High Specific Recognition Research Articles

Molecularly Imprinted Polymer Nanospheres with Hydrophilic Shells for Efficient Molecular Recognition of Heterocyclic Aromatic Amines in Aqueous Solution.

Heterocyclic aromatic amine molecularly imprinted polymer nanospheres with surface-bound dithioester groups (haa-MIP) were firstly synthesized via reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization. Then, a series of core-shell structural heterocyclic aromatic amine molecularly imprinted polymer nanospheres with hydrophilic shells (MIP-HSs) were subsequently prepared by grafting the hydrophilic shells on the surface of haa-MIP via on-particle RAFT polymerization of 2-hydroxyethyl methacrylate (HEMA), itaconic acid (IA), and diethylaminoethyl methacrylate (DEAEMA). The haa-MIP nanospheres showed high affinity and specific recognition toward harmine and its structural analogs in organic solution of acetonitrile, but lost the specific binding ability in aqueous solution. However, after the grafting of the hydrophilic shells on the haa-MIP particles, the surface hydrophilicity and water dispersion stability of the polymer particles of MIP-HSs greatly improved. The binding of harmine by MIP-HSs with hydrophilic shells in aqueous solutions is about two times higher than that of NIP-HSs, showing an efficient molecular recognition of heterocyclic aromatic amines in aqueous solution. The effect of hydrophilic shell structure on the molecular recognition property of MIP-HSs was further compared. MIP-PIA with carboxyl groups containing hydrophilic shells showed the highest selective molecular recognition ability to heterocyclic aromatic amines in aqueous solution.

Bimetallic MOF synergy molecularly imprinted ratiometric electrochemical sensor based on MXene decorated with polythionine for ultra-sensitive sensing of catechol

Dual-signal ratiometric molecularly imprinted polymer (MIP) electrochemical sensors with bimetallic active sites and high-efficiency catalytic activity were fabricated for the sensing of catechol (CC) with high selectivity and sensitivity. The amino-functionalization bimetallic organic framework materials (Fe@Ti-MOF-NH2), coupled with two-dimensional layered titanium carbide (MXene) co-modified glassy carbon electrode provides an expanded surface while amplifying the output signal through the electropolymerization immobilization of polythionine (pTHi) and MIP. The oxidation of CC and pTHi were presented as the response signal and the internal reference signal. The oxidation peak current at +0.42 V rose with increased concentration of CC, while the peak currents of pTHi at −0.20 V remained constant. Compared to the common single-signal sensing system, this one (MIP/pTHi/MXene/Fe@Ti-MOF-NH2/GCE), a novel ratiometric MIP electrochemical sensor exhibited two segments wide dynamic range of 1.0–300 μM (R2 = 0.9924) and 300–4000 μM (R2 = 0.9912), as well as an ultralow detection limit of 0.54 μM (S/N = 3). Due to the specific recognition function of MIPs and the advantages of built-in correction of pTHi, the prepared surface imprinting sensor presented an excellent performance in selectivity and reproducibility. Besides, this sensor possessed superior anti-interference ability with ions and biomolecules, excellent reproducibility, repeatability, and acceptable stability. Furthermore, the proposed sensing system exhibits high specific recognition in the determination of environmental matrices and biological fluids in real samples with satisfactory results. Therefore, this signal-enhanced ratiometric MIP electrochemical sensing strategy can accurately and selectively analyze and detect other substances.

Selective Adsorption of Quercetin by the Sol-Gel Surface Molecularly Imprinted Polymer.

Quercetin, as one of the most biologically active natural flavonoids, is widely found in various vegetables, fruits and Chinese herbs. In this work, molecularly imprinted polymer (MIP) was synthesized through surface molecular imprinting technology with sol-gel polymerization mechanism on SiO2 at room temperature using quercetin as the template, SiO2 as the supporter, 3-aminopropyltriethoxysilane (APTES) as the functional monomer, and tetraethoxysilane (TEOS) as the cross-linker. The prepared MIP was characterized via scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR) and nitrogen adsorption measurements to validate its surface morphology, structure and functionality. SEM images revealed that the morphology of MIP was rough and spherical with the particle size of 260 nm larger than that of the support SiO2. In the FTIR spectra of MIP, the band around 1499 cm-1 and 2932 cm-1 were assigned to N-H and C-H groups, respectively. The results indicated that the imprinted polymer layers were grafted on the surface of SiO2 and the MIP had been successfully prepared. Since the specific surface area and pore volume of MIP were markedly higher than those of NIP and SiO2 and were 52.10 m2 g-1 and 0.150 cm3 g-1, respectively, it was evident that the imprinting process created corresponding imprinted cavities and porosity. The MIP for adsorbing quercetin was evaluated by static adsorption experiment. The results indicated that the adsorption equilibrium could be reached within 90 min and the maximum adsorption capacity was as high as 35.70 mg/g. The mechanism for adsorption kinetics and isotherm of MIP for quercetin was proved to conform the pseudo-second-order kinetics model (R2 = 0.9930) and the Freundlich isotherm model (R2 = 0.9999), respectively, revealing that chemical adsorption and heterogeneous surface with multilayer adsorption dominated. In contrast to non-imprinted polymer (NIP), the MIP demonstrated high selectivity and specific recognition towards quercetin whose selectivity coefficients for quercetin relative to biochanin A were 1.61. Furthermore, the adsorption capacity of MIP can be maintaining above 90% after five regeneration cycles, indicating brilliant reusability and potential application for selective adsorption of quercetin.

Synthesis of dummy-template molecularly imprinted polymers as solid-phase extraction adsorbents for N-nitrosamines in meat products

In this study, the dummy template molecularly imprinted polymers were firstly prepared by precipitation polymerization method using benzhydrol, 5-nonanol and N-formylpyrrolidine as template molecules and methacrylic acid as functional monomer. The obtained polymers were characterized and showed spherical particles with loose and porous surface. The dynamic and static adsorption experiments indicated that the polymers exhibited large adsorption capacity, high adsorption efficiency and specific recognition ability towards N-nitrosamines. The parameters of solid phase extraction process were carefully optimized. Under the optimized conditions, the solid phase extraction process based on dummy molecularly imprinted polymers coupled with high performance liquid chromatography-tandem mass spectrometry for analyzing N-nitrosamine in various processed meat products was established. This proposed method showed good linearity (R2 ≥ 0.9985), and the limits of detection and the limits of quantification were in the range of 0.03–0.65 ng/g and 0.1–1.95 ng/g, respectively. With three spiked concentrations (5, 10, and 20 ng/g), satisfactory recoveries (61.0–105.2 %) were also obtained with relative standard deviations between 1.20 and 9.76 %. Those results demonstrated that the method for N-nitrosamine possessed the advantages of high selectivity, good reproducibility and high sensitivity, providing great convenient method for enriching and detecting N-nitrosamine in complex food matrix.

Development and characterization of DNA aptamer against Retinoblastoma by Cell-SELEX

Retinoblastoma (RB) is the most common paediatric intraocular tumour. The management of RB has improved the survival and vision with recent advances in the treatment. Improved therapeutic approaches focussing on targeting tumours and minimizing the treatment-associated side effects are being developed. In this study, we generated a ssDNA aptamer against RB by cell-SELEX and high-throughput sequencing using Weri-RB1 cell line as the target, and Muller glial cell line Mio-M1 as the control. Three aptamers were selected based on the number of repetitions in NGS and phylogenetic relationship and evaluated by flow cytometry to assess their binding affinity and selectivity. The dissociation constant, Kd values of three selected aptamers were found to be in the nanomolar range. Aptamer VRF-CSRB-01 with the best binding affinity and a Kd value of 49.41 ± 7.87 nM was further characterized. The proteinase and temperature treatment indicated that VRF-CSRB-01 targets surface proteins, and has a good binding affinity and excellent selectivity under physiological conditions. The aptamer VRF-CSRB-01 was stable over 72 h in serum and 96 h in cerebral spinal fluid and vitreous. With the high affinity, specificity, stability and specific recognition of clinical RB tumours, VRF-CSRB-01 aptamer holds potential for application in diagnosis and targeting RB.

Probing Queuosine Modifications of Transfer RNA in Single Living Cells via Plasmonic Affinity Sandwich Assay.

Queuosine (Q) modification on tRNA plays an essential role in protein synthesis, participating in many tRNA functions such as folding, stability, and decoding. Appropriate analytical tools for the measurement of tRNA Q modifications are essential for the exploration of new roles of Q-modified tRNAs and the rationalization of their exact mechanisms. However, conventional methods for Q modification analysis suffer from apparent disadvantages, such as destructive cells, tedious procedure, and low sensitivity, which much hamper in-depth studies of Q modification-related biological questions. In this study, we developed a new approach called plasmonic affinity sandwich assay that allows for facile and sensitive determination of Q-modified tRNAs in single living cells. This method relies on the combination of plasmon-enhanced Raman scattering detection, base-paring affinity in-cell microextraction, and a set of boronate affinity and molecularly imprinted labeling nanotags for selective recognition of individual Q modifications, including queuosine, galactosyl queuosine (Gal-Q), and mannosyl queuosine (Man-Q). The developed method exhibited high affinity extraction and high specificity recognition. It allowed for the measurement of tRNA Q modifications in not only Q-rich cultured tumor cells but also Q-deficient primary tumor cells. Usefulness of this approach for investigation of the change of the Q modification level in single cells under oxidative stress was demonstrated. Because of its significant advantages over conventional methods, this approach provides a promising analytical tool for the exploration of more roles of Q-modified tRNAs and elucidation of their mechanisms.

Recent Trends in the Development of Carbon-Based Electrodes Modified with Molecularly Imprinted Polymers for Antibiotic Electroanalysis

Antibiotics are antibacterial agents applied in human and veterinary medicine. They are also employed to stimulate the growth of food-producing animals. Despite their benefits, the uncontrolled use of antibiotics results in serious problems, and therefore their concentration levels in different foods as well as in environmental samples were regulated. As a consequence, there is an increasing demand for the development of sensitive and selective analytical tools for antibiotic reliable and rapid detection. These requirements are accomplished by the combination of simple, cost-effective and affordable electroanalytical methods with molecularly imprinted polymers (MIPs) with high recognition specificity, based on their “lock and key” working principle, used to modify the electrode surface, which is the “heart” of any electrochemical device. This review presents a comprehensive overview of MIP-modified carbon-based electrodes developed in recent years for antibiotic detection. The MIP preparation and electrode modification procedures, along with the performance characteristics of sensors and analytical methods, as well as the applications for the antibiotics’ quantification from different matrices (pharmaceutical, biological, food and environmental samples), are discussed. The information provided by this review can inspire researchers to go deeper into the field of MIP-modified sensors and to develop efficient means for reliable antibiotic determination.

CRISPR/Cas12a-based electrochemical biosensor for highly sensitive detection of cTnI

The successful fabrication of the cTnI detection platform is very meaningful for instant diagnosis of the myocardialinjury and related cardiovascular diseases (CVDs). In this research work, the magnetic nanoparticles and aptamer collaboration with the Cas12a/crRNA are used for the electrochemical detection of cTnI. The aptamer is hybridized with its partially complementary DNA (probe 2, P2) and then is modified on the magnetic nanoparticles. In the presence of cTnI, the cTnI combines with the aptamer and P2 is released. The released P2 is hybridized with the crRNA and the trans-cleavage activity of CRISPR/Cas12a is triggered. Therefore, the methylene blue-modified DNA (probe1, P1) on the surface of the electrode is cleaved, resulting in the decrease of the electrochemical signal. Based on the synergy effect of the high specific target recognition of aptamer, target-specifically triggering trans-cleavage activity of CRISPR/Cas12a, as well as good separation ability of magnetic nanoparticles, the developed electrochemical biosensor enables to detect cTnI with high specificity and sensitivity. The detection limit is low down to 10 pg/mL with a linear range from 100 pg/mL to 50000 pg/mL. The developed sensing platform was successfully applied for the detection of cTnI in human serum. This fabricated CRISPR/Cas12a-based electrochemical biosensor can offer a valuable tool for the diagnosis, prognosis, and treatment of patient with CVDs.

Electrospun molecularly imprinted sodium alginate/polyethylene oxide nanofibrous membranes for selective adsorption of methylene blue

Molecular imprinting technique is an efficient method to improve the selective adsorption capacity for the target pollutant. In this study, sodium alginate/polyethylene oxide molecularly imprinted nanofibrous membrane (SA/PEO-MINM) with average diameter of 185 ± 20 nm was successfully synthesized by electrospinning for selective adsorption of methylene blue (MB). Benefiting from the molecular imprinted technology, the adsorption amount of SA/PEO-MINM for MB was increased by about 65%, significantly higher than the non-imprinted membrane. Results showed that the adsorption equilibrium could be well fitted with Langmuir isotherm model and the maximum adsorption capacity towards MB was 3186.7 mg/g. Kinetic experiments well complied with the Pseudo second order model. Reusability studies indicated that the removal efficiency of MB could maintain 93% of the original adsorption capacity after four consecutive adsorption/desorption cycles. More importantly, the SA/PEO-MINM with high surface area and specific adsorption recognition sites showed excellent selective adsorption capacity in the adsorption experiment of MB and methylene orange mixed dye solution. In general, the SA/PEO-MINM can be successfully applied for the selective removal of MB from dye wastewater.

A highly sensitive plastic optic-fiber with a molecularly imprinted polymer coating for selective detection of 4-chlorophenol in water

To realize the selective detection of 4-chlorophenol (4-CP) in water, a highly sensitive plastic optical fiber (POF) evanescent wave sensor was prepared using a D-shaped POF coated selective adsorption film of 4-CP. The film was composed of molecularly imprinted polymer (MIP) particles and PEBA2533 matrix. The MIP particles were employed to realize the selective adsorption and enrichment of 4-CP molecules, which lead to the changes in the refractive index of the MIP-PEBA2533 coating. These, in turn, resulted in changes in the light transmission in the D-shaped region and caused the attenuation of the evanescent field intensity on the surface of the sensing region. To illustrate the measurement principle, a theoretical model of the sensor was established. Furthermore, the performance parameters of the sensors, such as sensitivity, repeatability, selectivity and low limit of detection (LOD), were experimentally studied. The results highlighted that the proposed sensor exhibited a good sensitivity of -0.56 × 10-6 (μg/L)-1 and LOD of 100 μg/L for the detection of 4-CP in water. Moreover, this optic-fiber sensor has high specific recognition ability for 4-CP. The results of this study could inform the development of methods for on-line and accurate detection of 4-CP.

Fluorescence detection of carbofuran in aqueous extracts based on dual-emission SiO2 @Y2 O3 :(Eu3+ ,Tb3+ )@MIP core-shell structural nanoparticles.

A novel double-windows fluorescence sensor for carbofuran (CF) detection was successfully developed based on rare-earth Eu,Tb-doped Y2 O3 @SiO2 -based molecularly imprinted nanoparticles (MINs) with a multilayer core-shell structure. The recognition process of the MINs for CF was fairly fast and needed only ~8 min to reach a dynamic equilibrium. Interestingly, one fluorescence attenuation window was found with an increase in CF concentration (Q) from 0.1 to 10 μg ml-1 and with a limit of detection (LOD) of 0.04 μg ml-1 at 544 nm belonging to the Tb3+ emission, as well as another fluorescence enhanced window within the CF concentration range 10-100 μg ml-1 (LOD = 4 μg ml-1 ) at 617 nm of Eu3+ emission in the dispersed rare-earth-doped MIN colloidal aqueous solution. Luminescence resonance energy transfer from CF to Eu3+ and an inner filter effect of CF towards Tb3+ , as well from the two independent detection windows were clearly observed simultaneously. The competition experiment displayed hardly any marked interference during detection of CF following addition of its analogues (carbaryl, isoprocarb, aldicarb, methomyl, and etofenprox). Moreover, the MINs could also be applied to accurately detect CF in rhubarb and wolfberry samples with recoveries of 85.7-92.2%. This sensing system has high specific recognition and a wide detection range for CF and provides new opportunities for pesticide detection.

L-Cysteine modified metal-organic framework as a chiral stationary phase for enantioseparation by capillary electrochromatography.

A new kind of chiral zirconium based metal–organic framework, l-Cys-PCN-222, was synthesized using l-cysteine (l-Cys) as a chiral modifier by a solvent-assisted ligand incorporation approach and utilized as the chiral stationary phase in the capillary electrochromatography system. l-Cys-PCN-222 was characterized by X-ray diffraction, thermogravimetric analysis, X-ray photoelectron spectroscopy, Fourier-transform infrared spectra, nitrogen adsorption/desorption, circular dichroism spectrum, zeta-potential and so on. The results revealed that l-Cys-PCN-222 had the advantages of good crystallinity, high specific surface area (1818 m2 g−1), thermal stability and chiral recognition performance. Meanwhile, the l-Cys-PCN-222-bonded open-tubular column was prepared using l-Cys-PCN-222 particles as the solid phase by ‘thiol–ene’ click chemistry reaction and characterized by scanning electron microscopy, which proved the successful bonding of l-Cys-PCN-222 to the column inner wall. Finally, the stability, reproducibility and chiral separation performance of the l-Cys-PCN-222-bonded OT column were measured. Relative standard deviations (RSD) of the column efficiencies for run-to-run, day-to-day, column-to-column and runs were 1.39–6.62%, and did not obviously change after 200 runs. The enantiomeric separation of 17 kinds of chiral compounds including acidic, neutral and basic amino acids, imidazolinone and aryloxyphenoxypropionic pesticides, and fluoroquinolones were achieved in the l-Cys-PCN-222-bonded OT column. These results demonstrated that the chiral separation system of the chiral metal–organic frameworks (CMOFs) coupled with capillary electrochromatography has good application prospects.

Development of a microfluidic dispensing device for multivariate data acquisition and application in molecularly imprinting hydrogel preparation.

Molecularly imprinted polymers (MIPs) are superior materials with a molecular recognition ability that apply to various applications. In order to get high specificity recognition for target molecules, selecting polymerization conditions, which provide high interaction with the target template, is crucial. However, it requires time and labor to find the optimal polymerization composition, especially for large biomolecules. The advance in the microfluidic field enables researchers to control the flow rate and divide solutions based on the design of microfluidic devices for acquiring multivariate data by simultaneously preparing samples with different conditions. In this work, we fabricated microfluidic dispensing devices with different flow path widths that can give the solution of different flow rates. The accuracy of the flow rate was compared with the simulation value. As a result, the flow rate data showed almost the same data as the simulation value, and the dispensing volume ratio showed high reproducibility. Besides, the multivariate data from mixing the fluorescent molecule and protein solutions prepared by the dispensing device and a micropipette showed no significant difference with existing laboratory equipment. Finally, the dispensing device was used for preparing MIP hydrogels for lysozyme as a template protein. We successfully acquired multivariate data on the adsorption capacity of proteins, as a result, the hydrogels provided a high imprinting factor and adsorption specificity toward lysozymes.

Release of florfenicol in seawater using chitosan-based molecularly imprinted microspheres as drug carriers

Novel molecularly imprinted polymer (MIP) microspheres using functionalized chitosan as eco-friendly substrates were prepared by surface imprinting method and applied as drug delivery carriers to provide extended-release of florfenicol (FF) in seawater. The chitosan-based composites were characterized by scanning electron microscopy and Fourier transforms infrared spectroscopy analyses. The swelling behavior, adsorption capability, and selectivity for FF were investigated. The results show that the MIPs possessed high drug loading saturation capacity and specific recognition affinity for FF. The release studies of MIPs as drug delivery carriers were evaluated in natural seawater. The microspheres exhibited slow sustained release profiles of FF and the release behavior conformed to the first-order kinetic equation. The imprinted microspheres as drug delivery devices would be a promising application for improving the efficacy of the antibiotic without exposing the ecological system to excess FF in aquaculture.

Multiply-amplified strategy for the ultrasensitive detection of kanamycin via aptamer-triggered three-dimensional G-quadruplex/Ni–Fe layered double oxide frame networks

In this work, a multiply-amplified peroxidase-like colorimetric strategy was proposed for the high-specific recognition and ultrasensitive detection of kanamycin (Kana). Based on two Kana-aptamer triggered sequential reactions, G-quadruplex (G4) and DNA (hairpins) modified Ni–Fe layered double oxides (LDOs) could be obtained simultaneously. Later, a three-dimensional G4/LDO frame networks, as a novel DNAzyme, with enhanced peroxidase-like catalytic activity was assembled through electrostatic interaction. This DNAzyme catalyzed 3,3′,5,5′-tetramethylbenzidine oxidation for the colorimetric detection of Kana. The enhancement principle was discussed and the charge transfer process during the catalytic reaction was investigated. Under the optimal experiment conditions, the proposed method exhibited high sensitivity, where the linear range is from 10 fM to 10 nM (r2 = 0.992), and the limit of detection is 3 fM (S/N = 3). The practicability of this assay was demonstrated by successfully application of residual Kana detection in genuine milk and urine samples.

Photonic and Magnetic Dual-Responsive Molecularly Imprinted Sensor for Highly Specific Recognition of Enterovirus 71.

The specific identification and detection of a virus are the critical factors to identify and control an epidemic situation. In this study, a novel photonic-magnetic responsive virus-molecularly imprinted photochemical sensor was constructed for recognition of enterovirus 71. As designed, the double-bond-modified magnetic metal organic framework and 4-(4'-acryloyloxyazo) benzoic acid were used as a magnetic carrier and light-responsive functional monomer, respectively. The structure of the recognition site of the virus-molecularly imprinted nanospheres can be photo-switched between two different structures to achieve rapid release and specific binding to the target virus. Additionally, the introduction of a magnetic core enables a rapid separation and recycling of imprinted particles. The device achieves a performance with high-specificity recognition (imprinting factor = 5.1) and an ultrahigh sensitivity with a detection limit of 9.5 × 10-3 U/mL (3.9 fM). Moreover, it has good reproducibility and can be stored for as long as 6 months. Thus, the approach used in this work opens a new avenue for the construction of multiresponsive virus sensors.

Metastatic and non-metastatic melanoma imaging using Sgc8-c aptamer PTK7-recognizer

Melanoma is one of the most aggressive and deadly skin cancers, and although histopathological criteria are used for its prognosis, biomarkers are necessary to identify the different evolution stages. The applications of molecular imaging include the in vivo diagnosis of cancer with probes that recognize the tumor-biomarkers specific expression allowing external image acquisitions and evaluation of the biological process in quali-quantitative ways. Aptamers are oligonucleotides that recognize targets with high affinity and specificity presenting advantages that make them interesting molecular imaging probes. Sgc8-c (DNA-aptamer) selectively recognizes PTK7-receptor overexpressed in various types of tumors. Herein, Sgc8-c was evaluated, for the first time, in a metastatic melanoma model as molecular imaging probe for in vivo diagnostic, as well as in a non-metastatic melanoma model. Firstly, two probes, radio- and fluorescent-probe, were in vitro evaluated verifying the high specific PTK7 recognition and its internalization in tumor cells by the endosomal route. Secondly, in vivo proof of concept was performed in animal tumor models. In addition, they have rapid clearance from blood exhibiting excellent target (tumor)/non-target organ ratios. Furthermore, optimal biodistribution was observed 24 h after probes injections accumulating almost exclusively in the tumor tissue. Sgc8-c is a potential tool for their specific use in the early detection of melanoma.

A highly selective colorimetric and fluorescent probe for cysteine sensing: Application in live cell imaging and test strips

Here, a coumarin-based probe 4-chloro-3-formyl-2-oxo-2H-chromen-7-yl acrylate (named CFCA), which performed high efficient and specific recognition for cysteine (Cys), was successfully synthesized and characterized by NMR and MS. The CFCA probe showed colorimetric and “turn-on” fluorescent signals to Cys, and its color changes could be recognized by naked eyes. CFCA responded to Cys with the characteristics of rapid response time (less than 40 s), high selectivity, and excellent anti-interference. Among all kinds of samples, only Cys could significantly enhance the fluorescence intensity of the solution, which was not affected by the presence of other interferences. At the same time, within a certain concentration range, there was a good linear relationship between relative intensity of fluorescence and Cys concentration. The detection limit has been calculated to be as low as 0.33 μM. And the sensing mechanism of CFCA with Cys has been well confirmed by 1H NMR and HR-MS spectra. Moreover, the fluorescence probe CFCA was successfully used for imaging Cys in living cells and processed into test strips for on-site analysis and testing.

Combinatorial Virtual Library Screening Study of Transforming Growth Factor-β2-Chondroitin Sulfate System.

Transforming growth factor-beta (TGF-β), a member of the TGF-β cytokine superfamily, is known to bind to sulfated glycosaminoglycans (GAGs), but the nature of this interaction remains unclear. In a recent study, we found that preterm human milk TGF-β2 is sequestered by chondroitin sulfate (CS) in its proteoglycan form. To understand the molecular basis of the TGF-β2–CS interaction, we utilized the computational combinatorial virtual library screening (CVLS) approach in tandem with molecular dynamics (MD) simulations. All possible CS oligosaccharides were generated in a combinatorial manner to give 24 di- (CS02), 192 tetra- (CS04), and 1536 hexa- (CS06) saccharides. This library of 1752 CS oligosaccharides was first screened against TGF-β2 using the dual filter CVLS algorithm in which the GOLDScore and root-mean-square-difference (RMSD) between the best bound poses were used as surrogate markers for in silico affinity and in silico specificity. CVLS predicted that both the chain length and level of sulfation are critical for the high affinity and high specificity recognition of TGF-β2. Interestingly, CVLS led to identification of two distinct sites of GAG binding on TGF-β2. CVLS also deduced the preferred composition of the high specificity hexasaccharides, which were further assessed in all-atom explicit solvent MD simulations. The MD results confirmed that both sites of binding form stable GAG–protein complexes. More specifically, the highly selective CS chains were found to engage the TGF-β2 monomer with high affinity. Overall, this work present key principles of recognition with regard to the TGF-β2–CS system. In the process, it led to the generation of the in silico library of all possible CS oligosaccharides, which can be used for advanced studies on other protein–CS systems. Finally, the study led to the identification of unique CS sequences that are predicted to selectively recognize TGF-β2 and may out-compete common natural CS biopolymers.

Nanozyme Sensor Array Plus Solvent-Mediated Signal Amplification Strategy for Ultrasensitive Ratiometric Fluorescence Detection of Exosomal Proteins and Cancer Identification.

Tumor exosomes with molecular marker-proteins inherited from their parent cells have emerged as a promising liquid biopsy biomarker for cancer diagnosis. However, facile, robust, and sensitive detection of exosomal proteins remains challenging. Therefore, a nanozyme sensor array is constructed by using aptamer-modified C3N4 nanosheets (Apt/C3N4 NSs) together with a solvent-mediated signal amplification strategy for ratiometric fluorescence detection of exosomal proteins. Three aptamers specific to exosomal proteins are selected to construct Apt/C3N4 NSs for high specific recognition of exosomal proteins. The adsorption of aptamers enhances the catalytic activity of C3N4 NSs as a nanozyme for oxidation of o-phenylenediamine (oPD) to 2,3-diaminophenazine (DAP). In the presence of target exosomes, the strong affinity between aptamer and exosome leads to the disintegration of Apt/C3N4 NSs, resulting in a decrease of catalytic activity, thereby reducing the production of DAP. The ratiometric fluorescence signal based on a photoinduced electron transfer (PET) effect between DAP and C3N4 NSs is dependent on the concentration of DAP generated, thus achieving highly facile and robust detection of exosomal proteins. Remarkably, the addition of organic solvent-1,4-dioxane can sensitize the luminescence of DAP without affecting the intrinsic fluorescence of C3N4 NSs, achieving the amplification of the aptamer-exosome recognition events. The detection limit for exosome is 2.5 × 103 particles/mL. In addition, the accurate identification of cancer can be achieved by machine learning algorithms to analyze the difference of exosomal proteins from different patients' blood. We hope that this facile, robust, sensitive, and versatile nanozyme sensor array would become a promising tool in the field of cancer diagnosis.