Phenylboronic acid self-assembled layer on glassy carbon electrode for recognition of glycoprotein peroxidase

We have successfully fabricated a phenylboronic acid self-assembled layer on glassy carbon electrodes (GCE), where 3-aminophenylboronic acid (APBA) is covalently bound to the electrochemical pretreated GCE surface with glutaraldehyde linkage. The specific binding of glycoprotein peroxidase with the self-assembled layer has been studied using horseradish peroxidase (HRP) as a model glycoprotein. Cyclic voltammetric, electrochemical impedance studies and photometric activity assays show that the affinity interaction of HRP with the APBA modified GCE surface includes specific and nonspecific bonding. The specific binding is attributed to the boronic acid–diols interaction where the boronic acid specifically binds the glycosylation sites of the HRP. This specific binding is reversible and can be split by sorbitol and glucose or released in an acidic buffer. The catalytic current of the HRP-loaded electrode, due to the catalytic oxidation of thionine in the presence of hydrogen peroxide, is proportional to HRP concentrations of the incubation solution. This work offers a new way to build novel sensors by specific binding of glycoproteins to a boronic acid self-assembled layer for determination of glycated proteins.     

Properties of poly-aminophenylboronate coatings in capillary electrophoresis for the selective separation of diastereoisomers and glycoproteins

The polymerisation of 3-aminophenylboronic acid (APBA) in aqueous environment has been used for the open tubular modification of capillary electrophoresis (CE) capillaries. Being poly-APBA endowed with boronic acid, aromatic rings and secondary amines groups, it posses a variety of functional groups affecting selectivity. Diastereoisomers (e.g. ascorbic and isoascorbic acid) and proteins (e.g. haemoglobins) were successfully separated onto poly-APBA column, by means of a combination of electrophoresis and open tubular electrochromatography. The mechanism of selection was investigated: results indicate an interplay between enhancing or silencing the contribution of the protonable functionalities (amino groups, boronic acid). The properties of APBA polymer coating make it attractive for CE separation and for further application in affinity separations and chip technologies.     

Lactose tailored boronic acid conjugated fluorescent gold nanoclusters for turn-on sensing of dopamine

Dopamine being a neurotransmitter and chemical messenger plays a vivacious role in a number of significant medical conditions like Parkinson’s disease, Attention Deficit Hyperactivity Disorder, Schizophrenia, and drug addiction. As turn-on sensors have a superior level of selectivity than fluorescence quenching based sensors, we developed a fluorescence retrieval strategy for dopamine sensing. Here, highly fluorescent amino phenyl boronic acid (APBA)?conjugated gold nanocluster (Au?BSA?APBA probe) has been synthesised from bovine serum albumin?protected gold nanocluster (Au?BSA NCs). Boronic acid forms boronate ester with disaccharides such as lactose due to its affinity to polyols. Hence fluorescence of Au?BSA?APBA probe is quenched when it binds with lactose molecules through boronate ester formation. The fluorescence of Au?BSA?APBA?lactose system can be retrieved (turn-on) with dopamine by the competitive displacement of lactose from the probe surface which suggests the higher affinity of boronic acid to the catechol group of dopamine. Furthermore, real samples spiked with dopamine including human serum and urine were analysed using this turn-on sensor and showed excellent recovery percentage. The developed fluorescent sensor offered high selectivity for dopamine over other catecholamines and aminoacids with detection limit as low as 0.7 μM.     

Point‐of‐Care Amperometric Testing of Diabetic Marker (HbA1c) Using Specific Electroactive Antibodies

Specific immune detection of glycated hemoglobin is still a great challenge owing to the small epitopic difference between Hemoglobin (Hb) and HbA1c. We report a new electrochemical immunoassay format for point of care testing of HbA1c. A conducting self‐assembled monolayer of mercaptophenyl boronic acid (MPBA) was used as a capture layer for binding of glycated proteins and ferrocene tagged anti‐HbA1c antibody (FcAb) as a tracer molecule on a gold screen printed electrode. Validation of the new HbA1c assay was carried out using 6 clinical samples with known HPLC values and a correlation coefficient of 98 % was observed.     

Interference of ascorbic acid in the sensitive detection of dopamine by a nonoxidative sensing approach

The electrochemistry of a poly(anilineboronic acid)/carbon nanotube composite was studied in the presence of dopamine and ascorbic acid. To understand the binding affinity of dopamine and ascorbic acid to the boronic acid functional groups in the composite, the association constants between the diol groups in dopamine and ascorbic acid and the boronic acid were experimentally determined using a fluorescence-based binding assay. The results demonstrate that ascorbic acid could severely interfere with the detection of dopamine in nonoxidative boronic acid-binding approaches: Ascorbic acid was able to electrocatalytically reduce the fully oxidized polyaniline backbone during the electrochemical oxidation process; similarly to dopamine, ascorbic acid was also able to bind to the boronic acid groups through its planar diol group even though the binding affinity is much lower. The examination of the dopamine transduction mechanism and ascorbic acid interference mechanism in this nonoxidative approach will benefit the design of future boronic acid-based sensors.     

Indirect Electrochemical Determination of Ribavirin Using Boronic Acid-Diol Recognition on a 3-Aminophenylboronic Acid-Electrochemically Reduced Graphene Oxide Modified Glassy Carbon Electrode (APBA/ERGO/GCE)

A novel method for the indirect electrochemical determination of ribavirin based on boronic acid-diol recognition was developed using the platform as the sensing element. The device was constructed using a 3-aminophenylboronic acid (APBA)-electrochemically reduced graphene oxide (ERGO) modified electrode. When the electrode was immersed in a solution of ribavirin, complexation of boronic acid groups of APBA with ribavirin occurred at the surface of the electrode and simultaneously caused steric hindrance, resulting in a current decrease because the ferricyanide redox probe was unable access the surface. Under the optimized conditions, a linear relationship was obtained between the relative change in current (%Δi) of [Fe(CN)₆]^3?/4?and the concentration of ribavirin at levels from 10.0 to 7.50?×?10²?ng mL^?1. The proposed electrochemical sensor performed with acceptable sensitivity and reproducibility and was successfully used to determine the content of ribavirin in an injection with satisfactory results.     

Dynamic-covalent macromolecular stars with boronic ester linkages

Macromolecular stars containing reversible boronic ester linkages were prepared by an arm-first approach by reacting well-defined boronic acid-containing block copolymers with multifunctional 1,2/1,3-diols. Homopolymers of 3-acrylamidophenylboronic acid (APBA) formed macroscopic dynamic-covalent networks when cross-linked with multifunctional diols. On the other hand, adding the diol cross-linkers to block copolymers of poly(N,N-dimethylacrylamide (PDMA))-b-poly(APBA) led to nanosized multiarm stars with boronic ester cores and PDMA coronas. The assembly of the stars under a variety of conditions was considered. The dynamic-covalent nature of the boronic ester cross-links allowed the stars to reconfigure their covalent structure in the presence of monofunctional diols that competed for bonding with the boronic acid component. Therefore, the stars could be induced to dissociate via competitive exchange reactions. The star formation-dissociation process was shown to be repeatable over multiple cycles.     

A reusable electrochemical immunosensor for carcinoembryonic antigen via molecular recognition of glycoprotein antibody by phenylboronic acid self-assembly layer on gold

A reusable amperometric immunosensor based on the reversible boronic acid-sugar interaction is proposed. The immunosensor was prepared by self-assembling a thiol-mixed monolayer comprised of conjugates of 3-aminophenylboronic acid with 11-mercaptoundecanoic acid (APBA-MUA) and 11-mercapto-1-undecanol (MU) on gold. The resulting boronic acid coating layer can specifically bind with the glycoprotein antibody, enzyme conjugated carcinoembryonic antibody (HRP-anti-CEA). Voltammetric and electrochemical impedance spectroscopic (EIS) studies and surface plasmon resonance (SPR) measurements show that the binding of HRP-anti-CEA to the APBA interface is reversible and the HRP-anti-CEA can be removed with an acidic buffer or a solution containing sorbitol. The bound enzyme-conjugated antibody can retain its enzyme catalytic activity to the reduction of hydrogen peroxide (H(2)O(2)) and its immunoactivity while binding with CEA to form an immunocomplex. After the formation of the immunocomplex, the access of the active center of HRP to thionine was partially inhibited. This leads to a linear decrease in the electrocatalytic response of HRP-anti-CEA-modified electrode over a CEA concentration range of 2.5 to 40.0 ng mL(-1). After monitoring the immunoreaction signals, the immunocomplex can be easily removed from the APBA interface with a regeneration solution. This regenerated APBA interface can rebound with HRP-anti-CEA and be recognized by the antigen, through which a reusable immunosensor with an RSD of 7.1% for four cycles can be obtained. Under optimal conditions, the detection limit for the CEA immunoassay is 1.1 ng mL(-1), at three times background noise. Serum CEA determination results, obtained with the proposed method, shows that the immunosensor has an acceptable accuracy.     

Identification and relative quantification of specific glycation sites in human serum albumin

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys₅₄₉ (K_Am(Glc)QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.     

基于有机硼酸的葡萄糖荧光传感器的研究进展

有机硼酸类荧光传感器可与二羟基化合物进行高亲和性且可逆地结合, 故可用于糖类传感和识别. 综述了近10年来选择性识别葡萄糖的有机硼酸类荧光传感器的研究进展.     

Preparation of novel bovine hemoglobin surface-imprinted polystyrene nanoparticles with magnetic susceptibility

In this research, a surface imprinting strategy has been adopted in protein imprinting. Bovine hemoglobin surface-imprinted polystyrene (PS) nanoparticles with magnetic susceptibility have been synthesized through multistage core-shell polymerization system using 3-aminophenylboronic acid (APBA) as functional and cross-linking monomers. Superparamagnetic molecularly imprinted polystyrene nanospheres with poly(APBA) thin films have been synthesized and used for the first time for protein molecular imprinting in an aqueous solution. The magnetic susceptibility is imparted through the successful encapsulation of Fe₃O₄ nanoparticles. The morphology, adsorption, and recognition properties of superparamagnetic molecularly imprinted polymers (MIPs) have been investigated using transmission electron microscopy, X-ray diffraction, thermogravimetric analysis, and vibrating sample magnetometer. Rebinding experimental results show that poly(APBA) MIPs-coated superparamagnetic PS nanoparticles have high adsorption capacity for template protein bovine hemoglobin and comparatively low nonspecific adsorption. The imprinted superparamagnetic nanoparticles could easily reach the adsorption equilibrium and achieve magnetic separation in an external magnetic field, thus avoiding some problems of the bulk polymer.     

Enantioselective recognition of mandelic acid by a 3,6-dithiophen-2-yl-9H-carbazole-based chiral fluorescent bisboronic acid sensor

We have prepared chiral fluorescent bisboronic acid sensors with 3,6-dithiophen-2-yl-9H-carbazole as the fluorophore. The thiophene moiety was used to extend the π-conjugation framework of the fluorophore in order to red-shift the fluorescence emission and, at the same time, to enhance the novel process where the fluorophore serves as the electron donor of the photoinduced electron transfer process (d-PET) of the boronic acid sensors; i.e., the background fluorescence of the sensor 1 at acidic pH is weaker compared to that at neutral or basic pH, in stark contrast to the typical a-PET boronic acid sensors (where the fluorophore serves as the electron acceptor of the photoinduced electron transfer process). The benefit of the d-PET boronic acid sensors is that the recognition of the hydroxylic acids can be achieved at acidic pH. We found that the thiophene moiety is an efficient π-conjugation linker and electron donor; as a result, the d-PET contrast ratio of the sensors upon variation of the pH is improved 10-fold when compared to the previously reported d-PET sensors without the thiophene moiety. Enantioselective recognition of tartaric acid was achieved at acid pH, and the enantioselectivity (total response K(D)I(F)(D)/K(L)I(F)(L)) is 3.3. The fluorescence enhancement (I(F)(Sample)/I(F)(Blank)) of sensor 1 upon binding with tartaric acid is 3.5-fold at pH 3.0. With the fluorescent bisboronic acid sensor 1, enantioselective recognition of mandelic acid was achieved for the first time. To the best of our knowledge, this is the first time that the mandelic acid has been enantioselectively recognized using a chiral fluorescent boronic acid sensor. Chiral monoboronic acid sensor 2 and bisboronic acid sensor 3 without the thiophene moiety failed to enantioselectively recognize mandelic acid. Our findings with the thiophene-incorporated boronic acid sensors will be important for the design of d-PET fluorescent sensors for the enantioselective recognition of α-hydroxylic acids such as mandelic acid, given that it is currently a challenge to recognize these analytes with boronic acid fluorescent molecular sensors.     

The relationship among pKa, pH, and binding constants in the interactions between boronic acids and diols—it is not as simple as it appears

In our continuing efforts into designing boronic acid-based sensors that recognize cell-surface carbohydrates, it has been necessary to examine various factors that affect the binding affinity between a boronic acid moiety and a diol. The current prevailing view is that the strongest boronic acid/diol complexes are generated by a combination of high solution pH and a low boronic acid pK_a. However, there has never been a systematic examination of the relationship among the binding constants, boronic acid pK_a, and the pH of the solution. Herein we report our findings with a series of 25 arylboronic acids with various substituents and their binding affinities with diols. We have found that (1) the relationship between the pK_a of monosubstituted phenylboronic acid and its substituents can be described using a Hammet plot; (2) the optimal pH for binding is not always above the pK_a of the boronic acid, and is affected by the pK_a values of the boronic acid and the diol, and other unknown factors; and (3) the general belief that boronic acids with lower pK_a values show greater binding affinities for diols is not always true.     

Boronic acid–lectin affinity chromatography. 1. Simultaneous glycoprotein binding with selective or combined elution

We introduce a novel combination of boronic acid affinity chromatography with lectin affinity chromatography, dubbed as boronic acid–lectin affinity chromatography (BLAC). Concanavalin A and wheat germ agglutinin lectins were mixed with the pesudo-lectin boronic acid to form the BLAC affinity column and their performance was evaluated with standard glycoproteins. Optimization of the binding and elution buffers for the BLAC system is described. The BLAC columns were employed to isolate glycoproteins of interest using both selective and/or combined elution.     

Chiral donor photoinduced-electron-transfer (d-PET) boronic acid chemosensors for the selective recognition of tartaric acids, disaccharides, and ginsenosides

A modular approach was proposed for the preparation of chiral fluorescent molecular sensors, in which the fluorophore, scaffold, and chirogenic center can be connected by ethynyl groups, and these modules can easily be changed to other structures to optimize the molecular sensing performance of the sensors. This modular strategy to assembly chiral sensors alleviated the previous restrictions of chiral boronic acid sensors, for which the chirogenic center, fluorophore, and scaffold were integrated, thus it was difficult to optimize the molecular structures by chemical modifications. We demonstrated the potential of our new strategy by the preparation of a sensor with a larger scaffold. The photoinduced electron‐transfer (PET) effect is efficient even with a large distance between the N atom and the fluorophore core. Furthermore, the rarely reported donor‐PET (d‐PET) effect, which was previously limited to carbazole, was extended to phenothiazine fluorophore. The contrast ratio, that is, PET efficiency of the new d‐PET sensor, is increased to 8.0, compared to 2.0 with the previous carbazole d‐PET sensors. Furthermore, the ethynylated phenothiazine shows longer excitation wavelength (centered at 380 nm) and emission wavelength (492 nm), a large Stokes shift (142 nm), and high fluorescence quantum yield in aqueous solution (Φ=0.48 in MeOH/water, 3:1 v/v). Enantioselective recognition of tartaric acid was achieved with the new d‐PET boronic acid sensors. The enantioselectivity is up to 10 (ratio of the binding constants toward D ‐ and L ‐tartaric acid, k_D/k_L). A consecutive fluorescence enhancement/decrease was observed, thus we propose a transition of the binding stoichiometry from 1:1 to 1:2 as the analyte concentration increases, which is supported by mass spectra analysis. The boronic acid sensors were used for selective and sensitive recognition of disaccharides and glycosylated steroids (ginsenosides).     

“Clickable” affinity ligands for effective separation of glycoproteins

In this paper, we present a new modular approach to immobilize boronic acid ligands that can offer effective separation of glycoproteins. A new “clickable” boronic acid ligand was synthesized by introducing a terminal acetylene group into commercially available 3-aminophenyl boronic acid. The clickable ligand, 3-(prop-2-ynyloxycarbonylamino)phenylboronic acid (2) could be easily coupled to azide-functionalized hydrophilic Sepharose using Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction under mild condition. Compared to other boronic acid affinity gels, the new affinity gel displayed superior effectiveness in separating model glycoproteins (ovalbumin and RNase B) from closely related bovine serum albumin and RNase A in the presence of crude Escherichia coli proteins. Because of the simplicity of the immobilization through “click chemistry”, the new ligand 2 is expected to not only offer improved glycoprotein separation in other formats, but also act as a useful building block to develop new chemical sensors for analysis of other glycan compounds.     

All-small-molecule dynamic covalent gels with antibacterial activity by boronate-tannic acid gelation

Reversible boronate-catechol linkage was widely used to construct two-dimensional coatings and threedimensional nanostructures or hydrogels.The construction of these functional materials usually requires the pre-synthesis of macro molecular building blocks,and direct gelation between natural polyphenols and small molecule boranic acids is yet to be investigated.In this study,we fabricated a family of allsmall-molecule dynamic covalent gels consisting of tannic acid and boronic acids.Transparent and thixotropic gels were formed by boronate affinity towards catechol groups abundant on natural polyphenols.The gels showed multi-responsiveness,such as acid-,base-,reduction-and oxidantsensitive depending on the used boronic acid building blocks.The chemistry for gel formation and stimuli-responsiveness was characterized by11B NMR spectroscopy.The multi-stimuli responsiveness,green processing and facile modular design make the boronic acid-tannic acid gels promising candidates for the development of smart soft materials.     

Direct analysis of polyols using 3-nitrophenylboronic acid in capillary electrophoresis: thermodynamic and electrokinetic principles of molecular recognition

The design of boronic acid sensors for photometric detection of carbohydrates has relied on exploiting differences in the thermodynamic stability of complex formation for molecular recognition. Herein, we introduce a direct method for analysis of sugar alcohols using 3-nitrophenylboronic acid (NPBA) as an electrokinetic probe in capillary electrophoresis (CE). Dynamic complexation of neutral polyols by NPBA during electromigration allows for their simultaneous resolution and UV detection based on formation of an anionic ternary boronate ester complex in phosphate buffer. Unlike conventional boronic acid sensors, thermodynamic and electrokinetic processes in CE allow for improved selectivity for the resolution of sugar alcohol stereoisomers having different vicinal polyol chain lengths even in cases when binding affinity is similar due to differences in their complex mobility. Three complementary approaches were investigated to compare the thermodynamics of polyol chelation with NPBA, namely direct binding assays by CE, UV absorbance spectroscopy and an indirect pK _a depression method. Overall, CE offers a convenient platform for characterization of reversible arylboronic acid interactions in free solution while allowing for direct analysis of complex mixtures of neutral/UV-transparent polyols without complicated sample handling.     

A new boronic acid fluorescent reporter that changes emission intensities at three wavelengths upon sugar binding

The boronic acid moiety is a very useful functional group for the preparation of sugar sensors. Along this line, water-soluble boronic acids that change fluorescent properties upon sugar binding are especially useful as reporter units in fluorescent sensors for carbohydrates. Herein, we report the discovery of a new water-soluble boronic acid (1, dibenzofuran-4-boronic acid) that exhibits unique fluorescence changes at three wavelengths upon binding with sugars under near physiological conditions.     

The Development of Boronic Acids as Sensors and Separation Tools

Synthetic receptors for diols that incorporate boronic acid motifs have been developed as new sensors and separation tools. Utilizing the reversible interactions of diols with boronic acids to form boronic esters under new binding regimes has provided new hydrogel constructs that have found use as dye‐displacement sensors and electrophoretic separation tools; similarly, molecular boronic‐acid‐containing chemosensors were constructed that offer applications in the sensing of diols. This review provides a somewhat‐personal perspective of developments in boronic‐acid‐mediated sensing and separation, placed in the context of the seminal works of others in the area, as well as offering a concise summary of the contributions of the co‐authors in the area. DOI 10.1002/tcr.201200006