Simultaneous Determination Of Carcinoembryonic Antigen Research Articles

Salt-induced gold nanoparticles aggregation lights up fluorescence of DNA-silver nanoclusters to monitor dual cancer markers carcinoembryonic antigen and carbohydrate antigen 125

In clinical diagnosis of cancer, the monitoring of single tumor marker may result in many false and missed results, while simultaneous detection of multiple tumor markers should be more accuracy and effective. Here, we report a new strategy that salt-induced gold nanoparticles (AuNPs) aggregation lights up fluorescence of dual-color DNA-silver nanoclusters-aptamer (DNA-AgNCs-apta) for the simultaneous monitoring of carcinoembryonic antigen (CEA) and carbohydrate antigen 125 (CA125). The dual-color aptasensor system is composed of green-emitting DNA-AgNCs with CEA aptamer (gDNA1-AgNCs-apta1) and red-emitting DNA-AgNCs with CA125 aptamer (rDNA2-AgNCs-apta2) in the ratio of 1:1 in volume. Upon addition of AuNPs, gDNA1-AgNCs-apta1 and/or rDNA2-AgNCs-apta2 are flexibly adsorbed onto the surface of AuNPs by terminal aptamer(s), which prevents salt-induced AuNPs aggregation under high salt condition and results in fluorescence quenching based on surface plasmon enhanced energy transfer (SPEET). With the addition of CEA and/or CA125, the target(s) and corresponding aptamer(s) coordinate to form the complex, keeping DNA-AgNCs-apta(s) far away from the surface of AuNPs and making AuNPs aggregated in high salt medium. The AuNPs aggregation leads to the recovery of fluorescence signals of DNA-AgNCs-apta(s) due to weakened SPEET. Utilizing the fluorescence aptasensor system, the limit of detection of CEA and CA125 are as low as 7.5 pg·mL-1 and 0.015 U·mL-1, respectively. The proposed method can be applied to the selective and simultaneous determination of CEA and CA125 in human serum.

Simultaneous Determination of Carcinoembryonic Antigen (CEA), Carbohydrate Antigen 19-9 (CA19-9), and Serum Protein p53 in Biological Samples with Protoporphyrin IX (PIX) Used for Recognition by Stochastic Microsensors

Three of the most assessed biomarkers in the screening of biological samples for gastric cancer are carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and serum protein p53 (p53). Protoporphyrin IX immobilized in different nitrogen-doped graphene materials was used as the recognition molecule for the simultaneous determination of CEA, CA19-9, and p53 in various biological samples: whole blood, saliva, urine, and tissue samples. The proposed stochastic microsensors used as the recognition molecule protoporphyrin IX were reliable for the simultaneous determination of CEA, CA19-9, and p53 due to their high sensitivities and low limits of determination. The wide linear concentration ranges made possible the determination of the biomarkers in different stages of gastric cancer, from the earliest to the latest stages.

Fluorometric immunoassay for the simultaneous determination of the tumor markers carcinoembryonic antigen and cytokeratin 19 fragment using two kinds of CdSe/ZnS quantum dot nanobeads and magnetic beads.

A method is described for the simultaneous determination of the carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1). Two kinds of CdSe/ZnS quantum dot nanobeads (QBs), with emission maxima at 530 nm (green) and 585 nm (yellow), were used as labels, and magnetic beads (MBs) for separation. The MBs were used as substrates to couple CEA and CYFRA21-1 antibody for isolating the proteins. Then, the differently colored QBs were linked to the antibodies against CEA and CYFRA21-1, respectively. Following the formation of the immunocomplex, the intensities of the green and yellow emissions were measured at the same excitation wavelength of 340 nm. The detection limits are 0.1 ng⋅mL- 1 for CEA, and of 0.2 ng⋅mL- 1 for CYFRA21-1. The recoveries from spiked serum are 92.1 - 118.1% for CEA, and from 90.8% to 115.2% for CYFRA21-1, with the relative standard deviations of 6.3 - 12.3% and 7.1 - 11.8%. The method was successfully applied to the simultaneous determination of the two proteins in human serum sample (n = 45). The results correlated well with those of the chemiluminescent enzyme immunoassay kit. Graphical AbstractSchematic presentation of the fluorescence immunoassay for the simultaneous determination of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1) based on quantum dot nanobeads (QBs) and magnetic beads (MBs) is reported. The intensities of two kinds of CdSe/ZnS QBs, with the emission maxima at 530 nm (green) and 585 nm (yellow) were measured at the same excitation wavelength of 340 nm.

A dual-label time-resolved fluorescence immunoassay for the simultaneous determination of carcinoembryonic antigen and squamous cell carcinoma antigen.

Among women worldwide, cervical cancer is the second-most common cancer, and cervical smears and DNA detection have lowsensitivity or are too expensive.The concentrations of carcinoembryonic antigen (CEA) and squamous cell carcinoma (SCC) in the serum were detected using a sandwich immunoassay. The CEA and SCC in the serum were captured by anti-CEA and anti-SCC antibodies. After combining other anti-CEA- and anti-SCC-labeled antibodies with europium (III) (Eu3+ ) and samarium (III) (Sm3+ ) chelates, CEA and SCC were detected with time-resolved fluorometry (TRF). The linear correlation coefficients (R2 ) of the CEA and SCC standard curves were 0.9997 and 0.9997, respectively. The minimum detection level for CEA was 1.15ng/mL (the linear dynamic range was 3.24-543.67ng/mL), and the average recovery was 100.83%. The sensitivity for SCC detection was 0.54ng/mL (the linear dynamic range was 2.47-96.58ng/mL), and the average recovery was 101.02%. High R2 between the results of commercial assays and this method were obtained (R2 =0.9983 for CEA, R2 =0.9878 for SCC). These findings indicated that the dual-label TRFIA invented in this study has high sensitivity, accuracy, and specificity in clinical analysis, which indicates that this method could be used for the early diagnosis and follow-up surveillance of cervical cancer.

Magnetoimmunosensor for simultaneous electrochemical detection of carcinoembryonic antigen and α-fetoprotein using multifunctionalized Au nanotags

We report a sandwich-type electrochemical immunoassay for simultaneous determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), using multifunctionalized gold nanoparticles as signal tags. Two nanotags including thionine-Au nanoparticles and ferrocene-Au nanoparticles were synthesized and used as distinguishable indicators. The sensor was fabricated by means of co-immobilization of primary anti-CEA (AbCEA) and anti-AFP (AbAFP) antibodies on the surface of Fe3O4 nanoparticle, the strategy is based on observation distinct voltammetric peak derived from distinguish probe (AbCEA-Au-thionine and AbAFP-Au-ferrocene nanotags). Experimental results revealed that this immunosensor enabled simultaneous determination of CEA and AFP in the linear ranges of (0.05–120ngmL−1) and (0.05–100ngmL−1), respectively. The detection limits (DL) for AFP and CEA were 0.012ngmL−1 and 0.018ngmL−1, respectively (S/N=3). Furthermore, the methodology was also evaluated for the analysis of AFP and CEA in the clinical human serum specimens, the obtained results was in desirable agreement with clinical data.

Graphene oxide reduced directly by redox probes for multiplexed detection of tumor markers.

Redox probe modified reduced graphene oxide was fabricated through directly reducing graphene oxide using redox probes (toluidine blue (TB) or amino-functionalized ferrocene (NH2-Fc)). Self-assembling gold nanoparticles to fabricate redox probe modified reduced graphene oxide-Au nanocomposites via electrostatic interaction was then carried out. A sandwich-type electrochemical immunosensor, for simultaneous multianalyte detection (carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes), was designed. Redox probe modified reduced graphene oxide-Au nanocomposites are used as immunosensing probes and an ionic liquid functionalized reduced graphene oxide-Au (IL-rGO-Au) nanocomposite is used as an immunosensing substrate. The resulting immunosensor exhibited high selectivity and sensitivity during the simultaneous determination of CEA and AFP in a single run. The linear ranges were from 0.01 to 100 ng mL-1 for both CEA and AFP and the detection limits reached 0.003 ng mL-1 for CEA and 0.002 ng mL-1 for AFP. Importantly, the proposed immunosensor was evaluated for the analysis of clinical serum samples, producing a good correlation with the data determined by the ELISA. This indicates that the immunosensor provides a possible application for the simultaneous multianalyte determination of CEA and AFP in clinical diagnostics.

Simultaneous electrochemical detection of cervical cancer markers using reduced graphene oxide-tetraethylene pentamine as electrode materials and distinguishable redox probes as labels

A novel, highly sensitive electrochemical immunoassay was proposed for the simultaneous determination of carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCCA) for the diagnosis of cervical cancer. Using an electrochemical analysis technique, two well-separated peaks were generated by neutral red and thionine, making the simultaneous detection of the two analytes on the electrode possible. Reduced graphene oxide-tetraethylene pentamine (rGO-TEPA), containing more amino groups, was of benefit to immobilize the primary antibody (Ab1) through an amidation reaction. Au@mesoporous carbon CMK-3 was synthesized and incubated with two secondary antibodies (Ab2) and different redox probes (neutral red and thionine) to fabricate the electrochemical immunosensor label intending to improve the analytical performance of the immunosensor. The immunosensor was prepared with a sandwich structure based on the peak current change of neutral red and thionine before and after the antigen–antibody reaction. The results showed that the immunosensor had a wide linear range, low detection limit, good reproducibility and stability. The method has been applied to the analysis of serum samples with satisfactory results.

A label-free immunosensor based on graphene nanocomposites for simultaneous multiplexed electrochemical determination of tumor markers

Here we prepared a label-free electrochemical immunosensor employing indium tin oxide (ITO) sheets as working electrodes and graphene nanocomposites as supporting matrix for simultaneous determination of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Reduced graphene oxide/thionine/gold nanoparticles nanocomposites were synthesized and coated on ITO for the immobilization of anti-CEA while reduced graphene oxide/Prussian Blue/gold nanoparticles were used to immobilize anti-AFP. The immunosensor determination was based on the fact that due to the formation of antibody–antigen immunocomplex, the decreased response currents of thionine and Prussian Blue were directly proportional to the concentrations of corresponding antigens. Experimental results revealed that the multiplexed immunoassay enabled the simultaneous determination of CEA and AFP with linear working ranges of 0.01–300ngmL−1. The limit of detections for CEA is 0.650pgmL−1 and for AFP is 0.885pgmL−1. In addition, the methodology was evaluated for the analysis of clinical serum samples and received a good correlation with the enzyme linked immunosorbent assay.

Au–ionic liquid functionalized reduced graphene oxide immunosensing platform for simultaneous electrochemical detection of multiple analytes

In this work, an Au–ionic liquid functionalized reduced graphene oxide nanocomposite (IL-rGO–Au) was fabricated via the self-assembly of ionic liquid functionalized reduced graphene oxide (IL-rGO) and gold nanoparticles (AuNPs) by electrostatic interaction. The IL-rGO can be synthesized and stabilized by introducing the cations of the amine-terminated ionic liquids (IL-NH2) into the graphene oxide (GO). With the assistance of IL-NH2, AuNPs were uniformly and densely absorbed on the surfaces of the IL-rGO. The proposed IL-rGO–Au nanocomposite can be used as an immunosensing platform because it can not only facilitate the electrons transfer of the electrode surface but also provide a large accessible surface area for the immobilization of abundant antibody. To assess the performance of the IL-rGO–Au nanocomposite, a sandwich-type electrochemical immunosensor was designed for simultaneous multianalyte detection (carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes). The chitosan (CS) coated prussian blue nanoparticles (PBNPs) or cadmium hexacyanoferrate nanoparticles (CdNPs) and loaded with AuNPs were used as distinguishable signal tags. The resulting immunosensor exhibited high selectivity and sensitivity in simultaneous determination of CEA and AFP in a single run. The linear ranges were from 0.01 to 100ngmL−1 for both CEA and AFP. The detection limits reached 0.01ngmL−1 for CEA and 0.006ngmL−1 for AFP, respectively. No obvious nonspecific adsorption and cross-talk was observed during a series of analyses to detect target analytes. In addition, for the detection of clinical serum samples, it is well consistent with the data determined by the ELISA, indicating that the immunosensor provides a possible application for the simultaneous multianalyte determination of CEA and AFP in clinical diagnostics.

Electrochemical immunosensor for simultaneous detection of multiplex cancer biomarkers based on graphene nanocomposites

In this work, a sandwich-format electrochemical immunosensor for simultaneous determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was fabricated using biofunctional carboxyl graphene nanosheets (CGS) as immunosensing probes, which were fabricated by means of immobilization of toluidine blue (TB) and labeled anti-CEA (Ab2,1), Prussian blue (PB) and anti-AFP (Ab2,2) successively on CGS. The capture anti-CEA (Ab1,1) and anti-AFP (Ab1,2) were immobilized onto the chitosan-Au nanoparticles (CHIT-AuNPs) modified electrode through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxy succinimide (EDC/NHS). Experimental results revealed that this sandwich-type immunoassay enabled simultaneous detection of CEA and AFP with linear range of 0.5–60ngmL−1 for both analytes. The detection limit was 0.1ngmL−1 for CEA and 0.05ngmL−1 for AFP (S/N=3). The assay results of serum samples with the proposed method were in a good agreement with the reference values from the standard ELISA method. And the negligible cross-reactivity between the two analytes allows it to possess potential promise in clinical diagnosis.

CD66b, CD66c and carcinoembryonic antigen (CEA) are independently regulated markers in sera of tumor patients.

Non-specific cross-reacting antigens (NCA-95 = CD66b and NCA-50/90 = CD66c) are members of the CEA (carcinoembryonic antigen = CD66e) family. Analysis of mRNA levels of CD66c in colon tumors suggests that this antigen is strongly up-regulated compared to its normal counterpart and could, therefore, be of clinical interest. CD66c is also expressed in normal lung and spleen tissues and, above all, on granulocytes. The appearance of CD66b in serum, the only strictly granulocyte-specific antigen, could point to the involvement of granulocytes in disease. Specific sandwich ELISAs have been established to determine CEA, CD66b and CD66c levels in serum. Controls have been carried out by testing sera from patients with benign tumors or inflammatory diseases and from healthy individuals. In sera of most patients suffering from solid tumors, sensitivities for CD66c are comparable to or lower than those for CEA. CD66c showed a much higher sensitivity in early colon tumor stages. Sensitivities over 40% have been determined for CD66b in sera of patients with uterine and kidney carcinomas. CML patients revealed sensitivities of 84% for CD66c and 47% for CD66b. Investigations of sera from patients with inflammatory colon diseases which are negative for CEA showed high sensitivity for CD66c but not for the granulocyte-specific CD66b. Patients with mastopathy revealed sensitivities of over 40% for both CD66c and CD66b. CD66b, CD66c and CEA are independently regulated proteins in a high percentage of patients. The simultaneous determination of CEA and CD66b/c can increase the sensitivities for malignant tumors but high sensitivities of CD66b/c for benign diseases limit their usefulness as tumor markers. CD66b may be interesting as a marker for kidney and corpus carcinomas, for which good markers are not yet available.

Limited improvement of tumour diagnosis by the simultaneous determination of carcinoembryonic antigen (CEA) and of a tumour-associated CEA-related antigen of Mr 128,000 in serum.

Two biotin-avidin based enzyme immunoassays were developed using three monoclonal anti-CEA antibodies with distinct epitope- and antigen-specificities. A broadly cross-reactive monoclonal anti-CEA antibody (T84.1) was immobilized on a solid support. Either monoclonal antibody T84.66 or CEA.11 was used as the second, biotin-labeled monoclonal antibody. Both antibodies do not cross-react with normal granulocytes or bile canaliculi. Monoclonal antibody CEA.11 binds to CEA and to an antigen with a relative molecular mass of 128,000 present in extracts from solid carcinomas. Monoclonal antibody T84.66 does not cross-react with the antigen of Mr 128,000. After adsorption of tumour extracts to a CEA-specific immunosorbent, residual activity was measurable by the CEA.11 assay, but not by the T84.66 assay. Serum samples (n = 726) from patients with malignant and non-malignant disease, as well as from healthy volunteers, were analysed by both immunoassays and by two commercial CEA immunoassays. In comparison with the T84.66 assay and the commercial assays, the CEA.11 assay did not significantly increase the sensitivity or specificity of tumour diagnosis.