Recognition Of Carcinoembryonic Antigen Research Articles

A label-free and signal-amplifiable fluorescent biosensor based on aptamer-conjugated gold nanoparticles and hybridization chain reaction for determination of carcinoembryonic antigen.

The sensitive detection of cancer biomarkers is crucial for early accurate diagnostics and therapy of cancer patients. Carcinoembryonic antigen (CEA) is a tumor-associated antigen derived from colon cancer and embryonic tissues. In this study, we have developed a label-free fluorescence biosensing platform for the quantification of CEA with the "turn-on" signal output. This platform employs a label-free strategy that incorporates an aptamer-modified gold nanoparticle (Apt@AuNP) probe for the recognition of CEA, in combination with hybridization chain reaction (HCR) amplification. In the presence of target CEA, Apt@AuNPs selectively capture CEA, resulting in a reduction of subsequent complementary chains (CP) binding on Apt@AuNPs. The remaining CP, acting as the initiator sequence for HCR, triggers the HCR, leading to the formation of abundant G-quadruplex structures. By employing Thioflavin T (ThT) for the formation of G-quadruplex/ThT complexes, the biosensor exhibits a significant enhancement of the fluorescence signal. Under optimized conditions, the biosensor platform demonstrates a limit of detection of 0.03 nM and a linear range from 0.1 to 2.5nM. Additionally, the specificity investigation reveals the high selectivity of this fluorescent biosensor. Finally, the performance of this method has been validated by successfully detecting CEA in real-life samples, highlighting its potential for clinical applications.

Label-free electrochemical biosensor based on Ng-PCL polymer signal amplification for the detection of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is the related antigen first extracted from embryonic tissue and is an acidic glycoprotein with human embryonic antigenic properties. It is an important biomarker for the detection of many malignancies. In this paper, we have developed a signal amplified sandwich-type electrochemical impedance biosensor based on naringin initiated poly(ε-caprolactone) (Ng-PCL) polymer for highly sensitive and rapid determination of CEA. The polymer Ng-PCL is a macromolecular polymer that can increase the sensitivity of biosensor. First, SH-functionalized CEA-aptamer 1 (Apt1) was attached to the electrode surface as a recognition probe by Au-S bonds in order to form a self-assembly monolayer (SAM). Then, the one end of CEA antigen could recognize the Apt1 modified on the electrode surface, the other end could recognize the COOH-functionalized CEA-aptamer 2 (Apt2) forming a sandwich structure. Finally, polymer Ng-PCL as a signal amplification unit, which was connected to Apt2 by ester bonds. As a result, the electron transfer in the electrode interface was hindered effectively and a label-free electrochemical impedance sensor was constructed successfully. The high sensitivity and selectivity of biosensor relied on the SAM, polymer Ng-PCL, and specific recognition of CEA. Under the optimum experimental conditions, we proposed electrochemical impedance biosensor had wide detection range between 5 pg/mL and 200 ng/mL, and the sensitivity can reach 0.675 pg/mL. Moreover, the electrochemical impedance biosensor performed high specificity, good stability and satisfactory reproducibility as well. Thus, this strategy will provide new path for the detection of other biomarkers in early clinical diagnosis.

A highly sensitive sensor for carcinoembryonic antigen based on AlGaN/GaN high-electron-mobility transistors

Carcinoembryonic antigen (CEA) is a well-known biomarker and validated serum biomarker for lung cancer. We introduce a simple label-free method for CEA detection. Specific recognition of CEA was made possible by immobilizing CEA antibodies in the sensing region of AlGaN/GaN high-electron-mobility transistors. The biosensors have a detection limit of 1 fg ml−1 in phosphate buffer solution. This approach has advantages of integration, miniaturization, low cost, and rapid detection compared to other testing methods for lung cancer and could be used in future medical diagnostics.

Chemical binding of molecular-imprinted polymer to biotinilated antibody: Utilization of molecular imprinting polymer as intelligent synthetic biomaterials toward recognition of carcinoma embryonic antigen in human plasma sample.

In this study, a novel biosensor based on molecular imprinting polymer (MIP) methodology was fabricated toward recognition of carcinoembryonic antigen (CEA). For this purpose, poly (toluidine blue) (PTB) was electropolymerized on the surface of gold electrode in the absence and presence of CEA. So, the target molecules were entrapped into the imprinted specific cavities of MIP. Obtained results show that, the binding affinity of the MIP system was significantly higher than that of revealed for the nonimprinted polymer (NIP) system, MIP-based biosensor revealed linear response from (0.005 to 75μg/L) and low limit of quantification of (0.005μg/L) by using chronoamperometry technique, leading to CEA monitoring in real and clinical samples. Thus, a novel technique for rapid, simple, sensitive and affordable monitoring of CEA (LLOQ=0.005 μg/L) has provided through developed biosensor. From a future perspective, moreover, this method can be considered as an applicable candidate in biomedical and clinical analysis for point-of-care usages.

Selection and characterisation of Affimers specific for CEA recognition

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.

Nucleic acid-functionalized metal-organic framework for ultrasensitive immobilization-free photoelectrochemical biosensing

This work established an immobilization-free photoelectrochemical (PEC) biosensor for ultrasensitive determination of carcinoembryonic antigen (CEA) based on the DNA-functionalized metal-organic frameworks (MOFs) and T7 exonuclease-aided recycling amplification. In this proposal, MOFs were served as nanocarriers for efficient encapsulation of electron donors, while an ingeniously designed hairpin probe (HP) employed as the recognition element. The recognition of CEA by its aptamer sequence in HP triggered the conformational change and the T7 exonuclease-aided recycling amplification, which opened the pore of MOFs to release a large number of electron donors, producing a significantly increased photocurrent. Benefitting from the high loading ability of MOFs and the excellent amplification efficiency of the T7 exonuclease-assisted recycling process, the proposed biosensor is capable of ultrasensitive and highly selective determination of CEA with a detection limit down to 0.36 fg mL-1 and a wide linear range from 1.0 fg mL-1 to 10 ng mL-1. Moreover, the proposed biosensor can also apply to measure CEA in spiked serum samples, indicating that this PEC biosensor holds excellent potential for application in bioanalysis and early disease diagnosis.

A novel sandwich-type SERS immunosensor for selective and sensitive carcinoembryonic antigen (CEA) detection

Monitoring the malignant tumors via cancer biomarkers is very significant process. Nonetheless, the practical clinical applications need selective and sensitive analytical methods/techniques. In this study, a novel sandwich type immunosensor based on surface-enhanced raman scattering (SERS) was presented including 4-mercaptobenzoic acid labeled MoS2 nanoflowers@Au nanoparticles (MoS2 NFs@Au NPs/ MBA) as CEASERS tag and Fe3O4@Au nanoparticles functionalized delaminated Ti3C2Tx MXene (Fe3O4 NPs@Au NPs/d-Ti3C2TX MXene) as SERS magnetic supporting substrate for carcinoembryonic antigen (CEA) detection. Especially, the determination of single molecule by using SERS method enables early diagnosis of major diseases. In addition, this technique can be utilized for multiplex analyzes owing to narrow well-resolved peaks. The prepared CEASERS tag and SERS magnetic supporting substrate were characterized by scanning electron microscope (SEM), x-ray diffraction (XRD) method, x-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR). A linearity of 0.0001–100.0 ng mL−1 was observed with high sensitivity. Finally, sandwich type immunosensor demonstrated good selectivity and stability for target CEA recognition in plasma sample.

Amperometric aptasensor for carcinoembryonic antigen based on a reduced graphene oxide/gold nanoparticles modified electrode

Abstract We report the construction of a novel amperometric aptasensor for the highly specific detection of carcinoembryonic antigen (CEA). The sensing interface was assembled by electrodeposition of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs) on a carbon screen-printed electrode (SPE) and further functionalization with a biotin and thiol-modified anti-CEA DNA hairpin aptamer. The sensing approach relies on the specific recognition of CEA by the folded aptamer, causing unfolding of the DNA hairpin structure and unmasking the biotin residues at the aptamer chain. Further incubation with a sptreptavidin-peroxidase conjugate allows the amperometric detection of the cancer biomarker. This aptasensor was able to detect CEA in the broad range from 20 pg mL−1 to 2 μg mL−1 (112 fM–11 μM) with a detection limit of 16 pg mL−1 (90 fM). The aptasensor also showed high reproducibility, specificity and stability, and was successfully validated in diluted human serum samples.

A novel bioassay for the monitoring of carcinoembryonic antigen in human biofluid using polymeric interface and immunosensing method.

Carcinoembryonic antigen (CEA) is a member of a family of cell surface glycoproteins. Recognition of CEA is needed to monitor the physiological status of the patient for treatment and also it is important to assess the severity of the disease. In this work, we reported a novel sandwich-type electrochemical immunosensor based on gold nanoparticles functionalized cysteamine-glutaraldehyde (AuNPs-CysA-GA) and it successfully designed to detection of the CEA biomarker in a human plasma sample. The AuNPs-CysA-GA provides a large surface area for the effective immobilization of CEA antibody, as well as it ascertains the bioactivity and stability of immobilized CEA antigens. Biotinylated-anti-CEA antibody (Ab1) was immobilized on the surface of glassy carbon electrode (GCE) modified AuNPs-CysA-GA. Also, secondary antibody (HRP-Ab2) was costed immobilized to complete the sandwich part of immunosensor. Field emission scanning electron microscope (FE-SEM and EDS), was employed to monitor the sensor fabrication procedure. The immunosensor was used for the detection of CEA using differential pulse voltammetry (DPVs) technique. The proposed interface led to enhancement of accessible surface area for immobilizing high amount of anti-CEA antibody, increasing electrical conductivity, boosting stability, and biocompatibility. Finally, the low limit of quantitation (LLOQ) of the proposed immunosensor was obtained as 7 ng/mL with the linear range of 0.001-5 μg/L. The proposed immunoassay was successfully applied for the monitoring of the CEA in unprocessed human plasma samples. Obtained results paved that the proposed bioassay can be used as a novel bioassay for the clinical diagnosis of cancer based on CEA monitoring.

Drug-loaded dual targeting graphene oxide-based molecularly imprinted composite and recognition of carcino-embryonic antigen

Despite extensive research on functional graphene oxide for anticancer drug delivery, the sensitivity of traditional protein targeting ligands to the environment limits the practical applications of targeted drug delivery. A unique molecularly imprinted magnetic graphene oxide was used as a novel drug delivery system for the treatment of tumors. Molecularly imprinted polymers (MIPs) synthesized by molecular imprinting technology have the advantages of good stability against chemical and enzymatic attacks, high specificity for a target template, and resistance to harsh environments. In our work, the MIP was used for specificity to tumor cells with carcino-embryonic (CEA) tumor markers as the template, and dopamine as the functional monomer was grafted on boronic acid-functionalized magnetic graphene oxide. The structure of the nanoparticles was optimized and characterized in detail by vibrating sample magnetometry, X-ray diffraction analysis, UV-vis spectroscopy, and flow cytometry. The prepared polymer has magnetic properties, specific recognition to CEA, biocompatibility and pH sensitivity for drug delivery. Cell culture research was carried out on the tumor cells and normal cells. The composites exhibited dual targeting properties that not only magnetically target but also specifically increase the drug cytotoxicity to the tumor cells by selectively binding to CEA. On the basis of these results, this study developed a novel approach for targeting tumor cells for drug delivery without needing to modify the protein ligand.

Avidin-gated mesoporous silica nanoparticles for signal amplification in electrochemical biosensor

We report herein a novel sensing strategy for electrochemical biosensors, by using mesoporous silica nanoparticles loaded with the redox probe methylene blue and capped with an avidin/imminobiotin stimulus-responsive gate-like ensemble as signal amplification element. As a proof of concept, an aptasensor for carcinoembryonic antigen (CEA) was constructed by attaching a biotin and thiol-functionalized anti-CEA DNA hairpin aptamer on gold nanoparticles modified carbon screen-printed electrodes. The biosensing approach relied on the unfolding of the aptamer molecule after specific recognition of CEA, unmasking the biotin residue and allowing further association with the avidin-capped mesoporous nanocarrier. Incubation with H2SO4 trigger the release of the encapsulated redox probe allowing the detection of the cancer biomarker from 1.0 pg/mL to 160 ng/mL.

Amperometric aptasensor for carcinoembryonic antigen based on the use of bifunctionalized Janus nanoparticles as biorecognition-signaling element

We report herein the design of a novel biosensing strategy for the detection of carcinoembryonic antigen (CEA), based on the use of Janus-type nanoparticles having Au and silica opposite faces as integrated electrochemical biorecognition-signaling system. The Janus nanoparticles were properly functionalized with horseradish peroxidase on the silica surface to act as signaling element, and a biotin thiol-modified anti-CEA DNA hairpin aptamer the Au face to assemble the biorecognition element. The sensing approach relies on the first specific recognition of CEA by the bifunctionalized Janus nanoparticles, causing unfolding of the DNA hairpin structure and unmasking the biotin residues at the aptamer chain. This CEA-Janus nanoparticle complex was then captured by avidin-modified Fe3O4@SiO2 NanoCaptors®, allowing further magnetic deposition on carbon screen printed electrodes for the amperometric detection of the cancer biomarker. The Janus nanoparticles-based aptasensor was able to detect CEA in the range from 1 to 5000 ng mL−1 (5.5 pM–28 nM) with a detection limit of 210 pg mL−1 (1.2 pM). The aptasensor also showed high reproducibility and storage stability, and was successfully validated in human serum.

New stochastic microsensors based on oleamides

Six oleamides: oleoylethanolamide, (Z)-N-[(1S)-2-hydroxy-1-(phenylmethyl)ethyl]-9octadecenamide, N-phenethyloleamide, N-[2-(4-methoxyphenyl)ethyl]oleamide, N-[1]naphthyloleamide, N-cyclohexyloleamide were synthesized and used as modifiers for the design of new stochastic microsensors based on graphite paste. Carcinoembryonic antigen (CEA) was used as a model analyte to prove the stochastic behavior of the microsensors. CEA was determined directly from whole blood samples with recoveries higher than 94.00%, favorizing pattern recognition of CEA in whole blood samples. The most sensitive stochastic microsensor was the one based on the physical immobilization of N-[2-(4-methoxyphenyl)ethyl]oleamide in graphite paste.

A Sandwich Electrochemical Immunosensor Using Magnetic DNA Nanoprobes for Carcinoembryonic Antigen

A novel magnetic nanoparticle-based electrochemical immunoassay of carcinoembryonic antigen (CEA) was designed as a model using CEA antibody-functionalized magnetic beads [DNA/Fe3O4/ZrO2; Fe3O4 (core)/ZrO2 (shell) nano particles (ZMPs)] as immunosensing probes. To design the immunoassay, the CEA antibody and O-phenylenediamine (OPD) were initially immobilized on a chitosan/nano gold composite membrane on a glassy carbon electrode (GCE/CS-nano Au), which was used for CEA recognition. Then, horseradish peroxidase (HRP)-labeled anti-CEA antibodies (HRP-CEA Ab2) were bound to the surface of the synthesized magnetic ZMP nanoparticles as signal tag. Thus, the sandwich-type immune complex could be formed between secondary antibody (Ab2) modified DNA/ZMPs nanochains tagged by HRP and GCE/CS-nano Au. Unlike conventional nanoparticle-based electrochemical immunoassays, the recognition elements of this immunoassay included both electron mediators and enzyme labels, which obviously simplifies the electrochemical measurement process. The sandwich-type immunoassay format was used for online formation of the immunocomplex of CEA captured in the detection cell with an external magnet. The electrochemical signals derived from HRP during the reduction of H2O2 with OPD as electron mediator were measured. The method displayed a high sensitivity for CEA detection in the range of 0.008–200 ng/mL, with a detection limit of 5 pg/mL (estimated at a signal-to-noise ratio of 3). The precision, reproducibility, and stability of the immunoassay were good. The use of the assay was evaluated with clinical serum samples, and the results were in excellent accordance with those obtained using the standard enzyme-linked immunosorbent assay (ELISA) method. Thus, the magnetic nanoparticle-based assay format is a promising approach for clinical applications, and it could be further developed for the detection of other biomarkers in cancer diagnosis.

Characterization of Genetically Modified T-Cell Receptors that Recognize the CEA:691-699 Peptide in the Context of HLA-A2.1 on Human Colorectal Cancer Cells

Carcinoembryonic antigen (CEA) is a tumor-associated protein expressed on a variety of adenocarcinomas. To develop an immunotherapy for patients with cancers that overexpress CEA, we isolated and genetically modified a T-cell receptors (TCRs) that specifically bound a CEA peptide on human cancer cells. HLA-A2.1 transgenic mice were immunized with CEA:691-699. A CEA-reactive TCR was isolated from splenocytes of these mice and was genetically introduced into human peripheral blood lymphocytes via RNA electroporation or retroviral transduction. Amino acid substitutions were introduced throughout the complementarity determining regions (CDR1, CDR2, and CDR3) of both TCR alpha and beta chains to improve recognition of CEA. Murine lymphocytes bearing the CEA-reactive TCR specifically recognized peptide-loaded T2 cells and HLA-A2.1(+) CEA(+) human colon cancer cells. Both CD8(+) and CD4(+) human lymphocytes expressing the murine TCR specifically recognized peptide-loaded T2 cells. However, only gene-modified CD8(+) lymphocytes specifically recognized HLA-A2.1(+) CEA(+) colon cancer cell lines, and tumor cell recognition was weak and variable. We identified two substitutions in the CDR3 of the alpha chain that significantly influenced tumor cell recognition by human peripheral blood lymphocytes. One substitution, T for S at position 112 (S112T), enhanced tumor cell recognition by CD8(+) lymphocytes, and a second dually substituted receptor (S112T L110F) enhanced tumor cell recognition by CD4(+) T cells. The modified CEA-reactive TCRs are good candidates for future gene therapy clinical trials and show the power of selected amino acid substitutions in the antigen-binding regions of the TCR to enhance desired reactivities.

Antibody response against neem leaf preparation recognizes carcinoembryonic antigen

An immune serum generated in Swiss mice against an aqueous preparation from neem leaf was reactive with carcinoembryonic antigen (CEA) and a peptide sequence derived from it. Using ELISA, we have demonstrated that CEA reactive antibody titer (chiefly IgG2a) was significantly decreased after absorption of the immune sera with CEA. Neem leaf preparation (NLP) generated immune sera was also reactive with CEA in immunoblotting and CEA reactive component in the NLP sera can be immunoprecipitated. Identical recognition of CEA expressed on human colorectal cancer specimens, by anti-CEA monoclonal antibody and NLP sera was documented by immunohistochemistry. NLP generated sera could also react with NLP in ELISA and this reactivity was decreased after absorption of the NLP with anti-CEA antibody. Estimation of protein in NLP revealed the presence of it, at least 10% of the total dry weight. In addition, existence of flavone and quercetin was also evidenced from LC-MS analysis. Further studies are needed to identify the antigenic component may have some homology with CEA molecule. This unique property of neem may be utilized for the immunotherapy of CEA positive tumors.

Targeting strategy for gene delivery to carcinoembryonic antigen-producing cancer cells by retrovirus displaying a single-chain variable fragment antibody.

Cancer-specific antigens are promising targets for the specific delivery of certain drugs or genes to cancer cells in cancer therapy. Carcinoembryonic antigen (CEA) is one of the cancer-associated antigens predominantly detected in the gastrointestinal cancer of the colon and stomach. Targeting strategies for CEA-producing cancer cells have been thoroughly developed mainly by the production of monoclonal antibodies to CEA and further single-chain variable fragment (scFv) antibodies. Here, we have generated Moloney murine leukemia virus-derived retroviral vectors co-displaying an anti-CEA scFv-envelope chimeric protein and an unmodified envelope protein to deliver a gene for herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase. The harvested viruses successfully incorporated the chimeric envelope protein as well as the unmodified envelope into the viral particles, and specifically bound to and infected human CEA-producing cancer cells via recognition of CEA, depending on the CEA-producing phenotype of the target cells. These results may have significant implications for the use of scFv directed against tumor-specific antigens for targeting specific antigen-producing cancer cells, a potential step toward in vivo cancer therapy.