the first half of next year. Vertex recently received a Commissioner’s National Priority Voucher for CASGEVY in the 5-11 years age group from the U.S. Food and Drug Administration to accelerate the review of the sBLA once submitted. Products under the program will be subject to a 1–2-month review clock from the start of FDA’s review and will also benefit from enhanced communication opportunities with the agency.

U.S. INDICATIONS AND IMPORTANT SAFETY INFORMATION FOR CASGEVY

WHAT IS CASGEVY?

CASGEVY is a one-time therapy used to treat people ages 12 years and older with:

• sickle cell disease (SCD) who have frequent vaso-occlusive crises or VOCs
• beta thalassemia (β-thalassemia) who need regular blood transfusions

CASGEVY is made specifically for each patient, using the patient’s own edited blood stem cells, and increases the production of a special type of hemoglobin called hemoglobin F (fetal hemoglobin or HbF). Having more HbF increases overall hemoglobin levels and has been shown to improve the production and function of red blood cells. This can eliminate VOCs in people with sickle cell disease and eliminate the need for regular blood transfusions in people with beta thalassemia.

IMPORTANT SAFETY INFORMATION

What is the most important information I should know about CASGEVY?

After treatment with CASGEVY, you will have fewer blood cells for a while until CASGEVY takes hold (engrafts) into your bone marrow. This includes low levels of platelets (cells that usually help the blood to clot) and white blood cells (cells that usually fight infections). Your doctor will monitor this and give you treatment as required. The doctor will tell you when blood cell levels return to safe levels.

• Tell your healthcare provider right away if you experience any of the following, which could be signs of low levels of platelet cells:
  • severe headache
  • abnormal bruising
  • prolonged bleeding
  • bleeding without injury such as nosebleeds; bleeding from gums; blood in your urine, stool, or vomit; or coughing up blood

• Tell your healthcare provider right away if you experience any of the following, which could be signs of low levels of white blood cells:

You may experience side effects associated with other medicines administered as part of the treatment regimen for CASGEVY. Talk to your physician regarding those possible side effects. Your healthcare provider may give you other medicines to treat your side effects.

How will I receive CASGEVY?

Your healthcare provider will give you other medicines, including a conditioning medicine, as part of your treatment with CASGEVY. It’s important to talk to your healthcare provider about the risks and benefits of all medicines involved in your treatment.

After receiving the conditioning medicine, it may not be possible for you to become pregnant or father a child. You should discuss options for fertility preservation with your healthcare provider before treatment.

STEP 1: Before CASGEVY treatment, a doctor will give you mobilization medicine(s). This medicine moves blood stem cells from your bone marrow into the blood stream. The blood stem cells are then collected in a machine that separates the different blood cells (this is called apheresis). This entire process may happen more than once. Each time, it can take up to one week.

During this step rescue cells are also collected and stored at the hospital. These are your existing blood stem cells and are kept untreated just in case there is a problem in the treatment process. If CASGEVY cannot be given after the conditioning medicine, or if the modified blood stem cells do not take hold (engraft) in the body, these rescue cells will be given back to you. If you are given rescue cells, you will not have any treatment benefit from CASGEVY.

STEP 2: After they are collected, your blood stem cells will be sent to the manufacturing site where they are used to make CASGEVY. It may take up to 6 months from the time your cells are collected to manufacture and test CASGEVY before it is sent back to your healthcare provider.

STEP 3: Shortly before your stem cell transplant, your healthcare provider will give you a conditioning medicine for a few days in hospital. This will prepare you for treatment by clearing cells from the bone marrow, so they can be replaced with the modified cells in CASGEVY. After you are given this medicine, your blood cell levels will fall to very low levels. You will stay in the hospital for this step and remain in the hospital until after the infusion with CASGEVY.

STEP 4: One or more vials of CASGEVY will be given into a vein (intravenous infusion) over a short period of time.

After the CASGEVY infusion, you will stay in hospital so that your healthcare provider can closely monitor your recovery. This can take 4-6 weeks, but times can vary. Your healthcare provider will decide when you can go home.

What should I avoid after receiving CASGEVY?

• Do not donate blood, organs, tissues, or cells at any time in the future

What are the possible or reasonably likely side effects of CASGEVY?

The most common side effects of CASGEVY include:

• Low levels of platelet cells, which may reduce the ability of blood to clot and may cause bleeding
• Low levels of white blood cells, which may make you more susceptible to infection

Your healthcare provider will test your blood to check for low levels of blood cells (including platelets and white blood cells). Tell your healthcare provider right away if you get any of the following symptoms:

• fever
• chills
• infections
• severe headache
• abnormal bruising
• prolonged bleeding
• bleeding without injury such as nosebleeds; bleeding from gums; blood in your urine, stool, or vomit; or coughing up blood

These are not all the possible side effects of CASGEVY. Call your doctor for medical advice about side effects. You may report side effects to FDA at 1-800-FDA-1088.

General information about the safe and effective use of CASGEVY

Talk to your healthcare provider about any health concerns.

Please see full Prescribing Information including Patient Information for CASGEVY.

About Vertex

Vertex is a global biotechnology company that invests in scientific innovation to create transformative medicines for people with serious diseases and conditions. The company has approved therapies for cystic fibrosis, sickle cell disease, transfusion-dependent beta thalassemia and acute pain, and it continues to advance clinical and research programs in these areas. Vertex also has a robust clinical pipeline of investigational therapies across a range of modalities in other serious diseases where it has deep insight into causal human biology, including neuropathic pain, APOL1-mediated kidney disease, IgA nephropathy, primary membranous nephropathy, autosomal dominant polycystic kidney disease, type 1 diabetes and myotonic dystrophy type 1.

Vertex was founded in 1989 and has its global headquarters in Boston, with international headquarters in London. Additionally, the company has research and development sites and commercial offices in North America, Europe, Australia, Latin America and the Middle East. Vertex is consistently recognized as one of the industry’s top places to work, including 16 consecutive years on Science magazine’s Top Employers list and one of Fortune’s 100 Best Companies to Work For. For company updates and to learn more about Vertex’s history of innovation, visit www.vrtx.com or follow us on LinkedIn, Facebook, Instagram, YouTube and X.

Vertex Special Note Regarding Forward-Looking Statements

This press release contains forward-looking statements as defined in the Private Securities Litigation Reform Act of 1995, as amended, including, without limitation, statements made by Carmen Bozic, M.D., and Haydar Frangoul, M.D., M.S., and statements regarding expectations for the clinical benefits of CASGEVY, plans to initiate global regulatory submissions for children 5-11, including in the U.S., in the first half of 2026, expectations that the use of a Priority Voucher will accelerate the review of the sBLA, expectations for the design of the CLIMB studies, including plans to follow patients after infusion, expectations that each patient will be asked to participate in the CLIMB-131 study and expectations that the studies will be extended to children 2-4 years of age. While Vertex believes the forward-looking statements contained in this press release are accurate, these forward-looking statements represent the company’s beliefs only as of the date of this press release and there are a number of risks and uncertainties that could cause actual events or results to differ materially from those expressed or implied by such forward-looking statements. Those risks and uncertainties include, among other things, that data from the company’s research and development programs may not support registration or further development of its potential medicines in a timely manner, or at all, due to safety, efficacy or other reasons, that the company may be unable to make the anticipated regulatory submissions on the expected timeline, or at all, and other risks listed under the heading “Risk Factors” in Vertex‘s most recent annual report and subsequent quarterly reports filed with the Securities and Exchange Commission at www.sec.gov and available through the company’s website at www.vrtx.com. You should not place undue reliance on these statements, or the scientific data presented. Vertex disclaims any obligation to update the information contained in this press release as new information becomes available.

(VRTX-GEN)

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Source: Vertex Pharmaceuticals Incorporated