Artificial Antibodies Research Articles

Lossy Mode Resonance Based Fiber Optic Creatinine Sensor Fabricated Using Molecular Imprinting Over Nanocomposite of MoS2/SnO2

Creatinine (CR) produced through the muscle metabolism acts as biomarker to monitor the kidney functioning of the human body. In this study, a successful effort has been made to develop a fast and easy detection method for the monitoring of CR concentration in an aqueous solution as well as in the artificial urine sample. The sensor has been developed over a lossy mode resonance (LMR) based optical fiber platform using MoS2@SnO2 nanocomposite as LMR supporting material and MoS2@SnO2 nanocomposite along with CR imprinted polymer film as artificial antibodies. The sensor’s performance has been studied for the CR concentration range from 0 to $2000~\mu \text{g}$ /mL which lies within the physiological range found in human blood and urine. The maximum sensitivity and detection limit of the sensor have been found to be 0.41 nm/( $\mu \text{g}$ /mL) and $1.86~\mu \text{g}$ /mL, respectively. The sensor has several advantages such as high selectivity, long-term stability, repeatability and fast response. The recovery of the sensor probes close to 100% with the artificial urine sample shows its potential use in the biomedical application.

Molecularly Imprinted Synthetic Antibodies: From Chemical Design to Biomedical Applications.

Billions of dollars are invested into the monoclonal antibody market every year to meet the increasing demand in clinical diagnosis and therapy. However, natural antibodies still suffer from poor stability and high cost, as well as ethical issues in animal experiments. Thus, developing antibody substitutes or mimics is a long-term goal for scientists. The molecular imprinting technique presents one of the most promising strategies for antibody mimicking. The molecularly imprinted polymers (MIPs) are also called "molecularly imprinted synthetic antibodies" (MISAs). The breakthroughs of key technologies and innovations in chemistry and material science in the last decades have led to the rapid development of MISAs, and their molecular affinity has become comparable to that of natural antibodies. Currently, MISAs are undergoing a revolutionary transformation of their applications, from initial adsorption and separation to the rising fields of biomedicine. Herein, the fundamental chemical design of MISAs is examined, and then current progress in biomedical applications is the focus. Meanwhile, the potential of MISAs as qualified substitutes or even to transcend the performance of natural antibodies is discussed from the perspective of frontier needs in biomedicines, to facilitate the rapid development of synthetic artificial antibodies.

Antibody-Free Hydrogel with the Synergistic Effect of Cell Imprinting and Boronate Affinity: Toward the Selective Capture and Release of Undamaged Circulating Tumor Cells.

The selective and highly efficient capture of circulating tumor cells (CTCs) from blood and their subsequent release without damage are very important for the early diagnosis of tumors and for understanding the mechanism of metastasis. Herein, a universal strategy is proposed for the fabrication of an antibody-free hydrogel that has a synergistic effect by featuring microinterfaces obtained by cell imprinting and molecular recognition conferred by boronate affinity. With this artificial antibody, highly efficient capture of human hepatocarcinoma SMMC-7721 cells is achieved: as many as 90.3 ± 1.4% (n = 3) cells are captured when 1 × 105 SMMC-7721 cells are incubated on a 4.5 cm2 hydrogel, and 99% of these captured cells are subsequently released without any loss of proliferation ability. In the presence of 1000 times as many nontarget cells, namely, leukaemia Jurkat cells, the SMMC-7721 cells can be captured with an enrichment factor as high as 13.5 ± 3.2 (n = 3), demonstrating the superior selectivity of the artificial antibody for the capture of the targeted CTCs. Most importantly, the SMMC-7721 cells can be successfully captured even when spiked into whole blood, indicating the great promise of this approach for the further molecular characterization of CTCs.

Gold star–carbon nanotube composite for analysing preeclampsia during pregnancy

Preeclampsia is the condition identified with a high blood pressure and protein in the urine during the period of pregnancy, which affects 6–8% of pregnant women after 20 weeks of pregnancy. Untreated preeclampsia causes serious health issues to the mother and baby. Diagnosing followed by treating preeclampsia with a suitable biomarker is mandatory for healthy delivery. Cortisol, the steroid hormone secreted from the adrenal glands, helps to regulate various physiological processes, such as immune response, metabolism and blood pressure. In this research, cortisol was quantified on carbon nanotube (CNT)-modified interdigitated electrode sensor. Aptamer, an artificial antibody having a high binding affinity to the target, was utilized for quantifying the cortisol. Further, to enhance the detection, gold star was conjugated with aptamer and immobilized on CNT. The limit of cortisol detection was found in the range of 7–15 pg/mL. Selective detection of cortisol was analysed with two different hormones, namely norepinephrine and progesterone, and it was found that aptamer recognizes only cortisol. This strategy with the nanocomposite helps in diagnosing preeclampsia during the period of pregnancy.

Handheld Aptasensor for Sandwiched Detection of Chloramphenicol

Chloramphenicol(CAP) is an antifungal agent approved for use in aquarium. However, CAP residue imparts serious adverse effect on human health as well as the ecology. Thus, keeping track of CAP in aquatic products and its environmental metabolites are of great importance. Herein, we developed a novel and neat electrochemical aptasensor for the detection of simulated CAP residual in a sandwich fashion. The screen-printed carbon electrode was employed as the sensing platform and electrodeposited with peculiar nanogolds to enhance the electrochemical performance. For the specific recognition of such small target, a pair of chloramphenicol-binding aptamers, the primary Apt1 and the secondary Apt2, were tailored as an artificial single-chained antibody couple, of which the thiolated Apt1 stood on the coated working substrate to capture the analyte, while the biotinylated Apt2 rallied the signal output. In the presence of CAP, Apt1 at the lower place cooperated with the upper Apt2 pinched CAP together. After that, the reporter avidin-conjugated horseradish peroxidase associated atop the mounted biotin labels, resulting in an electrocatalytic amplification. In whole, this modular biosensing construction would set a paradigm not just for practicable checkpoint quarantine or point-of-care testing, but potentially adaptable for technological fusion with “Internet Plus” and “Internet of Things”.

Immunological tests and their interpretation in uveitis.

Uveitis is a complex disorder including both infectious and non-infectious etiologies. Clinical diagnosis is a challenge because many diseases share common clinical signs. Laboratory support is crucial for confirming the clinical diagnosis. Laboratory diagnosis includes direct tests and indirect tests. For example smear, culture, and molecular diagnostics demonstrate the pathogens, hence they are direct tests. Immunologic tests employ an antigen to detect presence of antibodies to a pathogen, or an antibody to detect the presence of an antigen, of the pathogen in the specimens. The immunological tests used in laboratories are made by producing artificial antibodies that exactly “match” the pathogen in question. When these antibodies come into contact with a sample they bind to the matching pathogen if found in the sample. Hence they are grouped under indirect evidence. There are several investigations in uveitis to reach the confirmed diagnosis including microbiological, immunological, imaging and molecular diagnostic testing. In this section we will discuss immunological investigations of infectious and non-infectious uveitis.

A fluorescence microplate assay based on molecularly imprinted silica coated quantum dot optosensing materials for the separation and detection of okadaic acid in shellfish.

Molecularly imprinted polymers (MIPs) are attracting substantial interest as artificial plastic antibodies because of their biometric capability for targeting small molecules. In this study, molecularly imprinted silica material-coated quantum dots (MIS-QDs) with selective recognition capability to okadaic acid (OA) were developed and characterized. The synthesized MIS-QDs with specific imprinting cavities exhibited excellent recognition capability similar to those of biological antibodies and high fluorescence (FL) quenching selectivity for OA. Furthermore, the MIS-QDs with unsaturated bonds were immobilized onto the surface of 96-well microplates by cold plasma-induced grafting. A novel direct competitive microplate assay strategy was then proposed. The FL quenching properties of the developed microplate assay showed an excellent linear relationship with OA in the range of 10.0-100.0μg/kg with a correlation coefficient of 0.9961. The limit of detection for OA was 0.25μg/kg in the shellfish samples. The mean quantitative recoveries were 92.5%-101.0% and 92.9%-101.3%, with relative standard deviations of <7.7% and 7.6% for pure solvents and purified shellfish samples, respectively. The established microplate assay strategy can be used as a rapid and high-throughput method for analyzing OA marine toxins in biological samples.

Perspective on the Future Role of Aptamers in Analytical Chemistry.

It has been almost 30 years since the invention of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology and the description of the first aptamers. In retrospect over the past 30 years, advances in aptamer development and application have demonstrated that aptamers are potentially useful reagents that can be employed in diverse areas within analytical chemistry, biotechnology, biomedicine, and molecular biology. While often touted as artificial antibodies with an ability to be selected for any target, aptamer development, unfortunately, lags behind development of analytical methodologies that employ aptamers, hindering deeper integration into the application of analytical tool development. This perspective covers recent advances in SELEX methodology for improving efficiency of the SELEX procedure and enhancing affinity and specificity of the selected aptamers, what we view as a critical barrier in the future role of aptamers in analytical chemistry. We discuss postselection modifications that can be used for enhancing performance of the selected aptamers in an analytical device by including understanding intermolecular interaction forces in the binding domain. While highlighting promising properties of aptamers that enable several analytical advances, we provide discussion on the challenges of penetration of aptamers in the analytical field.

High-Affinity Detection of Metal-Mediated Nephrotoxicity by Aptamer Nanomaterial Complementation

: Nephrotoxicity, a chronic renal disease that results from the accumulation of endogenous and exogenous toxins in the kidney, disturbs the excretion and detoxification function of the kidney. Metal-mediated nephrotoxicity is induced by toxic metals/metalloids such as mercury, lead, arsenic, chromate, uranium, and cadmium. These materials become concentrated in the kidneys and injure the nephrons. Developing strategies to detect these metal ions will enable the earlier identification of kidney damage. An aptamer, an artificial antibody generated against a wide range of targets including metal ions, may be the right tool for the detection of metal ions associated with renal injury. The use of a detection system consisting of an aptamer and metallic nanoparticles is a potential way to overcome nephrotoxicity. Here, we discuss the detection of metal-mediated nephrotoxicity caused by metals/metalloids using the aptamer and nanomaterial-conjugated system.

Recent Advances in Identifying Biomarkers and High-Affinity Aptamers for Gynecologic Cancers Diagnosis and Therapy.

Cancer is the uncontrollable abnormal division of cell growth, caused due to the varied reasons. Cancer can be expressed in any part of the body, and it is one of the death-causing diseases. Human reproductive organs are commonly damaged by cancer. In particular, the women reproductive system is affected by various cancers including ovarian, cervical, endometrial, vaginal, fallopian tube, and vulvar cancers. Identifying these cancers at earlier stages prevents the damage to the organs. Aptamer is the potential probe that can identify these cancers. Aptamer is an artificial antibody selected from the randomized library of molecules and has a high binding affinity to the target biomarker. Targeting cancers in the reproductive organs using aptamers showed an excellent efficiency of detection compared to other probes. Different aptamers have been generated against the gynaecological cancer biomarkers, which include HE4, CA125, VEGF, OCCA (for ovarian cancer), EGFR, FGFR1, K-ras (for endometrial cancer), HPV E-16, HPV E-7, HPV E-6, tyrosine, and kinase (for cervical cancer), which help to identify the cancers in woman reproductive organs. In this overview, the biomarkers for gynecologic cancers and the relevant diagnosing systems generated using the specific aptamers are discussed. Furthermore, the therapeutic applications of aptamer with gynaecological cancers are narrated.

Epitope Imprinting Technology: Progress, Applications, and Perspectives toward Artificial Antibodies.

Epitope imprinting is a promising tool to generate antibody-like specific recognition sites. Recently, because of the ease of obtaining templates, the flexibility in selecting monomers, their resistance to harsh environments, and the high specificity toward targets, epitope-imprinted materials have attracted much attention in various fields, such as bioanalysis, clinical therapy, and pharmacy. Here, the discussion is focused on the current representative epitope imprinting technologies, including epitope bulk imprinting and epitope surface imprinting. Moreover, the application of epitope-imprinted materials to the recognition of peptides, proteins, and cells is reviewed. Finally, the remaining challenges arising from the intrinsic properties of epitope imprinting are discussed, and future development in the field is prospected.

Artificial Immune System-Based Learning Style Stereotypes

This paper addresses the problem of extracting fundamental learning style stereotypes through the exploitation of the biologically-inspired pattern recognition paradigm of Artificial Immune Systems (AIS). We present an unsupervised computational mechanism which exhibits the ability to reveal the inherent group structure of learning patterns that pervade a given set of educational profiles. We rely on the construction of an Artificial Immune Network (AIN) of learning style exemplars by proposing a correlation-based distance metric. This choice is actually imposed by the categoric nature of the underlying data. Our work utilizes an original dataset which was derived during the conduction of an extended empirical study involving students of the Hellenic Open University. The educational profiles of the students were built by collecting their answers on a thoroughly designed questionnaire taking into account a wide range of personal characteristics and skills. The efficiency of the proposed approach was assessed in terms of cluster compactness. Specifically, we measured the average correlation deviation of the students’ education profiles from the corresponding artificial memory antibodies that represent the acquired learning style stereotypes. Finally, the unsupervised learning procedure adopted in this paper was tested against a correlation-based version of the k-means algorithm indicating a significant improvement in performance for the AIS-based clustering approach.

Recent advances and application of epitope imprinted materials

As artificial antibodies, protein-imprinted materials offer many advantages such as long-term storage stability, potential reusability, resistance to harsh environments and low cost. However, they suffer from obvious disadvantages, when whole proteins are used as templates. First, it is difficult to retain the native conformation of template proteins during the polymerization process. Second, it is unavailable to obtain pure proteins, especially low abundance proteins as the templates. Third, the relatively large imprinted sites are able to bind a range of smaller non-targets, resulting in reduced selectivity. Epitopes are the regions within the structures of globular proteins that can be classified as antigenic determinants. The epitope’s structural specificity usually represents the entire protein. And, they can be easily obtained through artificial synthesis. Currently, as an alternative to the target protein, epitopes have been successfully employed as templates to fabricate the recognition sites for several proteins. Compared with protein templates, epitope templates are much more robust and stable during the imprinting process. Moreover, epitope templates could be obtained in large scale with low cost, even for the rare proteins. In this review, we critically and comprehensively survey recent advances and applications in epitope imprinting. The epitope imprinting techniques have been developed into several formats, epitope bulk imprinting, grafted epitope surface imprinting, constrained epitope surface imprinting and synergic epitope surface imprinting according to how the template is presented in the imprinted materials. For the epitope bulk imprinting, binding sites were formed throughout a bulk material. The grafted epitope surface imprinting is achieved by immobilizing epitope on the film or particle surface and then polymerizing with monomers and crosslinkers to form a state-of-the-art recognition sites on the polymer or silica surface. In the constrained epitope surface imprinting, the epitope is confined between the matrix support and the final polymer matrix. Then the matrix support is removed to expose the recognition sites, usually by simply peeling imprinted polymer from the matrix or dissolving the matrix. To improve the affinities and selectivity, additional affinities were integrated into the epitope imprinted surface, which results in the technique of synergic epitope surface imprinting. More recently, application of epitope-imprinted materials has also increased substantially with the rapid development of the life sciences, although they are not as widely used as whole protein molecule imprinting. These are demonstrated in the aspect of the peptide recognition, protein recognition and cell recognition. Finally, we summarized the remaining challenges arising from the intrinsic properties of epitope imprinting, and proposed its future development direction.

Artificial Antibody with Site-Enhanced Multivalent Aptamers for Specific Capture of Circulating Tumor Cells.

Isolation of circulating tumor cells (CTCs) from blood holdsgreat potential to diagnose cancers and discover therapeutic targets. Herein, we reported a novel kind of artificial antibody, the cell-imprinted hydrogel with site-directed modification of aptamers (APT-CIH) to achieve the specific capture of CTCs. Cell-imprinted sites not only could be used to recognize target cells but also could be used as efficient scaffolds for assembling aptamers to enhance the capture efficiency and selectivity. Due to the synergistic effect of conformation recognition and multivalent interaction between the aptamers and target cells, APT-CIH showed high capture efficiency and selectivity to SMMC-7721 cells. In the coexistence of the 1000 times leukemia Jurkat cells, the enrichment factor of APT-CIH could reach as high as 21.6 ± 3.1 toward target cells, while that relied only on cell imprinting or aptamer affinity was 8.1 ± 5.0 or 10.1 ± 1.3, respectively. Furthermore, the capture efficiency could reach 58.2% ± 10.9% with 1000 SMMC-7721 cells spiked in 1 mL of blood. Moreover, 92% of the captured cells could be released, beneficial to carry out further biological and clinical study of CTCs. These results demonstrated that APT-CIH might have great potential in CTCs analysis.

Advances in capture methods of circulating tumor cells

Since circulating tumor cells (CTCs) in the blood carry the genetic and phenotypic information of tumor tissues, they are regarded as important markers for liquid biopsy, especially for the early diagnosis of cancer, determination of treatment options and evaluation of prognosis. However, the capture and detection of CTCs is challenging because of their extremely low concentration in blood. In this review, recent advances in capture methods of CTCs based on their biophysical properties, affinity, and artificial antibodies are summarized. The advantages and drawbacks of these methods are discussed from the aspects of capture efficiency, capture purity and maintenance of activity after release. Future trends in CTCs capture are also discussed.

Molecularly imprinted polymer based sensor directly responsive to attomole bovine serum albumin

Through years of extensive research and development, molecularly imprinted polymers (MIPs) are still inferior to their biological rivals such as antibodies, enzymes etc. In this study we report a protein-imprinted cryogel, showing antibody-like affinity and selectivity against the protein template (bovine serum albumin, BSA). The MIP was synthesized from the co-polymerization of acrylamide, N,N-methylenebisacrylamide, acrylic acid and diallylamine. Due to the participation of the ionizable monomers (acrylic acid and diallylamine), imprinted cavities with inner surface-clung charged groups were created to recognize BSA. Therefore each cavity appears like a molecular capacitor charged by carboxyl and amino groups. As the cavities are all of a molecule-size volume, a membrane made of the MIP contains a huge array of the molecular capacitors. This will produce a synergistic effect and greatly amplify the impedance signal deviations when template sorption/desorption takes place on the sensor. When the MIP was used as an artificial antibody to make an electrochemical sensor, high sensitivity and selectivity were achieved at the same time. Results indicate that BSA could be determined in a linear range of 1.5 × 10–16–10–12 mol–L−1. Meanwhile a low limit of detection was achieved at 7.2 × 10–18 mol L−1. Conclusively protein-imprinted amphoteric polyacrylamide cryogels are materials of a great potential to sense and determine charged objects like molecules, cells, microorganisms or other particles.

Aptamer: A Versatile Probe in Medical Diagnosis

The aptamer is a single standard nucleic acid, also known as an artificial antibody, has been selected from the randomized library of oligonucleotide molecules by the process of “systematic evaluation of ligands by exponential enrichment.” Since the selected aptamers have displayed advantages and rival the antibodies, potentials of aptamer are widely spread to different fields such as medicine, therapeutics, environmental, and biosensor. In particular, aptamers have been focused in the field of medical diagnosis due to its higher sensitivity and specificity against the target molecules. Various kinds of sensors were utilized to diagnose the different kinds of diseases using aptamer as the probe. In this overview, we discussed the detailed applications of aptamers with the field of medical diagnosis.

Targeted Live Cell Raman Imaging and Visualization of Cancer Biomarkers with Thermal‐Stimuli Responsive Imprinted Nanoprobes

AbstractPrecise visualization of cancer biomarkers within live cells is significantly vital in oncology that requires techniques with high specificity and selectivity. Herein, a new imprinted plasmonic nanoprobe with thermosensitivity is fabricated, which features multifunctional roles as an “artificial antibody” for the active targeting of cancer biomarkers triggered by temperature and can also achieve their in situ Raman detection and imaging within living cells. A thiol‐containing poly(N‐isopropylacrylamide) linear polymer is first synthesized using reversible addition–fragmentation chain transfer polymerization followed by further reduction with NaBH4 to produce free thiol groups at one end. Then, plasmonic nanoprobes are constructed by creating protein‐imprinted poly(N‐isopropylacrylamide) self‐assembled monolayers on the surface of gold nanorods. High specificity and selectivity are demonstrated for the target protein, with an extracellular Raman detection limit of 10−14 mol L−1. Thermally responsive behavior is achieved by modulating the external temperature. The plasmonic properties enable in situ live cell intracellular Raman detection and imaging of endogenous cancer biomarker epidermal growth factor receptor. This technique offers significant promise for visualizing cancer biomarkers and monitoring molecular mechanisms both in vitro and in vivo, which are significant in clinical diagnosis and medical theranostics.

Synthetic Receptors for the Recognition and Discrimination of Post-Translationally Methylated Lysines.

Post-translational modifications (PTMs) describe the chemical alteration of proteins after their biosynthesis in ribosomes. PTMs play important roles in cell biology, including the regulation of gene expression, cell-cell interactions and the development of different diseases. A prominent class of PTMs is the side-chain methylation of lysine. For the analysis and discrimination of differently methylated lysines, antibodies are widely used, although methylated peptide and protein targets are known to be particularly difficult to differentiate by antibody-based affinity reagents; an additional challenge can be batch-to-batch reproducibility. The application of mass spectrometry techniques for methyllysine discrimination requires a complex sample preparation procedure and is not suited for working in cells. The desire to overcome the above-mentioned challenges has promoted the development of synthetic receptor molecules that recognise and bind methyllysines. Such "artificial antibodies" are of interest for a number of applications, for example, as reagents in biochemical assays, for the isolation and purification of post-translationally methylated proteins and for the tracking of signalling pathways. Moreover, they offer new approaches in diagnostics and therapy. This review delivers an overview of the broad field of methyllysine binding and covers a wide range of synthetic receptors used for the recognition of methylated lysines, including calixarenes, resorcinarenes, pillararenes, disulfide cyclophanes, cucurbiturils and acyclic receptors.

VALIS: an evolutionary classification algorithm

VALIS is an effective and robust classification algorithm with a focus on understandability. Its name stems from Vote-ALlocating Immune System, as it evolves a population of artificial antibodies that can bind to the input data, and performs classification through a voting process. In the beginning of the training, VALIS generates a set of random candidate antibodies; at each iteration, it selects the most useful ones to produce new candidates, while the least, are discarded; the process is iterated until a user-defined stopping condition. The paradigm allows the user to get a visual insight of the learning dynamics, helping to supervise the process, pinpoint problems, and tweak feature engineering. VALIS is tested against nine state-of-the-art classification algorithms on six popular benchmark problems; results demonstrate that it is competitive with well-established black-box techniques, and superior in specific corner cases.