Oriented Immobilization Of Antibodies Research Articles

Fabrication of a Heptapeptide-Modified Poly(glycidyl Methac-Rylate) Nanosphere for Oriented Antibody Immobilization and Immunoassay

Oriented antibody immobilization has been widely employed in immunoassays and immunodiagnoses due to its efficacy in identifying target antigens. Herein, a heptapeptide ligand, HWRGWVC (HC7), was coupled to poly(glycidyl methacrylate) (PGMA) nanospheres (PGMA-HC7). The antibody immobilization behavior and antigen recognition performance were investigated and compared with those on PGMA nanospheres by nonspecific adsorption and covalent coupling via carbodiimide chemistry. The antibodies tested included bovine, rabbit, and human immunoglobulin G (IgG), while the antigens included horseradish peroxidase (HRP) and β-2-Microglobulin (β2-MG). The nanospheres were characterized using zeta potential and particle size analyzers, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and reversed-phase chromatography, proving each synthesis step was succeeded. Isothermal titration calorimetry assay demonstrated the strong affinity interaction between IgG and PGMA-HC7. Notably, PGMA-HC7 achieved rapid and extremely high IgG adsorption capacity (~3 mg/mg) within 5 min via a specific recognition via HC7 without nonspecific interactions. Moreover, the activities of immobilized anti-HRP and anti-β2-MG antibodies obtained via affinity binding were 1.5-fold and 2-fold higher than those of their covalent coupling counterparts. Further, the oriented-immobilized anti-β2-MG antibody on PGMA-HC7 exhibited excellent performance in antigen recognition with a linear detection range of 0–5.3 μg/mL, proving its great potential in immunoassay applications.

Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application

BackgroundThrombosis is a common cause of vascular prosthesis failure. Antibody coating of prostheses to capture circulating endothelial progenitor cells to aid endothelialization on the device surface appears a promising solution to prevent thrombus formation. Compared with random antibody immobilization, oriented antibody coating (OAC) increases antibody-antigen binding capacity and reduces antibody immunogenicity in vivo. Currently, few OAC methods have been documented, with none possessing clinical application potential.ResultsDopamine and the linker amino-PEG8-hydrazide-t-boc were successfully deposited on the surface of cobalt chromium (CC) discs, CC stents and expanded polytetrafluoroethylene (ePTFE) grafts under a slightly basic condition. CD34 antibodies were immobilized through the reaction between aldehydes in the Fc region created by oxidation and hydrazides in the linker after t-boc removal. CD34 antibody-coated surfaces were integral and smooth as shown by scanning electron microscopy (SEM), had significantly reduced or no substrate-specific signals as revealed by X-ray photoelectron spectroscopy, were hospitable for HUVEC growth as demonstrated by cell proliferation assay, and specifically bound CD34 + cells as shown by cell binding testing. CD34 antibody coating turned hydrophobic property of ePTFE grafts to hydrophilic. In a porcine carotid artery interposition model, a confluent monolayer of cobblestone-shaped CD31 + endothelial cells on the luminal surface of the CD34 antibody coated ePTFE graft were observed. In contrast, thrombi and fibrin fibers on the bare graft, and sporadic cells on the graft coated by chemicals without antibodies were seen.ConclusionA universal, OAC method was developed. Our in vitro and in vivo data suggest that the method can be potentially translated into clinical application, e.g., modifying ePTFE grafts to mitigate their thrombotic propensity and possibly provide for improved long-term patency for small-diameter grafts.

Resorc[4]arene-Modified Gold-Decorated Magnetic Nanoparticles for Immunosensor Development.

In recent years, several efforts have been made to develop selective, sensitive, fast response, and miniaturized immunosensors with improved performance for the monitoring and screening of analytes in several matrices, significantly expanding the use of this technology in a broad range of applications. However, one of the main technical challenges in developing immunosensors is overcoming the complexity of binding antibodies (Abs) to the sensor surface. Most immobilizing approaches lead to a random orientation of Abs, resulting in lower binding site density and immunoaffinity. In this context, supramolecular chemistry has emerged as a suitable surface modification tool to achieve the preorganization of artificial receptors and to improve the functional properties of self-assembled monolayers. Herein, a supramolecular chemistry/nanotechnology-based platform was conceived to develop sensitive label-free electrochemical immunosensors, by using a resorcarene macrocycle as an artificial linker for the oriented antibody immobilization. To this aim, a water-soluble bifunctional resorc[4]arene architecture (RW) was rationally designed and synthesized to anchor gold-coated magnetic nanoparticles (Au@MNPs) and to maximize the amount of the active immobilized antibody (Ab) in the proper "end-on" orientation. The resulting supramolecular chemistry-modified nanoparticles, RW@Au@MNPs, were deposited onto graphite screen printed electrodes which were then employed to immobilize three different Abs. Furthermore, an immunosensor for atrazine (ATZ) analysis was realized and characterized by the differential pulse voltammetry technique to demonstrate the validity of the developed biosensing platform as a proof of concept for electrochemical immunosensors. The RW-based immunosensor improved AbATZ loading on Au@MNPs and sensitivity toward ATZ by almost 1.5 times compared to the random platform. Particularly, the electrochemical characterization of the developed immunosensor displays a linearity range toward ATZ within 0.05-1.5 ng/mL, a limit of detection of 0.011 ng/ml, and good reproducibility and stability. The immunosensor was tested by analyzing spiked fortified water samples with a mean recovery ranging from 95.7 to 108.4%. The overall good analytical performances of this immunodevice suggest its application for the screening and monitoring of ATZ in real matrices. Therefore, the results highlighted the successful application of the resorc[4]arene-based sensor design strategy for developing sensitive electrochemical immunosensors with improved analytical performance and simplifying the Ab immobilization procedure.

A colorimetric lateral flow immunoassay based on oriented antibody immobilization for sensitive detection of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The high human-to-human transmission and rapid evolution of SARS-CoV-2 have resulted in a worldwide pandemic. To contain SARS-CoV-2, it is essential to efficiently control the transmission of the virus through the early diagnosis of infected individuals, including asymptomatic people. Therefore, a rapid and accurate assay is vital for the early diagnosis of SARS-CoV-2 in suspected individuals. In this study, we developed a colorimetric lateral flow immunoassay (LFIA) in which a CBP31-BC linker was used to immobilize antibodies on a cellulose membrane in an oriented manner. The developed LFIA enabled sensitive detection of cultured SARS-CoV-2 in 15 min with a detection limit of 5 × 104 copies/mL. The clinical performance of the LFIA for detecting SARS-CoV-2 was evaluated using 19 clinical samples validated by reverse transcription-polymerase chain reaction (RTPCR). The LFIA detected all the positive and negative samples accurately, corresponding to 100% accuracy. Importantly, patient samples with low viral loads were accurately identified. Thus, the proposed method can provide a useful platform for rapid and accurate point-of-care testing of SARS-CoV-2 in infected individuals to efficiently control the COVID-19 pandemic.

Electrochemical detection of cystatin C by oriented antibody immobilization on streptococcal protein G–modified ZIF-8-Cu1−xNix(OH)2@Cu core-shell nanostructured electrode

Herein, we prepared a novel nanostructure involving Cu shell on Zeolitic imidazolate frameworks (ZIF-8) and Cu1−xNix(OH)2 composite (ZIF-8-Cu1−xNix(OH)2@Cu) combining sol-gel and co-precipitation method. The morphology, stoichiometry, and structure of the nanocomposite were elucidated by various physicochemical analyses. A poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) coated indium tin oxide (ITO) was modified with the synthesized ZIF-8-Cu1−xNix(OH)2@Cu nanoparticles (NPs) to obtain an efficient electrode for further antibody immobilization. The ZIF-8-Cu1−xNix(OH)2@Cu/PEDOT:PSS/ITO was applied for the detection of cystatin C, a promising biomarker of chronic kidney disease (CKD). The electrode was functionalized by streptococcal protein G (SPG) to bind the Fc region of anti-cystatin C in an oriented manner. The synergistic catalytic activities, high surface coverage, enhanced electroactive sites, and excellent redox properties of the proposed electrode lead to excellent electrochemical sensing. The proposed sensor obtained a much lower detection limit (33 pg/mL) for a linear range of 0.1 ng/mL to 1,000 ng/mL with high selectivity, stability, and reproducibility compared with bare ZIF-8/PEDOT:PSS/ITO-based immunosensor. The clinical feasibility of the sensor was confirmed by measuring the human serum in the presence of different concentrations of cystatin C. This work demonstrates a new and facile approach to fabricating a metal-organic framework (MOF) –based nanoimmunosensor for cystatin C, which has significant importance in diagnosing the renal failure.

Advanced Lab-on-Fiber Optrodes Assisted by Oriented Antibody Immobilization Strategy.

Lab-on-fiber (LoF) optrodes offer several advantages over conventional techniques for point-of-care platforms aimed at real-time and label-free detection of clinically relevant biomarkers. Moreover, the easy integration of LoF platforms in medical needles, catheters, and nano endoscopes offer unique potentials for in vivo biopsies and tumor microenvironment assessment. The main barrier to translating the vision close to reality is the need to further lower the final limit of detection of developed optrodes. For immune-biosensing purposes, the assay sensitivity significantly relies on the capability to correctly immobilize the capture antibody in terms of uniform coverage and correct orientation of the bioreceptor, especially when very low detection limits are requested as in the case of cancer diagnostics. Here, we investigated the possibility to improve the immobilization strategies through the use of hinge carbohydrates by involving homemade antibodies that demonstrated a significantly improved recognition of the antigen with ultra-low detection limits. In order to create an effective pipeline for the improvement of biofunctionalization protocols to be used in connection with LoF platforms, we first optimized the protocol using a microfluidic surface plasmon resonance (mSPR) device and then transferred the optimized strategy onto LoF platforms selected for the final validation. Here, we selected two different LoF platforms: a biolayer interferometry (BLI)-based device (commercially available) and a homemade advanced LoF biosensor based on optical fiber meta-tips (OFMTs). As a clinically relevant scenario, here we focused our attention on a promising serological biomarker, Cripto-1, for its ability to promote tumorigenesis in breast and liver cancer. Currently, Cripto-1 detection relies on laborious and time-consuming immunoassays. The reported results demonstrated that the proposed approach based on oriented antibody immobilization was able to significantly improve Cripto-1 detection with a 10-fold enhancement versus the random approach. More interestingly, by using the oriented antibody immobilization strategy, the OFMTs-based platform was able to reveal Cripto-1 at a concentration of 0.05 nM, exhibiting detection capabilities much higher (by a factor of 250) than those provided by the commercial LoF platform based on BLI and similar to the ones shown by the commercial and well-established bench-top mSPR Biacore 8K system. Therefore, our work opened new avenues into the development of high-sensitivity LoF biosensors for the detection of clinically relevant biomarkers in the sub-ng/mL range.

Characterization of a heptapeptide‐modified microsphere for oriented antibody immobilization

Oriented immobilization of antibodies is important for the effective recognition of target antigens. In this paper, a heptapeptide ligand, HWRGWVC (HC7), was modified onto non‐porous monosized poly(glyceryl methacrylate) (pGMA) microspheres (named pGMA‐HC7) to explore the antibody immobilization behaviors. Characterization of the microspheres by particle size analyzer, scanning electron microscopy, Fourier transform infrared spectroscopy, and reversed‐phase chromatography proved the success of each fabrication step. The capacity and activity of antibody immobilization through HC7 were studied using immunoglobulin G (IgG) as a model antibody and horseradish peroxidase (HRP) as a model antigen. Additionally, IgG immobilizations on pGMA microspheres by nonspecific adsorption and covalent coupling through carbodiimide chemistry were conducted for comparison. pGMA‐HC7 exhibited an IgG adsorption capacity of 3–4 mg/g in 10 min by the specific binding of HC7 without nonspecific interactions. Notably, the ligand HC7 showed a by two orders of magnitude stronger affinity for IgG than its original hexapeptide ligand HWRGWV. Moreover, the capacity and activity of the immobilized anti‐HRP antibody on pGMA‐HC7 were 1.6‐fold and 3‐fold higher than those of the covalent coupling, respectively. The results proved the superior role of HWRGWVC in the affinity binding of antibody and the potential of pGMA‐HC7‐25 in immunoassay and immunodiagnostic applications.

An oriented antibody immobilization based electrochemical platform for detection of leptin in human with different body mass index

Obesity diseases have drawn our attention attributing to unhealthy dietary habit. Leptin, as a peptide hormone produced primarily in adipose tissue, is a biomarker associated with obesity diseases. Here, we presented an electrochemical biosensor used reduced graphene oxide-gold nanomaterials (rGO-Au) to modify the glassy carbon electrode (GCE) and bionanocapsule-ZZ (ZZ-BNC) to vertically orient the antibody for detection of leptin. The linear correction curves of leptin concentration ranged from 0.001 to 1000 pg/mL with a low detection limit (0.00087 pg/mL). Compared with reported leptin sensors, the biosensor displayed low detection limit, higher selectivity and anti-interference. Notably, our biosensor enabled to detect the leptin in diet-induced obese mouse model with a good agreement of enzyme linked immunosorbent assay (ELISA). The distinct detection results also showed in human with different body mass index (BMI) using the biosensor. Therefore, our study demonstrated that the oriented antibody immobilization based electrochemical biosensor is an effective platform for detection of leptin in actual samples and may have broad applications in assessment of obesity diseases.

Facile engineered polymeric microdevice via co-coupling of phenylboronic acid and Protein A for oriented antibody immobilization enables substantial signal enhancement for an enhanced fluorescence immunoassay

The powerful immunoassay has attracted increasing attention for detecting trace substances. Herein, based on the co-coupling of phenylboronic acid and Protein A for oriented antibodies immobilization, we proposed a polymeric device-based fluorescence immunoassay with enhanced assay performance for Alpha-fetoprotein (AFP) analysis. Unlike traditional physical absorption or chemical covalent conjugation to immobilize antibodies, in this immunoassay, the bioreactivity of AFP antibody was maximumly obtained owing to the oriented immobilization manner. The package density of AFP antibody on the polymethylmethacrylate (PMMA) surface is significantly improved because of the co-coupling case associated distance effect. In addition, the non-specific absorption is greatly inhibited with the combined using of zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC) and bovine serum albumin (BSA). These integrated features facilitate the output of strong fluorescence in the presence of AFP but emit very weak background in the absence of AFP. After a typical sandwich immunoreaction, the recorded fluorescence intensity of the immunosensor is proportional to the AFP concentration, showing excellent sensitivity, specificity, and reliability for AFP determination. Comparative studies show the co-coupling is superior than its single coupling counterparts. We envision this unique immunoassay can provide a new avenue for building immunosensors in the fields of disease diagnosis, food safety detection, and environmental monitoring.

Design and Development of Antibody Functionalized Gold Nanoparticles for Biomedical Applications.

Antibody-functionalized gold nanoparticle constitutes a powerful interface biosystem for biomedical applications where the properties of gold nanoparticles and the specificity of antibody-antigen interactions are combined. This study provides insight into the key factors for the development of antibody functionalized gold nanoparticles focusing on the immobilization of the antibody. Here, we address an oriented antibody immobilization procedure on gold nanoparticles. It comprises chelatemodified gold nanoparticles that are designed for oriented immobilization of IgG antibodies (end on spatial orientation) through the metal-chelation to histidine-rich metal binding site in the heavy chain (Fc) of the antibody.

Preparation and application of immunoaffinity in-tube solid phase microextraction column with oriented antibody-immobilized porous layer open tubular capillary for high sensitive quantification of serum extracellular domain of human epidermal growth factor receptor 2 levels

Human epidermal growth factor receptor 2 (HER2) gene expresses a transmembrane glycoprotein that is over-expressed in 15–30% breast, 3% lung, and other several digestive cancers. So HER2 is a good biomarker for tumor diagnostic and treatment monitoring. Clinically, detection of HER2 often employs invasive approaches with tissue samples, which at large extent limit its universal application. Shedding of the extracellular domain (ECD) of the HER2 (HER2-ECD) into the circulation has led to the development of a serum test of HER2-ECD as an additional approach to probe the HER2 overexpression. However, few methods were developed due to the high sensitivity required by the serum HER2-ECD determination. In this work, we prepared a novel immunoaffinity in-tube solid phase microextraction (IT-SPME) sorbent for selective enrichment of HER2-ECD. Two clinical available monoclonal antibodies against to HER2, trastuzumab and pertuzumab, were selected as immunoaffinity ligands. Porous layer open tubular capillary with oriented antibody immobilization were fabricated and systematically optimized to afford a higher extraction capacity. The capacity was reached to 120.4 μg/m, which is more than 1000 times higher than that obtained by a common method (directly antibody immobilization on a naked capillary). After sample extraction and enrichment by the IT-SPME, the eluent were determined by a particle-enhanced turbidimetric immunoassay (PETIA). Sensitive quantification of HER2-ECD by the PETIA was thereby accomplished. HER2-ECD concentrations in 82 clinical serum samples were determined by the developed IT-SPME/PETIA method, and the results were well-correlated with that by the clinical used chemiluminescence immunoassay (CLIA). Besides, the IT-SPME/PETIA method was found providing 5 times higher sensitivity than the CLIA, and 500 times higher than the PETIA without IT-SPME. The results indicate that the developed method is suitable for high-sensitive quantification of HER2-ECD in clinical samples.

Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol.

The sensing properties of immunosensors are determined not only by the amount of immobilized antibodies but also by the number of effective antigen-binding sites of the immobilized antibody. Protein A (PA) exhibits a high degree of affinity with the Fc part of IgG antibody to feasibly produce oriented antibody immobilization. This work proposes a simple method to control the PA surface density on gold nanostructure (AuNS)-deposited screen-printed carbon electrodes (SPCEs) by mixing concentration-varied PA and bovine serum albumin (BSA), and to explore the effect of PA density on the affinity attachment of anti-salbutamol (SAL) antibodies by electrochemical impedance spectroscopy. A concentration of 100 μg/mL PA and 100 μg/mL BSA can obtain a saturated coverage on the 3-mercaptoproponic acid (MPA)/AuNS/SPCEs and exhibit a 50% PA density to adsorb the amount of anti-SAL, more than other concentration-varied PA/BSA-modified electrodes. Compared with the randomly immobilized anti-SAL/MPA/AuNS/SPCEs and the anti-SAL/PA(100 μg/mL):BSA(0 μg/mL)/MPA/AuNS/SPCE, the anti-SAL/PA(100 μg/mL): BSA(100 μg/mL)/MPA/AuNS/SPCE-based immunosensors have better sensing properties for SAL detection, with an extremely low detection limit of 0.2 fg/mL and high reproducibility (<2.5% relative standard deviation). The mixture of PA(100 μg/mL):BSA(100 μg/mL) for the modification of AuNS/SPCEs has great promise for forming an optimal protein layer for the oriented adsorption of IgG antibodies to construct ultrasensitive SAL immunosensors.

Regenerable myoglobin biosensor based on protein G immobilized on interdigitated electrodes

A simple, sensitive and regenerable biosensor was developed for determining the concentration of myoglobin (Myo). Protein G was employed as an antibody-linking agent, which affords oriented antibody immobilization and the ability to regenerate the sensor. The Myo-antibody of anti-Myo-IgG was immobilized on the protein G layer with its Fc binding domain facing the solution side and thus its orientation allowed easy access for Myo molecules. Electrochemical impedance spectroscopy was used to evaluate the Myo sensor performance. The sensor was able to detect Myo between 10 pg ml−1 and 1 μg ml−1. Regeneration of the sensor was achieved by immersing the electrodes in 0.2 M glycine buffer and by immobilizing fresh antibodies in neutral buffer. The response of the regenerated sensor to Myo was 65% that of the response of the initial sensor.

Highly Sensitive SPR Biosensor Based on Graphene Oxide and Staphylococcal Protein A Co-Modified TFBG for Human IgG Detection

A highly sensitive optical fiber surface plasmon resonance (SPR) biosensor based on graphene oxide (GO) and staphylococcal protein A (SPA) co-modified tilted fiber Bragg grating (TFBG) is proposed and demonstrated for the detection of human immunoglobulin G (IgG) for the first time. The gold film on the surface of the sensor was first fixed with GO and then modified with an SPA to improve the sensitivity of the sensor. Large specific surface area and abundant functional groups of GO can adsorb more antibodies. The combination of SPA and the antibody molecule Fc region makes the Fab area with antigen-binding sites extend outward, resulting in highly oriented antibody immobilization on the sensor surface and high antigen–antibody binding efficiency. The experimental results show that the sensitivity as well as the limit of detection of GO-SPA-modified TFBG-SPR biosensor is around 0.096 dB/( $\mu \text{g}$ /mL) and $0.5~\mu \text{g}$ /mL, showing better responses to human IgG solutions with a concentration range of 30– $100~\mu \text{g}$ /mL compared with the TFBG-SPR biosensors modified singly with GO or SPA. The biosensor exhibits the advantages of small size, ease of fabrication, high sensitivity, label-free, and rapid response, and provides a new solution for detecting low concentration of biological solution, presenting great application potential in the biochemistry field.

Erratum to “Comparative Assessment of Affinity-Based Techniques for Oriented Antibody Immobilization towards Immunosensor Performance Optimization”

Erratum to “Comparative Assessment of Affinity-Based Techniques for Oriented Antibody Immobilization towards Immunosensor Performance Optimization”

A novel oriented antibody immobilization based voltammetric immunosensor for allergenic activity detection of lectin in kidney bean by using AuNPs-PEI-MWCNTs modified electrode

As a well-known allergenic indicator in kidney beans, lectins have always been the serious threats for human health. Herein, we introduced a new label-free voltammetric immunosensor for the direct determination of kidney bean lectin (KBL) with potential allergenic activity. Gold nanoparticles-polyethyleneimine-multiwalled carbon nanotubes nanocomposite was one-pot synthesized and modified onto the glass carbon electrode to enhance catalytic currents of oxygen reduction reaction. The KBL polyclonal antibody, acquired from rabbit immunization, was orientedly immobilized on the electrode modified with recombinant staphylococcal protein A via fragment crystallizable (Fc) region of antibody. Under the optimized condition, the immunosensor displayed a good linear response (R2 = 0.978) to KBL with a range from 0.05 to 100 μg/mL and a detection limit of 0.023 μg/mL. Simultaneously, the immunosensor exhibited well selectivity, interference-resistant ability, stability (4 °C) and reproducibility. Compared with the conventional enzyme-linked immunosorbent assay (ELISA) method, the immunosensor was successfully applied to quantify allergenic activity of lectin in raw and cooked (boiled for 30 min) kidney bean milk samples. This new approach provides new perspectives both for rapid quantification of lectin in kidney beans-derived foodstuffs and as a real-time monitoring tool for the allergenic potential during the whole production and consumption process.

Comparative Assessment of Affinity-Based Techniques for Oriented Antibody Immobilization towards Immunosensor Performance Optimization

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.

Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range

There is a great need for targeted protein assays with the capacity of sensitive measurements in complex samples such as plasma or serum, not the least for clinical purposes. Proteomics keeps generating hundreds of biomarker candidates that need to be transferred towards true clinical application through targeted verification studies and towards clinically applicable analysis formats. The immunoaffinity assay AFFIRM (AFFInity sRM) combines the sensitivity of recombinant single chain antibodies (scFv) for targeted protein enrichment with a specific mass spectrometry readout through selected reaction monitoring (SRM) in an automated workflow. Here we demonstrate a 100 times improved detection capacity of the assay down to pg/ml range through the use of oriented antibody immobilization to magnetic beads. This was achieved using biotin-tagged scFv coupled to streptavidin coated magnetic beads, or utilizing the FLAG tag for coupling to anti-FLAG antibody coated magnetic beads. An improved multiplexing capacity with an 11-plex setup was also demonstrated compared to a previous 3-plex setup, which is of great importance for the analysis of panels of biomarker targets.

Development of hierarchical Fe3O4 magnetic microspheres as solid substrates for high sensitive immunoassays.

Improving the detection sensitivity of enzyme linked immunosorbent assay (ELISA) is of the utmost importance for meeting the demand of early disease diagnosis. In this work, a sensitive solid substrate for ELISA, i.e., hierarchical iron oxide magnetic microspheres, Fe3O4@mSiO2@poly[poly(ethylene glycol) methacrylate-co-glycidyl methacrylate], was developed via a novel surface-initiated photoiniferter-mediated polymerization (SI-PIMP) strategy. The magnetic microspheres consist of a magnetic Fe3O4 core that gives a high magnetic response, a 3D backbone, a mesoporous SiO2 middle layer, that facilitates microsphere stability and provides anchoring sites, and polymer brushes, that serve as an antifouling and oriented antibody immobilization layer. As a result, the as-prepared microspheres possess a high antibody loading capacity, an enhanced detection signal and a dramatically improved sensitivity, resulting in a 25-fold improvement over conventional ELISA solid substrates.

Oriented Antibody Immobilization and Immunoassay Based on Boronic Acid-containing Polymer Brush

High sensitive immunoassay platforms have gained intense attention due to their vital roles in early-stage disease diagnosis and therapeutic information feedback. Although random covalent-binding of antibody has been widely adopted in immunoassays due to its simplicity and effectiveness, it readily loses its activity and fails to exhibit high antigen-binding capacity. In this work, copolymer of zwitterionic sulfobetaine methacrylate (SBMA) and glycidyl methacrylate (GMA) brushes were immobilized onto inert polypropylene (PP) via surface-initiated atom transfer radical polymerization (ATRP) based on biomimetic dopamine pretreatment. Subsequently, boronic acid (BA) groups were covalently bonded via active GMA units, followed by the introduction of oriented immobilization of antibody. Owing to the oriented immobilization of antibody facilitated by BA groups in polymer brush, the bioactivity of antibody is well preserved, which endows the surface with significantly enhanced antigen-binding capacity. Moreover, the existence of SBMA segments in polymer brushes renders the surface high resistance to nonspecific protein adsorption, significantly alleviating the signal interference of antigen recognition. This strategy could find potential applications in developing high sensitive immunoassay platforms based on the different substrates.