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A facial and rapid method for glycoproteins enrichment by capillary electrophoresis was developed. The 3-aminophenylboronic acid-functionalized poly(glycidyl methacrylate) microparticles (PGMA@APBA) were attached to the capillary inlet (length of â¼1.5 cm) by electrostatic self-assemble action to prepare a partially coated capillary column. The process is simple and reversible, allowing for easy renewal of the PGMA@APBA coating when its enrichment efficiency decreases. By utilizing the coated column, glycoproteins can be enriched within 2 min. The column exhibits a specific enrichment for glycoproteins and can be consecutively used for approximately 60 runs. The relative standard deviations (RSDs) of peak area of run-to-run (n = 5) and batch-to-batch (n = 3) were 1.5 % and 1.0%, respectively. The method was successfully applied to enrich glycoproteins from 1 × 1012-fold diluted real egg white sample, indicating its practical applicability.
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Electroforesis Capilar , Compuestos Epoxi , Metacrilatos , Ácidos Polimetacrílicos , GlicoproteínasRESUMEN
Orientationally immobilized enzyme microreactors (OIMERs), embedded in microfluidic paper-based analytical devices (µPADs) were developed for improved detection of pesticide residues in food. Acetylcholinesterase (AChE) was orientationally immobilized on the reusable Part I of the µPADs, using the specific affinity binding of concanavalin A (Con A) to a glycosyl group on AChE. Using the disposable Part II, facile colorimetric quantification was performed with a smartphone and software, or qualitative detection by a naked-eye visual test. The AChE immobilized in OIMERs not only had improved activity and stability, but also high sensitivity, with a limit of detection as low as (0.007 ± 0.003) µg/mL. The method was used to detect pesticides residues in real vegetable samples; the recovery (88.6-102.7%) showed high reliability for pesticide residues detection in foods. A molecular docking study and an enzyme kinetic analysis were conducted to characterize the mechanism of action of the OIMERs.
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Técnicas Analíticas Microfluídicas , Residuos de Plaguicidas , Residuos de Plaguicidas/análisis , Enzimas Inmovilizadas/química , Microfluídica , Acetilcolinesterasa/metabolismo , Reproducibilidad de los Resultados , Cinética , Simulación del Acoplamiento Molecular , Dispositivos Laboratorio en un Chip , PapelRESUMEN
Stem cells are known for their resilience and enhanced activity post-stress. The mammary gland undergoes frequent remodeling and is subjected to recurring stress during the estrus cycle, but it remains unclear how mammary stem cells (MaSCs) respond to the stress and contribute to regeneration. We discovered that cytotoxic stress-induced activation of CD11c+ ductal macrophages aids stem cell survival and prevents differentiation. These macrophages boost Procr+ MaSC activity through IL1ß-IL1R1-NF-κB signaling during the estrus cycle in an oscillating manner. Deleting IL1R1 in MaSCs results in stem cell loss and skewed luminal differentiation. Moreover, under cytotoxic stress from the chemotherapy agent paclitaxel, ductal macrophages secrete higher IL1ß levels, promoting MaSC survival and preventing differentiation. Inhibiting IL1R1 sensitizes MaSCs to paclitaxel. Our findings reveal a recurring inflammatory process that regulates regeneration, providing insights into stress-induced inflammation and its impact on stem cell survival, potentially affecting cancer therapy efficacy.
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Glándulas Mamarias Animales , Células Madre , Femenino , Animales , Diferenciación Celular/fisiología , Transducción de Señal , Paclitaxel/farmacología , Paclitaxel/metabolismoRESUMEN
Dynamic regulation of integrin activation and inactivation is critical for precisely controlled cell adhesion and migration in physiological and pathological processes. The molecular basis for integrin activation has been intensively studied; however, the understanding of integrin inactivation is still limited. Here, we identify LRP12 as an endogenous transmembrane inhibitor for α4 integrin activation. The LRP12 cytoplasmic domain directly binds to the integrin α4 cytoplasmic tail and inhibits talin binding to the ß subunit, thus keeping integrin inactive. In migrating cells, LRP12-α4 interaction induces nascent adhesion (NA) turnover at the leading-edge protrusion. Knockdown of LRP12 leads to increased NAs and enhanced cell migration. Consistently, LRP12-deficient T cells show an enhanced homing capability in mice and lead to aggravated chronic colitis in a T cell-transfer colitis model. Altogether, LRP12 is a transmembrane inactivator for integrins that inhibits α4 integrin activation and controls cell migration by maintaining balanced NA dynamics.
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Integrina alfa4 , Integrinas , Proteínas Relacionadas con Receptor de LDL , Animales , Cricetinae , Ratones , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células CHO , Integrina alfa4/metabolismo , Integrinas/metabolismo , Unión Proteica , Humanos , Proteínas Relacionadas con Receptor de LDL/metabolismoRESUMEN
Riblet and superhydrophobic surfaces are two typical passive control technologies used to save energy. In this study, three microstructured samples-a micro-riblet surface (RS), a superhydrophobic surface (SHS), and a novel composite surface of micro-riblets with superhydrophobicity (RSHS)-were designed to improve the drag reduction rate of water flows. Aspects of the flow fields of microstructured samples, including the average velocity, turbulence intensity, and coherent structures of water flows, were investigated via particle image velocimetry (PIV) technology. A two-point spatial correlation analysis was used to explore the influence of the microstructured surfaces on coherent structures of water flows. Our results showed that the velocity on microstructured surface samples was higher than that on the smooth surface (SS) samples, and the turbulence intensity of water on the microstructured surface samples decreased compared with that on the SS samples. The coherent structures of the water flow on microstructured samples were restricted by length and structural angles. The drag reduction rates of the SHS, RS, and RSHS samples were -8.37 %, -9.67 %, and -17.39 %, respectively. The novel established RSHS demonstrated a superior drag reduction effect and could improve the drag reduction rate of water flows.
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Revealing the turbulent drag reduction mechanism of water flow on microstructured surfaces is beneficial to controlling and using this technology to reduce turbulence losses and save energy during water transportation. Two microstructured samples, including a superhydrophobic and a riblet surface, were fabricated near which the water flow velocity, and the Reynolds shear stress and vortex distribution were investigated using a particle image velocimetry. The dimensionless velocity was introduced to simplify the Ω vortex method. The definition of vortex density in water flow was proposed to quantify the distribution of different strength vortices. Results showed that the velocity of the superhydrophobic surface (SHS) was higher compared with the riblet surface (RS), while the Reynolds shear stress was small. The vortices on microstructured surfaces were weakened within 0.2 times that of water depth when identified by the improved ΩM method. Meanwhile, the vortex density of weak vortices on microstructured surfaces increased, while the vortex density of strong vortices decreased, proving that the reduction mechanism of turbulence resistance on microstructured surfaces was to suppress the development of vortices. When the Reynolds number ranged from 85,900 to 137,440, the drag reduction impact of the superhydrophobic surface was the best, and the drag reduction rate was 9.48%. The reduction mechanism of turbulence resistance on microstructured surfaces was revealed from a novel perspective of vortex distributions and densities. Research on the structure of water flow near the microstructured surface can promote the drag reduction application in the water field.
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A facial and efficient method for the screening of acetylcholinesterase (AChE) inhibitors by capillary electrophoresis was developed. Based on the specific affinity of concanavalin A (Con A) for binding to the glycosyl group of AChE, enzyme molecules were oriented-immobilized on the surface of gold nanoparticles (AuNPs@Con A@AChE). Then, these modified nanoparticles were bounded to the capillary inlet (about 1.0 cm) by electrostatic self-assembly to obtain the oriented-immobilized enzyme microreactor (OIMER). Compared to an IMER with a free enzyme, the peak area of the product obtained by the OIMER increased by 52.6%. The Michaelis-Menten constant (Km) was as low as (0.061 ± 0.003) mmol/L. The method exhibits good repeatability with a relative standard deviation (RSD) of 1.3% for 100 consecutive runs. The system was successfully applied to detect the IC50 values of donepezil and four components from Chinese medicinal plants. This work demonstrates the potential of this method as a low cost, simple, and accurate screening method for other enzyme inhibitors.
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Inhibidores de la Colinesterasa , Nanopartículas del Metal , Inhibidores de la Colinesterasa/farmacología , Enzimas Inmovilizadas , Acetilcolinesterasa , Oro , Electroforesis Capilar , Concanavalina ARESUMEN
Tumor metastasis is the leading cause of cancer-associated mortality. Unfortunately, the underlying mechanism of metastasis is poorly understood. Expression of legumain (LGMN), an endo-lysosomal cysteine protease, positively correlates with breast cancer metastatic progression and poor prognosis. Here, we report that LGMN is secreted in the zymogen form by motile breast cancer cells. Through binding to cell surface integrin αvß3 via an RGD motif, the autocrine pro-LGMN activates FAK-Src-RhoA signaling in cancer cells and promotes cancer cell migration and invasion independent of LGMN protease activity. Either silencing LGMN expression or mutationally abolishing pro-LGMNâαvß3 interaction significantly inhibits cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Finally, we developed a monoclonal antibody against LGMN RGD motif, which blocks pro-LGMNâαvß3 binding, and effectively suppresses cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Thus, disruption of pro-LGMNâintegrin αvß3 interaction may be a potentially promising strategy for treating breast cancer metastasis.
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Neoplasias de la Mama , Integrina alfaVbeta3 , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Cisteína Endopeptidasas , Femenino , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Metástasis de la Neoplasia , OligopéptidosRESUMEN
A hydroxypropyl-ß-cyclodextrin (HP-ß-CD) imprinted coating based on polyhedral oligomeric silsesquioxane (POSS) for open tubular electrochromatography was prepared. The mixture of methacryl-POSS (MA0735), HP-ß-CD (template), methacrylic acid (MAA, monomer), N,N'-methylenebisacrylamide (MBA, crosslinker) and toluene-dimethyl sulfoxide (porogen) was used to synthesize the chiral selective coating. The influence of synthesis parameters on the imprinting effect and separation performance, including the amount of HP-ß-CD, POSS, and MAA, was investigated systemically. The optimum polymerization was prepared by mixing HP-ß-CD, MA0735, MAA, and MBA with the molar ratio of 1 : 1.87 : 1.60 : 1.60. Five racemates were separated by the modified capillary columns using aqueous buffer. Column efficiency on the POSS-based MIPs coating column was greater than 22 000 plates/m. MIPs-POSS hybrid coating capillaries had improved resolution (3.36 times) and the greatest resolution was up to 6.15 within 10 min.
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Insulin production is required for glucose homeostasis. Pancreatic islet ß cells are the only cells that produce insulin in humans; however, generation of functional ß cells in vitro from embryonic or adult tissues has been challenging. Here, we describe isolation of pancreatic islet progenitors from adult mice, which enables the efficient generation and long-term expansion of functional islet organoids in vitro. This protocol starts with purification of protein C receptor (Procr)-expressing islet progenitors. Coculture with endothelial cells generates islet organoids in vitro that can be expanded by passage. Functional maturation is achieved as a consequence of a prolonged culture period and cyclic glucose stimulation. Primary islet organoids form in 7-10 days. Subsequently, each passage takes 1 week, with the final maturation step requiring 3 weeks of additional culture. The resulting organoids are predominantly composed of ß cells but also contain small proportions of α, δ and pancreatic polypeptide cells. The organoids sense glucose and secrete insulin. This approach thus provides a strategy for ß cell generation in vitro and an organoid system to study islet regeneration and diseases.
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Islotes Pancreáticos , Organoides , Animales , Células Endoteliales/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , RatonesRESUMEN
The protein C receptor (Procr) has been implicated as a stem cell surface marker in several tissues. It is unknown whether Procr acts as a functional signaling receptor in stem cells. Here, by conditional knockout in mammary stem cells (MaSCs), we demonstrate that Procr is essential for mammary gland development and homeostasis. Through proteomics profiling, we identify that, upon stimulation by the ligand protein C, Procr interacts with heat shock protein 90 (HSP90AA1) via its short cytoplasmic tail, recruiting Src and IGF1R to the complex at the plasma membrane. We show that Procr acts as a signaling receptor of protein C in regulation of MaSCs through HSP90, Src, and IGF1R in vitro. In vivo, IGF1R deletion in MaSCs displays similar phenotypes to Procr deletion. These findings illustrate the essential role of Procr signaling in MaSC maintenance, shedding light onto the molecular regulation by Procr in tissue stem cells.
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Proteína C , Células Madre , Animales , Receptor de Proteína C Endotelial/genética , Receptor de Proteína C Endotelial/metabolismo , Glándulas Mamarias Animales/metabolismo , Proteína C/metabolismo , Transducción de Señal , Células Madre/metabolismoRESUMEN
Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and OSE stem cells rapidly generate new cells for the repair. How the stem cell activation is triggered by the rupture and promptly turns on proliferation is unclear. Our previous study has identified that Protein C Receptor (Procr) marks OSE progenitors. In this study, we observed decreased adherent junction and selective activation of YAP signaling in Procr progenitors at OSE rupture site. OSE repair is impeded upon deletion of Yap1 in these progenitors. Interestingly, Procr+ progenitors show lower expression of Vgll4, an antagonist of YAP signaling. Overexpression of Vgll4 in Procr+ cells hampers OSE repair and progenitor proliferation, indicating that selective low Vgll4 expression in Procr+ progenitors is critical for OSE repair. In addition, YAP activation promotes transcription of the OSE stemness gene Procr. The combination of increased cell division and Procr expression leads to expansion of Procr+ progenitors surrounding the rupture site. These results illustrate a YAP-dependent mechanism by which the stem/progenitor cells recognize the murine ovulatory rupture, and rapidly multiply their numbers, highlighting a YAP-induced stem cell expansion strategy.
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Células Epiteliales , Ovario , Animales , Receptor de Proteína C Endotelial/genética , Células Epiteliales/fisiología , Epitelio/metabolismo , Femenino , Ratones , Ovario/metabolismo , Células Madre/metabolismo , Proteínas Señalizadoras YAPRESUMEN
This publisher's note contains corrections to Opt. Lett.46, 685 (2021)OPLEDP0146-959210.1364/OL.415087.
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This Letter proposes a novel phase-sensitive optical time domain reflectometry (Φ-OTDR) with continuous chirped-wave (CCW), which can make full use of both time and frequency domain resources. The principle and benefits of CCW Φ-OTDR are elaborated. With the merit of CCW Φ-OTDR, 1.042 MHz sensing bandwidth and 5pε/Hz strain sensitivity are achieved along a 1013 m fiber with 4.4 m spatial resolution. To the best of the authors' knowledge, this is the first time that a Φ-OTDR achieves megahertz sensing bandwidth with metric spatial resolution, and without limiting the frequency feature of the disturbance. The good performance in long-range sensing is also verified over a 49.7 km fiber. More than that, the digital domain flexibility of the proposed scheme can be used to optimize the measured acoustic signal according to its feature and the practical needs.
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Quasi-distributed acoustic sensing (Q-DAS) based on ultra-weak fiber Bragg grating (UWFBG) is currently attracting great attention, due to its high sensitivity and excellent multiplexing capability. Phase-sensitive optical time-domain reflectometry (Φ-OTDR) based on phase demodulation is one of the most promising interrogation schemes for Q-DAS. In this article, a novel interleaved identical chirped pulse (IICP) approach is proposed on the basis of pulse compression Φ-OTDR with coherent detection. Different from the frequency-division-multiplexing (FDM) method, the identical pulses are used for multiplexing in the IICP scheme, and the mixed reflection signals can be demodulated directly, so the inconsistent phase offsets in FDM can be avoided. As a result, this scheme can enlarge the measurement slew-rate (SR) of Q-DAS by times compared with traditional single pulse scheme. In the proof-of-principle experiment, the SR of 28.9 mÉ/s has been achieved with an 860 m sensing range, which is 5 times as that of the traditional single pulse scheme; meanwhile, the response bandwidth has been enlarged by 5 times. The 277 kHz response bandwidth has been achieved, with 5 m spatial resolution and 2.8 pε/Hz strain sensitivity.
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A method is described for cysteine (Cys) determination on paper-based analytical devices using aspartic acid modified gold nanoparticles (Asp-AuNPs). The Asp-AuNPs were characterized by their size, zeta potential, and UV-visible absorption spectrum. After the addition of Cys, it will interact with Asp-AuNPs selectively and leads to the aggregation of Asp-AuNPs. A color change from red to blue can be observed on the paper-based analytical devices. The results were recorded using a cell phone and subsequently analyzed using the Photoshop software. The ratiometric color intensity at red channel and blue channel (Red/Blue) increased linearly in the range 99.9-998.7 µM for Cys (R = 0.9984), and the limit of detection was 1.0 µM. The effects of assay conditions have been investigated and are discussed. The Cys concentration was determined as (0.27 ± 0.02 mM) in human plasma, and the recovery was from 99.2 to 101.1%. Graphical abstract Schematic representation of the paper-based assay system using aspartic acid modified gold nanoparticles (Asp-AuNPs). The ratiometric color intensity method was used for the cysteine (Cys) determination.
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Ácido Aspártico/química , Colorimetría/métodos , Cisteína/sangre , Nanopartículas del Metal/química , Papel , Secuencia de Carbohidratos , Teléfono Celular , Colorimetría/instrumentación , Oro/química , Humanos , Límite de Detección , Programas InformáticosRESUMEN
From small unicellular organisms to large mammals, swallowing is an important way for them to interact with their external environment. The majority of these animals swallow their targets for the purpose of hunting, and some fish and amphibians protect their cubs from external injury by swallowing them. Thus, swallowing can produce an efficient capture, keep the integrity of targets, and provide effective protection for swallowed objects. Inspired by this, we propose a novel soft swallowing robot (SSR) capable of swallowing various objects that have different shapes and stiffnesses, protecting objects from squeeze and collision, and withstanding high temperature, which are enabled by a compliant guiding structure consisting of a double thin-walled capsule filled with fluid and a linearly movable traction body. In this article, we study the SSR supported by air and water, respectively; furthermore, we experimentally conclude that the working medium has a great influence on the inherent characteristics of the SSR. Our study helps lay the foundation for the research of soft robotic systems with swallowing characteristics, and the SSR is expected to enter the practical application field from the laboratory.
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Robótica , Animales , Deglución , MamíferosRESUMEN
A fluorescent probe for the colorimetric detection of cysteine (Cys) was synthesized by combining resorufin with 7-nitrobenzofurazan. The resultant probe was characterized by FT-IR spectroscopy, fluorescence detection, mass spectrometry and NMR spectroscopy. After reacting with Cys, resorufin is released and the color changes from light yellow to red on paper-based analytical devices. The results were recorded using a common cell phone and subsequently analyzed using Photoshop software. The color intensity of the RGB channel increased linearly in the 0.04 to 70.04 µM Cys concentration range when the probe concentration was 2.6 mM (R = 0.9965), and the limit of detection was 16 nM. The effects of detection conditions have been investigated and are discussed. The interference deviations of 13 substances to Cys were in the range of -2% to +2%. The Cys concentration was determined as 270.8 ± 19.3 µM in human plasma, and the recovery was from 99.7% to 100.4%. The work demonstrates the potential of the method to detect Cys in real samples with low cost and high sensitivity.
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Alzheimer's disease (AD) is the most common cause of dementia in elderly individuals. Currently, acetylcholinesterase inhibitors (AChEI) are the most effective clinical treatment for AD. AChEIs in natural products may have therapeutic potential and should be screened for use in AD treatment. The authors describe a simple and reliable method for AChEI screening by capillary electrophoresis (CE). A hexadimethrine bromide (HDB) solution was pushed into a capillary (0.015 MPa×10 s) and incubated for 5 min. The capillary was flushed with deionized water for 5 min to remove free HDB, followed by plugging with an acetylcholinesterase (AChE) solution. After a 5 min incubation, the AChE was immobilized on the positively charged coating by ion binding, and the micro-reactor was created. The substrate solution, acetylthiocholine iodide (AThC), was injected into the capillary and incubated in the micro-reactor for 1 min. The unreacted substrate and the enzymolysis product were separated by CE. Gastrodin, an important component of Gastrodia elata, can inhibit AChE activity. After a certain amount of gastrodin was spiked into the substance solution, the peak area of the product decreased. Greater peak area reduction indicated stronger inhibition of AChEI. We observed good reproducibility of the product peak, with relative standard deviation (RSD) values less than 5.3%. The micro-reactor can be reused up to 300 times, which greatly improves efficiency. When the concentration of gastrodin was 5.24 µmol/L, the inhibition rate of AChE reached 64.8%. The IC50 of gastrodin was (2.26±0.14) µmol/L (R2=0.9983), which was consistent with the result of traditional UV method (2.09±0.18 µmol/L). If the function of the micro-reactor deteriorates, it can be conveniently renewed by flushing the column to remove the enzyme and repeating the AChE immobilization protocol. The proposed method is simple, efficient, and low cost, and can be used to screen AChEI from natural products, thus contributing to the improvement of AD treatment.
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Acetilcolinesterasa , Alcoholes Bencílicos/farmacología , Inhibidores de la Colinesterasa/farmacología , Electroforesis Capilar , Enzimas Inmovilizadas , Glucósidos/farmacología , Reproducibilidad de los ResultadosRESUMEN
Specific recognition of caffeic acid (CA) from Taraxacum mon-golicum Hand.-Mazz. was successfully performed using a new pH responsive magnetic molecularly imprinted polymers (pH-MMIPs) by simple surface molecular imprinting polymerization. The pH-MMIPs were prepared on the surface of the Fe3O4@SiO2@MPS particles using CA as a template, 2-(dimethylamino) ethyl methacrylate (DMA) as the pH responsive functional monomer, 4-vinylpyridine (4-VP) as an assisting functional monomer, ethylene glycol dimethyl acrylate (EGDMA) as cross-linker, 2,2'-azobisisobutyronitrile (AIBN) as initiator and methanol-H2O (1:1, v/v) as the porogen. The resultant polymers were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), vibrating sample magnetometry (VSM) and x-ray diffraction (XRD). The adsorption experiments revealed that the pH-MMIPs performed high adsorption ability (11.5â¯mgâ¯g-1)â¯by changing solution pH. Successful selective adsorption of CA was achieved with distribution coefficient of 0.12 and 0.21 towards ferulic acid and chlorogenic acid. Furthermore, pH-MMIPs were employed as adsorbents for extraction and enrichment of CA from Taraxacum mon-golicum Hand.-Mazz. extract. The recoveries of CA in the Taraxacum mon-golicum Hand.-Mazz. ranged from 90.47% to 98.97%. The results proved that the polymers have the potential to provide a selective recognition of CA in complex samples by simple pH regulation.