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FTIR spectra of PGMA (black), PGMA-APBA (red), and APBA (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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There still has a big challenge for low-abundance protein detection with antibody microarrays. In this work, phenylboronic acid (PBA) polymer brush-enabled oriented, high density, and covalent antibody immobilization was realized for sensitive antibody microarrays. PBA-enabled oriented antibody attachment via carbohydrate at Fc portion keeps antige...
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The MosaiQ® COVID-19 Antibody test fulfills the minimal requirements for serological testing according to the French regulation.
Citations
... 36 The freshly etched GaAs substrates were immersed in a 2 mM solution of MUBIB prepared in deoxygenated ethanol. In parallel, phenylboronic acid moieties were incorporated to MUBIB-Ep-GaAs samples following the procedure reported by Liu et al. 38 The samples were incubated in a solution containing 3-aminophenylboronic acid (50 mM) in methanol/ water (1:1, v/v) for 1 h at room temperature under agitation. Following the incubation, the obtained samples (MUBIB-PhB-GaAs) were thoroughly washed with (1:1) methanol/ water and dried under N 2 flow. ...
... The band at 1358 cm −1 has been previously assigned to B−O stretching, while the bands at 1604 and 1571 cm −1 correspond to the CC stretching of the benzene ring. 38 These results confirm the successful conjugation of phenylboronic acid to MUBIB-Ep. ...
This work reports on the potential of polymer brushes (PBs) grown on GaAs substrates (PB-GaAs) as a promising platform for the detection of Legionella pneumophila (Lp). Three functionalization approaches of the GaAs surface were used, and their compatibility with antibodies against Lp was evaluated using Fourier transform infrared spectroscopy and fluorescence microscopy. The incorporation of PBs on GaAs has allowed a significant improvement of the antibody immobilization by increased surface coverage. Bacterial capture experiments demonstrated the promising potential for enhanced immobilization of Lp in comparison with the conventional alkanethiol self-assembled monolayer-based biosensing architectures. Consistent with an eightfold improved capture of bacteria on the surface of a PB-functionalized GaAs/AlGaAs digital photocorrosion biosensor, we report the attractive detection of Lp at 500 CFU/mL.
... PEG moieties incorporated for A1 and A3 approaches are responsible for a decrease in the contact angle, while the decrease obtained upon phenylboronic acid incorporation can be attributed to the double hydroxyl groups. 30 Despite that, an increase in hydrophobicity is associated with better bacteria adhesion/ detection, and the obtained contact angles fall within the abovementioned range of values associated with an efficient bacteria adhesion. Furthermore, a slight increase in hydrophilicity has shown to efficiently prevent nonspecific adsorption. ...
... Incorporation of Phenylboronic Acid Moieties to MUBIB−Ep (MUBIB−Ep−PhB). MUBIB−Ep−PhB was prepared following a procedure previously reported where MUBIB−Ep was deposited on initiator-activated glass substrates.30 Briefly, MUBIB−Ep wafers were immersed in a solution containing 3-aminophenylboronic acid (50 mM) in methanol/water (1:1, v/v) followed by incubation for 1 h at room temperature under agitation. ...
Despite evidence showing that polymer brushes (PBs) are a powerful tool used in biosensing for minimizing nonspecific interactions, allowing for optimization of biosensing performance, and the fact that GaAs semiconductors have proven to have a remarkable potential for sensitive biomolecule detection, the combination of these two robust components has never been considered nor evaluated as a platform for biosensing applications. This work reports different methodologies to prepare and tune PBs on the GaAs interface (PB−GaAs) and their potential as useful platforms for antibody grafting, with the ultimate goal of demonstrating the innovative and attractive character of the PB−GaAs interfaces in the enhanced capture of antibodies and control of nonspecific interactions. Three different functionalization approaches were explored, one “grafting-to” and two “grafting-from,” in which atom transfer radical polymerization (ATRP) was performed, followed by their corresponding characterizations. Demonstration of the compatibility of Escherichia coli (E. coli) and Legionella pneumophila (Lp) antibodies with the PB−GaAs platform compared to the results obtained with conventional biosensing architectures developed for GaAs indicates the attractive potential for operation of a sensitive biosensor. Furthermore, these results showed that by carefully choosing the nature and preparation methodology of a PB−GaAs interface, it is possible to effectively tune the affinity of PB−GaAs-based sensors toward E. coli and Lp antibodies ultimately demonstrating the superior specificity of the developed biosensing platform.
... To passivate the remaining boronic acid groups in the areas adjacent to the drop-casted antibodies, dextran was employed, due to its large molecular weight and number of vicinal diols [32]. Based on the results obtained previously, PBS 1× pH 7.4 was employed as a buffer system so that it does not compete with the already immobilized secondary antibodies. ...
... Nevertheless, an immunosensor performance will also depend on the nature of the antigen targeted and its size as well as the constraints imposed by the employed surface functionalization chemistry. Therefore, and despite the fact that improved antibody loading and increased antigen binding were observed for protein A/G-modified surfaces, boronate ester chemistry can still find many applications as it can easily get incorporated in polymer brushes, hydrogel, or complex constructs [25,32,50], thus achieving surface loading of biomolecules in the three dimensions. Furthermore, the tunability of the interactions between boronate esters and the diols on antibodies through the adjustment of the buffer pH value can find numerous applications [51,52]. ...
Immunosensor sensitivity and stability depend on a number of parameters such as the orientation, the surface density, and the antigen-binding efficiency of antibodies following their immobilization onto functionalized surfaces. A number of techniques have been developed to improve the performance of an immunosensor that targets one or both of the parameters mentioned above. Herein, two widely employed techniques are compared for the first time, which do not require any complex engineering of neither the antibodies nor the surfaces onto which the former get immobilized. To optimize the different surface functionalization protocols and compare their efficiency, a model antibody-antigen system was employed that resembles the complex matrices immunosensors are frequently faced with in real conditions. The obtained results reveal that protein A/G is much more efficient in increasing antibody loading onto the surfaces in comparison to boronate ester chemistry. Despite the fact, therefore, that both contribute towards the orientation-specific immobilization of antibodies and hence enhance their antigen-binding efficiency, it is the increased antibody surface density attained with the use of protein A/G that plays a critical role in achieving maximal antigen recognition.
... Amino group was introduced on surface of the slides through the APTES silanization. P (GMA-co-PEGMA)-glass slides were prepared according to the procedures reported in our previous works [18,19]. Briefly, the aminated slides were placed into 25 ml of ice-cold methylene chloride containing 192.5 μL TEA, followed by dropwise addition of 160 μL BIB. ...
... As shown in Fig. 6, the gap closure rate under treatment of Thiamet G is significantly higher than the non-treated group at all testing time points, indicating that O-GlcNAcylation elevation promotes cell migration. The result is good consistent with the previously reported works using different cancer cells including prostate [19], ovarian [34] and cholangiocarcinoma [35]. ...
Although a variety of approaches have been developed to analyze protein O-GlcNAcylation, efficient investigations on O-GlcNAcylation of proteins of interest in high-throughput manner are still in high demand to further explore its functionality. In this work, we first develop a powerful microarray platform for a sensitive, specific and high-throughput analysis of protein O-GlcNAcylation. The developed array biochip is then utilized to parallelly analyze the O-GlcNAcylation of three oncogenic transcription factors C-Myc, NF-κB and p53 in normal prostate epithelial cell (RWPE-1) and prostate cancer cell line (PC-3). The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are also monitored by the microarray platform. The experimental results show that the total O-GlcNAcylation and OGT level are obviously elevated in PC-3 as compared to RWPE-1. The protein expression-normalized O-GlcNAcylation of C-Myc and NF-κB in PC-3 is significantly higher than that in RWPE-1, while opposite result is observed from p53. In addition, the biological behaviors including proliferation and migration of PC-3 cells are also studied when OGA inhibitor Thiamet G is applied to elevate the total O-GlcNAcylation level.
... An immunoassay based on a specific antibodyÀantigen interaction is one of the most popular analytical methods for the clinical diagnosis of various diseases. To date, many kinds of immunoassay schemes based on different signalÀtransducer principles including fluorescence [2,3], chemiluminescence [4], electrochemistry [5,6], surface Plasmon resonance (SPR) [7], quartz crystal microbalance (QCM) [8,9], and colorimetry [10,11] have been used for cancer diagnosis via detection of specific protein biomarkers. Among these, the colorimetric immunoassay is the most commonly used method for practical applications, particularly pointÀofÀcare diagnosis. ...
Colorimetric immunoassays have been attracting more attention for use in practical applications, especially in point−of−care diagnostics. In comparison with a single color immunoassay, the dose−dependent multicolor strategy greatly improves the detection resolution and accuracy of visual inspection. In the current study, a concentration−dependent multicolor conversion strategy was developed based on gold nanoparticle (AuNP)−mediated copper deposition for signal amplification and Prussian blue for color generation. Under optimal conditions, a dose−dependent multicolor from yellow through green to blue were successfully achieved, which was easier to be differentiated from each other by the naked eyes. With rabbit IgG and prostate specific antigen (PSA) as model analytes, semi−quantitative evaluations were demonstrated in lab buffer and serum by direct readout with the naked eyes. Quantitative detections were also accomplished by measurement of absorbance of Prussian blue with a common UV–Vis spectrophotometer. A limit of detection (LOD) down to sub−picogram per milliliter was determined. In addition, this newly developed colorimetric assay method can be easily adapted for the detection of other biomolecules by simply changing the recognition pairs.
... 27 Another approach can be use of an antibody and/or receptor molecule (red V-shape) end-attached to responsive chain. 20,51 Consequently, this receptor molecule will capture a specific target molecule when it is exposed to the surface. Simultaneously, non-specific adsorption of target molecules either to the surface or receptor molecule are prevented by the non-responsive, second brush (black curves) such as poly(ethylene oxide). ...
We propose the design of a novel mixed polymer brush system that could act as a selective sensor with a distinct on-off switch. In the proposed system, a (single) nanoparticle (such as an antibody) is end-attached to a responsive chain, which is surrounded by a brush of nonresponsive chains. The collapse of the responsive chain leads to a protected state, where the nanoparticle is hidden in the polymer brush, while swelling of the responsive chain brings the nanoparticle outside of the brush into an exposed and active state. We investigate this system by numerical self-consistent field theory and predict a first-order like transition between the active state and the protective state at a critical decrease in solvent quality for the responsive chain. We show that by careful design of the brush parameters such as grafting density and chain length, for a given particle size, it is possible to fine-tune the desired switching mechanism.
... At the same time, a high antigen-binding ability and low nonspecific protein adsorption are also observed from the site-specific immobilization of antibody on 3-D BA-containing zwitterionic polymer brush. More recently, Liu et al. reports a high sensitive antibody microarray on 3-D phenylboronic acid polymer brush-functionalized glass slide (Fig. 7) [109]. Because of the BA-containing polymer brush-enabled site-specific antibody immobilization and the high capture antibody loading capacity, immunoreaction efficiency and immunoassay sensitivity are significantly improved compared with that achieved from poly(glycidyl methacrylate)-brush modified surface. ...
... In addition, several IBA-containing Fig. 7 Schematic illustration of phenylboronic acid polymer brush-modified slide preparation and oriented antibody immobilization. (Reprinted with permission from Ref. [109]. Copyright (2014) Elsevier) functional ligands including IBA-fluorescein (IBA-FITC), IBA-iRGD cyclic peptide (IBA-iRGD), and IBA-paclitaxel (IBA-paclitaxel) were covalently and site-selectively introduced to antibodies at the NBS [119]. ...
... One of the most popular strategy is based on the evaluation of bioaffinity or bioactivity of the surface-tethered proteins. Take antibody as an example, if antibody is immobilized on a given surface, the antigen binding capability or efficiency can be masured via SPR or fluorescence [54,109]. Antigen/antibody ratios measured in experiment can be attributed mainly to the variations in antibody orientation, providing an indirect method to deduce the orientation of attached antibodies. ...
Immobilization of proteins on a solid support is critical with respect to the fabrication and performance of biosensors and biochips. Protein attachment with a preferable orientation can effectively avoid its denaturation and keeps its active sites fully exposed to solution, thus maximally preserving the bioaffinity or bioactivity. This review (with 140 refs.) summarises the recent advances in oriented immobilization of proteins with a particular focus on antibodies and enzymes. Following an introduction that describes reasons for oriented immobilization on (nano)surfaces, we summarize (a) methods for (bio)chemical affinity-mediated oriented immobilization (with sections on immunoglobulin G (IgG)-binding protein as the capture ligand, DNA-directed immobilization, aptamer- and peptide-mediated immobilization, affinity ligand and fusion tag-mediated immobilization, material-binding peptide-assisted immobilization); (b) methods for covalent oriented immobilization (with sections on immobilization via cysteine residues or cysteine tags, via carbohydrate moieties; via enzyme fusion or enzymatic catalysis, and via nucleotide binding sites of antibodies); (c) methods based on molecular imprinting techniques; (d) methods for characterization of oriented immobilized proteins; and then make conclusions and give perspectives.
Graphical Abstract
This review summarises recent advances in oriented immobilization of proteins based on strategies via bio−/chemical affinity, covalent bonding, and molecular imprinting techniques. Advantages and disadvantages of each approach are discussed.
... Our results affirmed clearly the potential impact of the oriented Ab immobilization strategy in improving the bioanalytical performance of an IA, in line with the literature reports. [58][59][60][61][62][63][64][65] In particular, a wide range of oriented Ab immobilization strategies 57,66 has been developed for different immunosensors and IA formats for a plethora of analytes. Therefore, the screening of an appropriate oriented Ab immobilization strategy would be a critical preliminary step in the development of analytically-superior biosensors, diagnostics, lab-on-a-chip and other assay formats. ...
A rapid and highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) has been developed and validated for detecting human C-reactive protein (CRP), a specific biomarker for inflammatory and metabolic disorders, and infections. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A/G (Pr A/G) was diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), dispensed on a KOH-treated gold (Au)-coated SPR chip, and incubated for 30 min. The Pr A/G functionalized Au SPR chip was then bound to anti-human CRP capture antibody (Ab), blocked with bovine serum albumin, and subsequently used for the detection of CRP. The highly-simplified oriented Ab immobilization strategy enabled the leach-proof binding of capture Ab in 5-fold shorter time than conventional procedures. The developed IA detected 1.2-80 ng mL(-1) of CRP with a limit of detection (LOD) and a limit of quantification (LOQ) of 1.2 ng mL(-1) and 4.6 ng mL(-1), respectively. It detected CRP spiked in diluted human whole blood, serum and plasma as well as the CRP levels in the ethylenediaminetetraacetic acid (EDTA) plasma samples of patients with the same precision as the clinically-accredited analyzer-based IA and conventional CRP sandwich ELISA. The Ab-bound SPR chips stored at 4 °C retained their functional activity for 10 weeks, resulting in significant reduction in the overall analysis time.
A BSTRACT
Background
Because of their sensitive and selective responses to a wide variety of analytes, colorimetric sensors have gained widespread acceptance in recent years. Gold nanoparticles (AuNPs) are widely employed in visual sensor strategies due to their high stability and ease of use. Combining AuNPs with a responsive polymer can result in distinct surface plasmon resonance (SPR) changes that can be utilized as colorimetric biosensors.
Objectives
The purpose of this research is to develop a colorimetric-based sensor through the utilization of the optical properties of gold nanoparticles (AuNPs) crosslinked with pH-responsive polymers poly (acrylic acid) (PAA) conjugated to 3-aminophenyl boronic acid (APBA).
Methods
The polymer (PAA) was synthesized via RAFT polymerization. The inversed Turkevic method was used to produce AuNPs, which were subsequently used in a self-assembly process using poly (acrylic acid)-aminophenyl boronic acid (PAA-APBA) to create the self-assembled AuNPs-APBA-PAA. The particle size, zeta potential, and reversibility of the polymer-modified gold nanoparticles were determined using a transmission electron microscope (TEM), a particle size analyzer (PSA), and an Ultraviolet-Visible spectrophotometer (UV-Vis spectrophotometer). Visual, UV-Vis spectrophotometer and TEM observations confirmed the system’s ability to identify bacteria. Statistical analysis was performed using a one-way analysis of variance using Excel software.
Results
Using UV-Vis spectrophotometry, the particle size of AuNPs was determined to be 25.7 nm, and the maximum absorbance occurred at 530 nm. AuNPs PAA APBA colloid exhibited an absorbance maximum of 532 nm, a zeta potential of -41.53, and a pH transition point between 4 and 5. At E. coli concentrations of 4.5 x 10 ⁷ CFU/mL, the color of the system sensors changed from red to blue after 15 hours of incubation, whereas at S. aureus concentrations of 1.2 x 10 ⁹ CFU/mL, the color changed to purple immediately after mixing. The TEM confirmed that the detection mechanism is based on the boronate-polyol bonding of saccharides on the outer membranes of Escherichia coli and Staphylococcus aureus .
Conclusions
The use of APBA in conjunction with pH-responsive PAA polymers containing AuNPs to detect E. coli and S. aureus bacteria induces a maximum wavelength transition, followed by a color change from red to blue. By the process of de-swelling of the responsive polymer, which induces the aggregation of the AuNPs, the established sensor system is able to alter the color. The conjugated polymer and gold nanoparticle-based sensor system demonstrated a promising method for bacterial detection.
