Efficient Antibody Immobilization Research Articles

A photopatterned SERS substrate with a sandwich structure for multiplex detection

Substrate photopatterning has provided versatile applications in biomedical fields. Herein, an universal and efficient photoligation reaction has been used to prepare a patterned capture substrate for a sandwich SERS immunoassay. Photoirradiation induces mild and efficient immobilization of antibodies at the desired region of a gold surface, and the antibody-antigen interaction helps the substrate to capture the antigens in solution specifically. After exposing to SERS probes, i.e., the gold nanoparticles labelled with both antibodies and intrinsically strong Raman reporters, multiple quantitative SERS determination of antigens can be achieved with high sensitivity and specificity. The limit of detection can be as low as 10−12 mol/L for four kinds of cancer biomarkers, which provides a promising method for the construction of highly sensitive and high-throughput SERS detection chip and the application of in vitro diagnosis.

Determination of Vascular Endothelial Growth Factor (VEGF) in Cell Culture Medium by Gold-Coated Magnetic Nanoparticle Based Label-Free Electrochemical Impedance Spectroscopy (EIS)

A gold-coated magnetic nanoparticle (MNPs@Au) based label-free electrochemical impedance spectroscopy (EIS) sensor is reported for determining vascular endothelial growth factor (VEGF) in cell culture media. VEGF antibodies (Ab) were modified on MNPs@Au to construct an Ab-bound magnetic complex (MNPs@Au-Ab) that was magnetically immobilized on a magnetic glassy carbon electrode (mGCE). The VEGF is captured on the MNPs@Au-Ab/mGCE, leading to increased electron-transfer resistance (R et) utilized for the determination of VEGF. Combining the highly efficient immobilization of antibodies on magnetic nanocomposites with the high sensitivity of electrochemical impedance spectroscopy, an excellent dynamic range for the VEGF concentration from 1.0 × 10−2 to 1.0 × 104 ng mL−1 was obtained with a detection limit of 2.43 pg mL−1. Quantitative determination and the investigation of the secretion of VEGF by various cells were also conducted. VEGF released by cancer cells (MDA-MB-231 cells and HCT116 cells) was significantly higher than for normal cells (L02 cells), which matched well with an enzyme linked immunosorbent assay (ELISA). These results show that the secretion of VEGF is associated with the metastatic level of cells. The reported sensor is expected to be applied in studies of cancer and tumor metastasis.

Impedimetric Immunosensor Utilizing Polyaniline/Gold Nanocomposite-Modified Screen-Printed Electrodes for Early Detection of Chronic Kidney Disease

The presence of small amounts of human serum albumin (HSA) in urine or microalbuminuria (30–300 µg/mL) is a valuable clinical biomarker for the early detection of chronic kidney disease (CKD). Herein, we report on the development of an inexpensive and disposable immunosensor for the sensitive, specific, and label-free detection of HSA using electrochemical impedance spectroscopy (EIS). We have utilized a simple one-step screen-printing protocol to fabricate the carbon-based three-electrode system on flexible plastic substrates. To enable efficient antibody immobilization and improved sensitivity, the carbon working electrode was sequentially modified with electropolymerized polyaniline (PANI) and electrodeposited gold nanocrystals (AuNCs). The PANI matrix serves as an interconnected nanostructured scaffold for homogeneous distribution of AuNCs and the resulting PANI/AuNCs nanocomposite synergically improved the immunosensor response. The PANI/AuNCs-modified working electrode surface was characterized using scanning electron microscopy (SEM) and the electrochemical response at each step was analyzed using EIS in a ferri/ferrocyanide redox probe solution. The normalized impedance variation during immunosensing increased linearly with HSA concentration in the range of 3–300 µg/mL and a highly repeatable response was observed for each concentration. Furthermore, the immunosensor displayed high specificity when tested using spiked sample solutions containing different concentrations of actin protein and J82 cell lysate (a complex fluid containing a multitude of interfering proteins). Consequently, these experimental results confirm the feasibility of the proposed immunosensor for early diagnosis and prognosis of CKD at the point of care.

Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure.

Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagents are expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via N-hydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.

Antibody biosensors for spoilage yeast detection based on impedance spectroscopy

Brettanomyces is a yeast species responsible for wine and cider spoilage, producing volatile phenols that result in off-odors and loss of fruity sensorial qualities. Current commercial detection methods for these spoilage species are liable to frequent false positives, long culture times and fungal contamination. In this work, an interdigitated (IDE) biosensor was created to detect Brettanomyces using immunological reactions and impedance spectroscopy analysis. To promote efficient antibody immobilization on the electrodes’ surface and to decrease non-specific adsorption, a Self-Assembled Monolayer (SAM) was developed. An impedance spectroscopy analysis, over four yeast strains, confirmed our device's increased efficacy. Compared to label-free sensors, antibody biosensors showed a higher relative impedance. The results also suggested that these biosensors could be a promising method to monitor some spoilage yeasts, offering an efficient alternative to the laborious and expensive traditional methods.

Water-dispersible graphene/amphiphilic pyrene derivative nanocomposite: High AuNPs loading capacity for CEA electrochemical immunosensing

In this work, a water-dispersible graphene/amphiphilic pyrene derivative nanocomposite has been synthesized for electrochemical immunosensing carcinoembryonic antigen (CEA). Water-soluble 4-armed poly(ethylene glycol)-NH2 is immobilized on graphene through noncovalent functionalization with the assistance of pyrene as an anchor group. The noncovalent functionalization strategy can significantly improve the hydrophilicity of graphene and loading capacity of gold nanoparticles (AuNPs) on graphene, resulting in efficient immobilization of antibodies on the nanocomposite. The morphology and conductivity of the as-prepared nanocomposites were characterized by scanning electron microscopy, Fourier transform infrared and UV–vis spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). CEA monoclonal antibody (anti-CEA) is immobilized on AuNPs via gold-thiol chemistry to construct the electrochemical immunosensing platform, and the surface binding events between anti-CEA and CEA is achieved by monitoring the change of the electrochemical impedance response. Under optimum conditions, the modified electrode exhibits a linear impedance response to CEA in a wide range of 0.1–1000ngmL−1 (R2=0.9959) with low detection limit of 0.06ngmL−1 (S/N=3).

A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10−4 to 102 ng/mL).

A porous silicon immunoassay platform for fluorometric determination of α-synuclein in human cerebrospinal fluid

Levels of total and/or oligomeric α-synuclein may be used as a biomarker tool to aid in the diagnosis and development of new disease-modifying therapies. We report here on a porous silicon antibody microarray for the fluorimetric determination of cerebrospinal fluid levels of total α-synuclein, a protein involved the pathology of Parkinson’s disease. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, and this offers a large binding capacity for capturing probe molecules. Porous silicon also warrants efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. The platform requires 10 μL of cerebrospinal fluid, and each test requires 4 h for assay only (including immobilization of capturing antibody). The limit of detection is 35 pg mL−1 of α-synuclein in cerebrospinal fluid, and the dynamic analytical range extends from 0.01 to 100 ng·mL−1.

In situ characterization of antibody grafting on porous monolithic supports

The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3±0.9mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2±0.2μg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65±6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab′)2 fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained.

A Mussel Adhesive Protein Fused with the BC Domain of Protein A is a Functional Linker Material that Efficiently Immobilizes Antibodies onto Diverse Surfaces

AbstractThe efficient immobilization of antibodies onto solid surfaces is vital for the sensitivity and specificity of various immunoassays and immunosensors. A novel linker protein, BC‐MAP, is designed and produced in Escherichia coli by genetically fusing mussel adhesive protein (MAP) with two domains (B and C) of protein A (antibody‐binding protein) for efficient antibody immobilization on diverse surfaces. Through direct surface‐coating analyses, it is found that BC‐MAP successfully coats diverse surfaces including glass, polymers, and metals, but the BC domain alone does not. Importantly, antibodies are efficiently immobilized on BC‐MAP‐coated surfaces, and the immobilized antibodies interact selectively with their corresponding antigen. Quartz‐crystal‐microbalance analyses show that BC‐MAP has excellent antibody‐binding ability compared to that of BC protein on gold surfaces. These results demonstrate that the MAP domain, with uniquely strong underwater adhesive properties, plays a role in the direct and efficient coating of BC‐MAP molecules onto diverse surfaces that lack additional surface treatment, and the BC domain of BC‐MAP contributes to the selective and oriented immobilization of antibodies on BC‐MAP‐coated surfaces. Thus, the BC‐MAP fusion protein could be a valuable novel linker material for the facile and efficient immobilization of antibodies onto diverse solid supports.

Noncovalent Antibody Immobilization on Porous Silicon Combined with Miniaturized Solid-Phase Extraction (SPE) for Array Based ImmunoMALDI Assays

This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30-60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 μL, 1 nM).

Specific antibody immobilization with biotin-poly( l-lysine)-g-poly(ethylene glycol) and protein A on microfluidic chips

Highly efficient antibody immobilization is crucial for conducting high-performance immunoassays such as enzyme-linked immunosorbent assay (ELISA) in microarray and microfluidic biochips. In this study, a biotin-poly( l-lysine)-g-poly(ethylene glycol) (biotin-PLL-g-PEG) and protein A-based technique was developed to immobilize antibody on the surface of poly(methyl methacrylate) (PMMA) microchannels. First, PMMA surface was activated by oxygen plasma, followed by poly(acrylic acid) (PAA) grafting to add functional carboxyl group for subsequent binding. After the biotin-PLL-g-PEG molecules reacted with carboxyl groups through the electrostatic interactions, biotinylated protein A was immobilized on the surface through a linking molecule, neutravidin. To evaluate the applicability of this novel immobilization strategy, human interferon-gamma (IFN-γ) was used as a model protein. Since protein A could better control the immobilization orientation, and the combination of biotin-PLL-g-PEG and PLL-g-PEG could adjust the conformation of antibodies, antigen capture efficiency and detection signals were significantly improved on the microchips by using this strategy. The optimal grafting conditions were also experimentally determined: the biotin grafting ratio of 0.189 in the PLL-g-PEG molecule and the mixture ratio of 85% (biotin-PLL-g-PEG to PLL-g-PEG). This surface modification can be applied for targeted drug delivery, biosensor and other immunoassay applications.

Protein A‐based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme‐linked immunosorbent assay

Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.

Self-Directed and Self-Oriented Immobilization of Antibody by Protein G−DNA Conjugate

A versatile biolinker for efficient antibody immobilization was prepared by site-specific coupling of protein G to DNA oligonucleotide. This protein G-DNA conjugate ensures the controlled immobilization of an antibody to the intended area on the surface of bioassay chips or particles, while maintaining the activity and orientation of the bound antibody. Streptococcus protein G tagged with a cysteine residue at the N-terminus was chemically linked to amine-modified, single-stranded DNA. SPR analysis indicated that the protein G-DNA conjugates sequence-specifically bind to complementary surface-bound DNA probes. More importantly, the resulting protein G, which is hybridized onto the DNA surface, possesses a greater antibody/antigen binding ability than even properly oriented protein G linked on the chip surface by chemical bonding. Antibody targeting on glass slides could also be achieved by using this linker system without modifying or spotting antibodies. Moreover, the protein G-DNA conjugate provided a simple but effective method to label DNA-functionalized gold nanoparticles with target antibodies. The DNA-linked protein G construct introduced in this study offers a useful strategy to manage antibody immobilization in many immunoassay systems.