Artificial Antibodies Research Articles

Copper mediated molecularly imprinted polymers for fast recognizing tylosin

As one kind of artificial antibody, molecularly imprinted polymers (MIPs) has been widely used to separate/enrich target molecules from samples with the complex matrix prior to instrumental analysis. In this work, one novel copper mediated magnetic MIPs was developed towards anti-bacteria macrolide antibiotic tylosin (TYL). The obtained microspheres had a lot of convexities distributed evenly on the surface. And it exhibited high hydrophilicity and superparamagnetic ability, as well as excellent selectivity and specificity. Notably, it only took 5 min to reach the absorption equilibrium for TYL, which made it highly feasible for high-throughput analysis. Furthermore, the microspheres was applied as the magnetic dispersed solid phase extraction material to enrich trace TYL residue in (spiked) human urine. At three spiking levels, mean recoveries are in the range of 76.42–93.72% with relative standard deviations of 2.75–8.24% (n = 3) with HPLC/UV-Vis. The work provided one promising reference to prepare novel solid phase extraction materials for enriching/separating trace component in complex matrix.

A Potential MDM2 Inhibitor Formed by Restoring the Native Conformation of the p53 α-Helical Peptide on Gold Nanoparticles.

Many efforts have been made to develop inhibitors of MDM2 as potential drugs for cancer therapy. In this work, we use our previous developed conformational engineering technique to stabilize the binding conformation of the p53 transcription activation domain (TAD) peptide on gold nanoparticles (AuNPs), and create an AuNP-based anti-MDM2 artificial antibody, denoted as anti-MDM2 Goldbody, that specifically binds MDM2. Though the free TAD peptide is unstructured, circular dichroism (CD) spectra confirm that its α-helical conformation in the original p53 protein is restored on the anti-MDM2 Goldbody, and surface plasmon resonance (SPR) experiments confirm that there is strong specific interaction between the anti-MDM2 Goldbody and MDM2, demonstrating the anti-MDM2 Goldbody as a potential inhibitor of MDM2. This work demonstrates that the conformational engineering technique is not limited to the antigen-antibody systems, but can also be applied more widely in other protein-protein interfaces to create increasingly more artificial proteins for various biomedical applications.

Sensitive and selective detection of carbamazepine in serum samples by bionic double-antibody sandwich method based on cucurbit[7]uril and molecular imprinted polymers

A novel bionic enzyme-linked immunosorbent assay (BELISA) based on double-antibody sandwich method is firstly designed for the detection of carbamazepine (CBZ) in human serum samples. In this BELISA system, cucurbit[7]uril (CB[7]) is employed as an artificial capture antibody (cAb), and molecularly imprinted polymers (MIPs) is used as an artificial detection antibody (dAb). Nanozymes (PdNPs) as signal generators are integrated with MIPs. This couple of bionic antibodies exhibits not only the excellent physical and chemical stability, but also the superior molecular recognition ability. Based on two bionic antibodies that can selectively recognize different sites of CBZ molecule, a new BELISA method has been constructed for the first time. The proposed BELISA method displays a good linear relationship ranging from 2 to 20 μg mL-1. The detection limit is 0.37 μg mL-1, which can well meet clinical testing demand. It provides a more stable and economical method for clinical therapeutic drug monitoring (TDM).

An artificial antibody for exosome capture by dull template imprinting technology.

Exosomes are extracellular vesicles with unique size distribution derived from the parent cells. They are involved in intercellular communication and transport, and are also biomarkers for early diagnosis and prognosis of disease. However, the isolation and characterization of exosomes face the challenges of large sample requirements and low enrichment efficiency of traditional methods. Herein, a simple method is proposed for the preparation of an artificial antibody that has a synergistic effect by featuring nanocavities obtained by dull template imprinting and molecular recognition conferred by electrostatic interaction. With this artificial antibody, highly efficient capture and proteome analysis of exosomes from urine and cell culture media are achieved: for urine, the abundance of Top100 exosomal proteins enriched by this artificial antibody increased from 1.85% to 9.66%. For the cell culture medium, the abundance of the Top100 proteins enriched by this artificial antibody was 28.4%. Moreover, this artificial antibody has a comparable effect to the commercial precipitation-based method in capturing exosomes and has advantages in removing contaminants such as prothymosin alpha (PTMA), demonstrating the superior selectivity of the artificial antibody. Overall, this artificial antibody holds promise to capture exosomes from biofluids and is compatible with subsequent proteome analysis.

Rational Design of Nanoparticle-Based Artificial Antibody Specifically Targeting Different Locations on the Same Protein

Rational Design of Nanoparticle-Based Artificial Antibody Specifically Targeting Different Locations on the Same Protein

PEGylation of Goldbody: PEG-aided conformational engineering of peptides on gold nanoparticles.

It is still a great challenge to engineer flexible non-functional molecules into special conformations to carry out novel functions. Previously, we successfully restored the native conformations and functions of the flexible complementary-determining regions (CDRs) of antibodies on the surface of gold nanoparticles (AuNPs), and created a class of AuNP-based artificial antibodies, denoted as Goldbodies. Yet, in these Goldbodies, there are dozens of CDRs on one Goldbody. Herein, we show that the number of CDRs per Goldbody could be reduced by more than one order of magnitude, by replacing the majority of the CDRs with polyethylene glycol (PEG) with a molecular weight around 600 Da, while the native conformations and functions of the CDRs could still be restored on AuNPs. Also, we find that the PEG with two terminal –SH groups is much better than the PEG with a single –SH group for aiding the restoration of the native conformation of the CDRs on AuNPs. To demonstrate the potential generic applicability of the PEGylation in aiding conformational engineering of peptides, two PEGylated Goldbodies have been created, which can specifically recognize lysozyme and epidermal growth factor receptor, respectively. The PEGylated Goldbodies further prove the mechanism of conformational engineering and the “Confined Lowest Energy Fragments” (CLEFs) hypothesis, and pave the way for future applications of Goldbodies.

Development of artificial antibody against receptor binding domain of SARS-CoV-2 spike protein.

Development of artificial antibody against receptor binding domain of SARS-CoV-2 spike protein.

Advances in the Determination of Anabolic-Androgenic Steroids: From Standard Practices to Tailor-Designed Multidisciplinary Approaches.

Anabolic-androgenic steroids (AASs), a group of compounds frequently misused by athletes and, unfortunately, also by the general population, have lately attracted global attention; thus, significant demands for more precise, facile, and rapid AAS detection have arisen. The standard methods ordinarily used for AAS determination include liquid and gas chromatography coupled with mass spectrometry. However, good knowledge of steroid metabolism, pretreatment of samples (such as derivatization), and well-trained operators of the instruments are required, making this procedure expensive, complicated, and not routinely applicable. In the drive to meet current AAS detection demands, the scientific focus has shifted to developing novel, tailor-made approaches leading to time- and cost-effective, routine, and field-portable methods for AAS determination in various matrices, such as biological fluids, food supplements, meat, water, or other environmental components. Therefore, herein, we present a comprehensive review article covering recent advances in AAS determination, with a strong emphasis on the increasingly important role of chemically designed artificial sensors, biosensors, and antibody- and fluorescence-based methods.

Nanostructured Shell-Layer Artificial Antibody with Fluorescence-Tagged Recognition Sites for the Trace Detection of Heavy Metal Ions by Self-Reporting Microsensor Arrays.

Herein, a strategy for a metal ion-imprinted artificial antibody with recognition sites tagged by fluorescein was carried out to construct the selective sites with a sensitive optical response signal to the specific metal ion. The synthesized silica nanoparticles were modified by the derivative residue group of 3-aminopropyltriethoxysilane conjugated with a 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) molecule through the hydrolysis and condensation reactions. The as-prepared silica nanoparticles were encapsulated by metal ion (Cu2+, Cd2+, Hg2+, and Pb2+)-imprinted polymers with nanostructured layers through the copolymerization of ethyl glycol dimethyl methacrylate (EGDMA) as a cross-linker, AIBN as an initiator, metal ions as template molecules, AA as a functional monomer, and acetonitrile as a solvent. The layers of molecular imprinted polymers (MIPs) with a core-shell structure removed template molecules by EDTA-2Na to retain the cavities and spatial sizes to match the imprinted metal ions. The microsensor arrays were achieved by the self-assembly technique of SiO2@MIP nanoparticles on the etched silicon wafer with regular dot arrays. The nanostructured-shell layers with fluorescence-tagged recognition sites rebound metal ions by the driving force of concentration difference demonstrates the high selective recognition and sensitive detection to heavy metal ions through the decline of fluorescence intensity. The LOD concentration for four metal ions is down to 10-9 mol·L-1. The method will provide biomimetic synthesis, analyte screen, and detection of highly dangerous materials in the environment for theoretical foundation and technological support.

Molecularly imprinted polymer as a synthetic antibody for the biorecognition of hazelnut Cor a 14-allergen

Artificial receptors that mimic their natural biological counterparts have several advantages, such as lower production costs and increased shelf-life stability/versatility, while overcoming the ethical issues related to raising antibodies in animals. In this work, the proposed tailor-made molecularly imprinted polymer (MIP)-allergen receptors aimed at substituting or even transcending the performance of biological antibodies. For this purpose, a MIP was proposed as an artificial antibody for the recognition of hazelnut Cor a 14-allergen. The target protein was grafted onto the conducting polypyrrole receptor film using gold screen-printed electrodes (Au-SPE). The electrochemical assessment presented a linear response for the dynamic range of 100 fg mL−1-1 μg mL−1 and a LOD of 24.5 fg mL−1, as determined by square wave voltammetry from the calibration curves prepared with standards diluted in phosphate buffer. Surface plasmon resonance (SPR) was used as a secondary transducer to evaluate the performance of the Cor a 14-MIP sensor, enabling a linear dynamic range of 100 fg mL−1- 0.1 μg mL−1 and a LOD of 18.1 fg mL−1. The selectivity of the tailored-made Cor a 14-MIP was tested against potentially cross-reactive plant/animal species based on the rebinding affinity (Freundlich isotherm-KF) of homologues/similar proteins, being further compared with custom-made polyclonal anti-Cor a 14 IgG immunosensor. Results evidenced that the MIP mimics the biorecognition of biological antibodies, presenting higher selectivity (only minor cross-reactivity towards walnut and Brazil nut 2S albumins) than the Cor a 14/anti-Cor a 14 IgG immunosensor. The application of electrochemical Cor a 14-MIP sensor to model mixtures of hazelnut in pasta enabled quantifying hazelnut down to 1 mg kg−1 (corresponding to 0.16 mg kg−1 of hazelnut protein in the matrix). To the best of our knowledge, Cor a 14-MIP is the first sensor based on an artificial/synthetic biorecognition platform for the specific detection of hazelnut allergens, while presenting high-performance parameters with demonstrated application in food safety management.

Graphene-Based Femtogram-Level Sensitive Molecularly Imprinted Polymer of SARS-CoV-2.

Rapid distribution of viral‐induced diseases and weaknesses of common diagnostic platforms for accurate and sensitive identification of infected people raises an urgent demand for the design and fabrication of biosensors capable of early detection of viral biomarkers with high specificity. Accordingly, molecularly imprinted polymers (MIPs) as artificial antibodies prove to be an ideal preliminary detection platform for specific identification of target templates, with superior sensitivity and detection limit (DL). MIPs detect the target template with the “lock and key” mechanism, the same as natural monoclonal antibodies, and present ideal stability at ambient temperature, which improves their practicality for real applications. Herein, a 2D MIP platform consisting of decorated graphene oxide with the interconnected complex of polypyrrole‐boronic acid is developed that can detect the trace of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) antigen in aquatic biological samples with ultrahigh sensitivity/specificity with DL of 0.326 and 11.32 fg mL–1 using voltammetric and amperometric assays, respectively. Additionally, the developed MIP shows remarkable stability, selectivity, and accuracy toward detecting the target template, which paves the way for developing ultraspecific and prompt screening diagnostic configurations capable of detecting the antigen in 1 min or 20 s using voltammetric or amperometric techniques.

Molecularly imprinted polymer-based sensors for cancer biomarker detection

Ultrasensitive, quick, and non-invasive recognition of biomarkers is very useful for detection, screening, and management of cancer effectively. In this regard, there have been enormous number of articles published each year comprising natural biomolecules e.g., antibodies, enzymes, receptors and aptamers as a recognition element of sensors for the detection of cancer biomarkers. However, due to stability issue and high-cost of natural biomolecules, polymeric material-based recognition element involving molecular imprinting has drawn a huge attention over the last couple of decades. The molecularly imprinted polymers (MIPs), also known as “artificial antibodies”, show sensitivity comparable to natural receptors. This review aims to deliver an insight and potential application of the MIPs towards cancer biomarker detection methods. Some discussion about classical recognition elements is also included here for better understanding the biomimicking approach. Many industries have already proved the potential of the new alterative sensing platform and if some of the drawbacks can be addressed carefully during the synthesis of the MIPs, we hope to see MIP-based diagnostic kits for cancer detection. The review is aimed to help the readers and the researchers to understand the potential of MIPs in cancer biomarker detection as well as recognition element for sensors.

Higher Order Protein Catenation Leads to an Artificial Antibody with Enhanced Affinity and In Vivo Stability.

The chemical topology is a unique dimension for protein engineering, yet the topological diversity and architectural complexity of proteins remain largely untapped. Herein, we report the biosynthesis of complex topological proteins using a rationally engineered, cross-entwining peptide heterodimer motif derived from p53dim (an entangled homodimeric mutant of the tetramerization domain of the tumor suppressor protein p53). The incorporation of an electrostatic interaction at specific sites converts the p53dim homodimer motif into a pair of heterodimer motifs with high specificity for directing chain entanglement upon folding. Its combination with split-intein-mediated ligation and/or SpyTag/SpyCatcher chemistry facilitates the programmed synthesis of protein heterocatenane or [n]catenanes in cells, leading to a general and modular approach to complex protein catenanes containing various proteins of interest. Concatenation enhances not only the target protein's affinity but also the in vivo stability as shown by its prolonged circulation time in blood. As a proof of concept, artificial antibodies have been developed by embedding a human epidermal growth factor receptor 2-specific affibody onto the [n]catenane scaffolds and shown to exhibit a higher affinity and a better pharmacokinetic profile than the wild-type affibody. These results suggest that topology engineering holds great promise in the development of therapeutic proteins.

Solid-phase synthesis of imprinted nanoparticles as artificial antibodies against the C-terminus of the cannabinoid CB1 receptor: exploring a viable alternative for bioanalysis

The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to Gi/o proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 ± 10.5 nm at pH 3 (25 ºC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 ± 15.2 nm at 35 °C. Lower critical solution temperature of this polymer was found to be ≈ 33.4 °C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1414-472 and GST-CB1414-442 were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR).Graphical abstract

Antibody Mimics as Bio-orthogonal Catalysts for Highly Selective Bacterial Recognition and Antimicrobial Therapy.

Bacterial infectious diseases seriously threaten public health and life. The specific interaction between an antibody and its multivalent antigen is an attractive way to defeat infectious disease. However, due to the high expense and strict storage and applied conditions for antibodies, it is highly desirable but remains an urgent challenge for disease diagnosis and treatment to construct artificial antibodies with strong stability and binding ability and excellent selectivity. Herein, we designed and synthesized antibody-like bio-orthogonal catalysts with the ability to recognize specific bacteria and accomplish in situ drug synthesis in captured bacteria by using improved bacterial imprinting technology. On one hand, the artificial antibody possesses a matching morphology for binding pathogens, and on the other hand, it acts as a bio-orthogonal catalyst for in situ synthesis of antibacterial drugs in live bacteria. Both in vitro and in vivo experiments have demonstrated that our designed antibody can distinguish and selectively bind to specific pathogens and eliminate them on site with the activated drugs. Therefore, our work provides a strategy for designing artificial antibodies with bio-orthogonal catalytic activity and may broaden the application of bio-orthogonal chemistry.

Template Theories, the Rule of Parsimony, and Disregard for Irreproducibility—The Example of Linus Pauling’s Research on Antibody Formation

In 1940, Linus Pauling proposed his template theory of antibody formation, one of many such theories that rejected Paul Ehrlich’s selective theory of preformed “receptors” (antibodies), assuming instead a direct molding of antibody shapes onto that of the antigen. Pauling believed that protein shapes—independently of amino acid sequences—determined antibody specificity and biological specificity in general. His theory was informed by his pioneering work on protein structure, and it was inspired by the intuitive “rule of parsimony” and simplicity. In 1942, Pauling published his alleged success in producing specific artificial antibodies through experiments based on his 1940 theory. However, his experiments could not be reproduced by prominent immunochemists at the time, and, later, it became generally accepted that antibody specificity was not generated according to Pauling’s and others’ “instruction” template theories. A citation analysis shows that Pauling’s papers on antibody generation continue to be cited as, among other things, pioneering studies of a chemical technology called “molecular imprinting.” The examples of Pauling and other protein chemists are used in this paper to demonstrate that scientific belief, philosophical concepts, and subjective theory preferences facilitated the occurrence of irreproducibility in immunochemistry and beyond. The article points to long-term consequences for the scientific community if irreproducible results are not acknowledged. It concludes by arguing that despite the risks, e.g., for the occurrence and perpetuation of irreproducible results that they entail, subjectivity and a commitment to scientific convictions have often been pre-requisites for the generation, and holding on to, scientific innovation in the face of doubt and rejection from the scientific community.

One year on: The impact of COVID-19 on clinical research

One year on: The impact of COVID-19 on clinical research

Transition-Metal Dichalcogenide Artificial Antibodies with Multivalent Polymeric Recognition Phases for Rapid Detection and Inactivation of Pathogens.

Antibodies are recognition molecules that can bind to diverse targets ranging from pathogens to small analytes with high binding affinity and specificity, making them widely employed for sensing and therapy. However, antibodies have limitations of low stability, long production time, short shelf life, and high cost. Here, we report a facile approach for the design of luminescent artificial antibodies with nonbiological polymeric recognition phases for the sensitive detection, rapid identification, and effective inactivation of pathogenic bacteria. Transition-metal dichalcogenide (TMD) nanosheets with a neutral dextran phase at the interfaces selectively recognized S. aureus, whereas the nanosheets bearing a carboxymethylated dextran phase selectively recognized E. coli O157:H7 with high binding affinity. The bacterial binding sites recognized by the artificial antibodies were thoroughly identified by experiments and molecular dynamics simulations, revealing the significance of their multivalent interactions with the bacterial membrane components for selective recognition. The luminescent WS2 artificial antibodies could rapidly detect the bacteria at a single copy from human serum without any purification and amplification. Moreover, the MoSe2 artificial antibodies selectively killed the pathogenic bacteria in the wounds of infected mice under light irradiation, leading to effective wound healing. This work demonstrates the potential of TMD artificial antibodies as an alternative to antibodies for sensing and therapy.

Catalytic Clusterbody for Enhanced Quantitative Protein Immunoblot.

To assess low-abundance protein biomarkers associated with tumor progression, we have developed artificial catalytic antibodies based on well-defined metal clusters modified with rationally designed peptides, termed clusterbodies. Such clusterbodies possess favorable integrated features of matched ultrasmall sizes, intrinsic fluorescence, and enzyme-like catalytic and selective recognition properties that are inaccessible to traditional antibodies. Consequently, a quantitative assay with high accuracy and high sensitivity is established by measuring the fluorescence and catalytic chemiluminescence of metal clusters preferentially recognizing the protein biomarker, which is confirmed by the molecular-weight marker references of immunoblotting. The results of quantitative immunoblotting are highly close to that derived from the enzyme-linked immunosorbent assay, implying the reliability of this protocol. Remarkably, the detection limit of the aimed protein achieved is as low as 1.0 pg, one magnitude lower than that of the conventional immunoassay. The significant variation of expression levels of the biomarker in tumor cells evidently indicates their distinguished invasion ability. This platform has potential application in analyzing low-abundance protein biomarkers in complex biological matrixes, which is essential to corroborate tumor malignancy in early stage. It inspires the construction of clusterbody-based precise bioprobes with customized structures and integrative functions for advanced quantitative biosensing.

Abiotic Mimic of Matrix Metalloproteinase-9 Inhibitor against Advanced Metastatic Cancer.

As the most representative family of proteinases related to tumorigenesis, matrix metalloproteinase-9 (MMP-9) represents a key player in cancer cell migration and regulation of the tumor microenvironment. The inhibition of MMP-9 activity has been pursued as a target for anticancer therapy. However, most synthetic MMP-9 inhibitors have failed in clinical trials because of their lack of selectivity. Here, an abiotic mimic based on molecularly imprinted nanoparticles has been designed as an inhibitor for MMP-9. To attain fast mass transfer and facilitate multifunctional roles, we synthesized the imprinted polymer thin layer on the surface of gold nanorods by reversible addition-fragmentation chain transfer polymerization using MMP-9 as the template, which captures MMP-9 selectively and inhibits its activity by providing steric hindrance to the activity-related domain of MMP-9. In vitro cell experiments and in vivo studies in mice demonstrate that the imprinted artificial antibody suppresses the migration and growth of metastatic tumors. The tumor growth inhibition rate reaches up to 54 ± 15%. Compared with the typical photothermal therapy induced by gold nanorods, the use of MMP-9-imprinted synthetic antibody could better inhibit the lung tumor metastasis by quenching the enzyme activity of MMP-9. This study offers a new paradigm in the engineering of imprinted nanoparticles as inhibitors for cancer therapy.