Constructuion Of Signal Enhanced Nano-immunosensor For The Assay Of Four Lung Cancer Related Tumor Markers

The death rate of lung cancer patient precedes the patient with other malignancy,which is one of most harmful malignancies for human health. The World HealthOrganization forecasts that there will be more than one million new lung cancer cases until2025. Early diagnosis and early therapy have positive effect on prolonging the lifetime ofpatients with pulmonary carcinoma. Therefore, the study on the diagnosis method andtechnology for the early-stage lung cancer is important in lung cancer control. Thequantities of tumor markers in serum indicate the nature change of malignancy, by whichcan assist in the cancer’s diagnosis, classification, prognosis and treatment guidelines. Andthe assay for tumor markers owing the virtue of sensitive, minimal invasive and availablespecimens is the high-efficient and necessary biochemical indicator for diagnosis ofmalignanc. At present, the detected tumor markers in the clinic are not specific forpulmonary carcinoma and the single marker usually confine the clinic diagnosis. The jointassay of multiplex tumor markers will improve the diagnostic sensitivity, specificity andaccuracy. Based on the retrospective analysis for the tumor markers level by protein chip inthe serum of patients and healthy volunteers, we found that carbohydrate antigen19-9(CA19-9), carcinoembryonic antigen (CEA), carbohydrate antigen125(CA125),carbohydrate antigen15-3(CA15-3), neuron specific enolase (NSE) and so on presented ahigh relevance with the pulmonary carcinoma. Among these, the joint assay for the fourbiomarkers of CA19-9, CA15-3, CA125and CEA distinguishingly improved the diagnosisspecificity and accuracy for the pulmonary carcinoma. Thus, developing the jointimmol/Lunoassay for the four markers is hopeful to improve the rate of early diagnosis forlung cancers and has important clinic valaue in the lung cancer control.Electrochemiluminescence (ECL), a kind of analytical technique combining theelectrochemistry and luminescence, involves the generation of species at electrode surfacesthat then undergo electron-transfer reactions to form excited states that emit light. The ECL with the wide detected scale, in which the ECL probes present non-interfering signal at awide potential interval, can realize the simultaneous detection for multiple ECL signal atthe same electrode surface. Combing the ECL or electrochemical technique with theimmunoassay to assemble the new immunosensor has the potentiality to realize the fast andportable joint immunoassay for multiple tumor markers, which play an important role toimprove the rate of early diagnosis for lung cancer. Nanomaterials with specialphysical-chemical properties and biocompatibility can be immobilized on the solid-statesurface or tagged on the antibody molecule. They can improve the conductivity andcatalyze the photon reaction so as to enhance the ECL or electrochemical signal, whichhave been widely employed in the ECL or electrochemical immunoassay field. Furthermore,the biological enzyme can react with the substrate to amplify the analysis signal andimprove the sensitivity, widely employing in the electrochemical immunosensor butnonetheless rarely employing in the ECL immunoassay. Multiplexing the nanomaterials andenzyme to exert the synergistic catalysis, the catalytical efficiency usually increasesexponentially at a dramatic rate. Based on the synergistic catalysis to constructimmunosensor, the analysis signal of sensor can be obviously enhanced, availing for theultrasensitive assay of low level tumor markers in early-state lung cancer patients.In our work, a retrospective study was firstly done on tumor markers data of1021patients with pulmonary carcinoma (adenocarcinoma392cases, squamous carcinoma386cases, small cell lung cancer243cases) and827normal cases (control group) viaprotein-chip assay. The motiviation was to find the tumor markers highly related withpulmonary carcinoma and analyze their specifity, sensitivity and accuracy for the jointimmunoassay. Four highly-related tumor markers were finally confirmed to act as theanalysis model. Nanotechnology, enzyme, electrochemiluminescence and electrochemistrywere combined to construct the immunosensor for the joint detection of four tumor markers.The experiments were respectively based on the synergistic catalysis of nicotinamideadenine dinucleotide hydride cross-linked silver nanoparticles for ECL signal of Rucomplex, and the the catalysis of horseradish peroxidase for the cathodic ECL signal ofperoxydisulfate to construct the ECL immunosensor for the ultrasensitive assay of CA125and CEA. Furthermore, the experiment employed two different electrochemical probes todop the TiO2nanoparticles, label two different antibodies of anti-CA19-9and anti-CA15-3, and construct the electrochemical immunosensor for the simultenious assay of CA19-9andCA15-3.Objective1． To retrospectively study the expression data of tumor markers in differentpathological types of pulmonary carcinoma, screen the tumor markers that were highlyrelated with lung cancer and fit to be jointly detected in the early state of lung cancer.2．To employ the synergistic catalysis of nicotinamide adenine dinucleotide hydride(NADH) and silver nanoparticles (nano-Ag) in electrochemiluminescence (ECL) signalamplification, and construct a signal enhanced electrochemiluminescence immunosensorfor tumor marker of carbohydrate antigen125(CA125) assay.3．A label-free ECL immunosensor based on the catalysis of horseradish peroxidase forthe the cathodic ECL of peroxydisulfate was constructed to quantify carcinoembryonicantigen (CEA).4．An electrochemical immunosensor with high diagnostic sensitivity and accuracywas constructed to simultenious quantify the multianalytes of carbohydrate antigen19-9(CA19-9) and carbohydrate antigen15-3(CA15-3) via cation-exchange labelingrecongnition antibody, with methylene blue (MB) or cobaltous dipyridine complex (Co)doped Nafion@TiO2nanocomposite as signal marker.Materials and Methods1．The expression and clinical significance of four tumor markers in differentpathological types of lung cancer1021patients with pulmonary carcinoma (adenocarcinoma392cases, squamouscarcinoma386cases, small cell lung cancer243cases) and827normal cases (control group)were collected for tumor markers detection via protein-chip. The expression levels of tumormarkers in sersum were retrospectively studied for evaluating their clinic value in differentpathological types of pulmonary carcinoma. By analyzing the data and combining otherdiagnostic methods, this work concluded the relationship between the species of tumormarkers and pathological type of lung cancer, while counting the diagnositic sensitivity,specificity and accuracy by joint multianalytes immunoassay. Finally, the four tumormarkers of CA19-9, CEA, CA125and CA15-3were screened as the model for thefollowing study on the diagnosis of early-state lung cancer via multianalytes immunosensor.2． Signal-enhanced electrochemiluminescence immunosensor based on thesynergistic catalysis of nicotinamide adenine dinucleotide hydride and silvernanoparticlesThe metal-organic nanocomposites were introduced into the biological labeltechnology. A new metal-organic nanocomposite with synergistic catalysis function wasprepared and developed to construct an electrochemiluminescence (ECL) immunosensor forultrasensitive detection of tumor biomarker CA125. Silver nanoparticles (AgNPs) andnicotinamide adenine dinucleotide hydride (NADH) that can participate and catalyze theECL reaction of Ru(bpy)32+were employed as the metal component and the organiccomponent to synthesize the metal-organic nanocomposite of NADH–AgNPs (NA). Thenovel ECL immunosensor was assembled via Ru(bpy)32+-doped silica nanoparticles(Ru–SiO2) modified electrode with the NA as immune labels. Based on the circularlysynergistic catalysis by the silver and NADH for the solid-phase ECL of Ru(bpy)32+, theproposed immunosensor ultrasensitively sensed the concentration of CA125antigen inserum sample.3．Horseradish peroxidase catalyzing the cathodic electrochemiluminescence ofperoxy-disulfate to construct the label-free immunosensor for CEA assayThis method employed the reaction between enzyme and substrate to generate amountsof dissolved oxygen for catalyzing the cathodic ECL signal of peroxydisulfate andconstructing the label-free immunosensor. Based on the immuconjugated antigen on theelectrode surface shielding the contact between the substrate and the solid-state mobilizedenzyme, the ECL signal decreased with the increasing of analyte level. Thus, the label-freeECL immunosensor built the dependent relationship between ECL signal and the CEAantigen for the marker assay in serum sample.4．Cation-exchange antibody labeling for simultaneous electrochemical detectionof tumor markers CA15-3and CA19-9The method employed a nanohybrid composed of an ionomer and conductive titaniumdioxide nanoparticles that act as a matrix support for the antibodies labeling. The twoantibodies (anti-CA15-3and anti-CA19-9) were labeled (a) with a cobaltous dipyridinecomplex, and (b) with methylene blue. Labeling was based on cation-exchange interaction rather than on covalent conjugation. The redox potentials of the two labels were separatedby an interval of0.3V. The resulting sandwich-type immunosensor was read out bydifferential pulse voltammetry. The potential sites and currents of the two redox probesreflected the concentration of the two analytes.Results1．The expression and clinical significance of four tumor markers in differentpathological types of tumors1) The expression of four tumor markers in the group of lung cancer and healthyvolunteers: The expression level of CA19-9, CEA, CA125and CA15-3in serum samplepresented distinguished direfference between the1021lung cancer patients and827healthycases (P<0.01).2) The positive rates comparison of tumor markers in different pathological types oflung cancer: the1021cases of lung cancer patients were classified as adenocarcinoma392cases (38.39%), squamous carcinoma386cases (37.80%), and small cell lung cancer243cases (23.80%). The positive rates of CA19-9, CEA, CA125and CA15-3in the type ofnon-small cell lung cancer was higher than that in small cell lung cancer. The differenceswere statically significant. The positive rates of CEA was highest in the adenocarcinomapatients (up to55.35%), which was higher than that in squamous carcinoma (26.68%) andsmall cell lung cancer (25.92%); The positive rates of CA125was smaller than that of CEA,but highest in the adenocarcinoma patients’ serum (up to41.07%) and higher than that insquamous carcinoma (27.72%) and small cell lung cancer (29.62%); The positiveexpression of CA19-9and CA15-3also highestly presented in the group of adenocarcinomapatients.3) The accuracy of four tumor markers for the diagnosis of different pathological typesof lung cance was assessed by ROC curve: CA19-9presented high accuracy for thediagnosis of adenocarcinoma and lung cancer with the area under ROC curve of0.810and0.617(with the area of0.5as the control value); CEA presented good accuracy for thediagnosis of adenocarcinoma and non-small lung cancer, respectively with the area underROC curve of0.697and0.446; CA125presented acceptable accuracy for the diagnosis oflung cancer, adenocarcinoma and small lung cance, reapectively with the area under ROCcurve of0.612,0.600and0.440; CA15-3only presented acceptable accuracy for the diagnosis of adenocarcinoma.4) The sensitivity and specificity of tumor markers for the diagnosis of differentpathological types of lung cancer: The detection of single tumor marker all presented lowsensitivity, CEA (37.5%), CA125(33.3%), CA19-9(14.8%), CA15-3(6.8%); and thesensitivity presented higher in the diagnosis of adenocarcinoma than in the diagnosis ofsquamous carcinoma and small-cell lung cancer.5) The clinic value of joint immunoassay for four tumor markers: the joint detection bytwo analytes, three analytes and four analytes were respectively counted to analyze theirsensitivity, specificity and accuracy for the diagnosis of lung cancer. For the jointimmunoassay of two analytes, the joint of CEA and CA125presented the highest sensitivity19.01%; the joint immunoassay of three analytes did not present the obvious difference; thejoint of four analytes presented the highest specificity than other matrix. The accuracy ofthe joint immunoassay increased with the increasing analyte items. But the sensitivity of thejoint immunoassay decreased with the increasing analyte items. And the joint immunoassayof the highest specificity for diagnosis of lung cancer2． Signal-enhanced electrochemiluminescence immunosensor based on thesynergistic catalysis of nicotinamide adenine dinucleotide hydride and silvernanoparticles1) The ECL intensity was linearly correlated to the concentrations of CA125onlogarithmic scales in the range of0.1-150U/mL. The synergistic ECL catalysis ofmetal-organic nanocomposite amplified response signal and pushed the detection limitdown to0.03U/mL, which initiated a new ECL labeling field and has great significance forECL immunoassays.2) The proposed immunosensor presented the acceptable specificity, repeatablility andstability.3) Comparing the detected data of CA125by the proposed immunosensor with thedata by ELISA, the results all showed the correlation. The lower detection limit presented atthe proposed ECL immunosensor. It can be further employed in the detection of serumsample. 3．Horseradish peroxidase catalyzing the cathodic electrochemiluminescence ofperoxy-disulfate (S2O82-) to construct the free-labeled immunosensor for CEA assay1) The enzyme catalysis remarkably amplified cathodic ECL signal of peroxydisulfate.With this catalysis, a free-label ECL immunosensor even can detect the low limit1.04ng/mL for the CEA.2) The proposed immunosensor presented the acceptable specificity and selecivity.3) Comparing with prophase work， this work successfully employed theenzyme-linked immunoassay in the ECL immunoassay, which improved the sensitivity ofimmunoassay and could be used to detect the trace tumor markers in the early-state lungcancer.4．Cation-exchange antibody labeling for simultaneous electrochemical detectionof tumor markers CA15-3and CA19-91) The nanohybrids of Nafion-coated TiO2nanoparticles that had both virtues ofNafion and nanoparticles were prepared as the matrix support for cation-exchange labeling,which avoid the negative effect of covalent labeling and avail the immune recognition.2) Based on the electrochemical assay of differencial pulse voltage (DPV), thepotential sites and currents of the two redox probes reflected the concentration of the twoanalytes. The two analytes were determined with a detection limit of1.6U/mL for CA19-9,and0.3U/mL for CA15-3.3) The proposed immunosensor was employed for the recovery determination of dualanalytes CA19-9and CA15-3in human serum. The results showed that the proposedimmunosensor presented the good specificity and repeatability for dual analytes assay.Conclusion1．The expression and clinical significance of four tumor markers in differentpathological types of tumorsFour tumor markers of CA19-9, CEA, CA125and CA15-3were with high accuracyfor the diagnosis of lung cancer and adenocarcinoma. The joint detection for dual tumormarkers can obviously improve the sensitivity and accuracy in the diagnosis. Therefore, thestudies on the joint assay of these tumor markers availed to the fast diagnosis of early-statelung cancer with important significance for the control of lung cancer.2． Signal-enhanced electrochemiluminescence immunosensor based on the synergistic catalysis of nicotinamide adenine dinucleotide hydride and silvernanoparticlesThe new metal-organic solid nanocomposites of NA were successfully developed asECL labels to construct a sandwich-type immunosensor. We inoculated the synergisticcatalysis of the catalytical-material field into the ECL signal amplification strategy. Thenew material family exerts the ECL signal-enhanced properties via the coreacted catalysismechanism. The synergism between the metal of silver and the biocompatible organic ofNADH accelerates the electron transport and facilitates the generation of the excite state,making the nanocomposites exhibit better ECL catalysis properties than either of thecomponents. Based on the heterogeneous catalysis of NA for the solid-phase ECL ofRu(bpy)32+, the proposed immunosensor successfully realized the ultrasensitive assay fortumor marker CA125, exceeding other reported electrochemical or piezoelectricimmunosensors.3．Horseradish peroxidase catalyzing the cathodic electrochemiluminescence ofperoxy-disulfate (S2O82-) to construct the free-labeled immunosensor for CEA assayA free-labeled ECL enzyme-linked immunoassay was successfully designed for theassay of CEA. The proposed immunosensor employed the enzyme to catalyze the cathodicECL signal, which intiated a new enzyme-linked immunoassay method for ECLimmunoassay and have the potentiality to detect the trace tumor markers in serum sample.4．Cation-exchange antibody labeling for simultaneous electrochemical detectionof tumor markers CA15-3and CA19-9A multianalysis strategy with cation-exchange labeling was successfully designedusing Nafion coated TiO2nanoparticles as matrix support. The tumor mark of CA19-9andCA15-3were chosen as the synchronous analysis models. The labeling route of antibodyentrapment following by signal molecules adsorption effectively avoids the site competitionon matrix support and prevents the covalence-caused deactivation from the recognitionantibody. Coupling DPV technique, the distinct potential positions and current intensitiesrespectively reflected the identities and quantization of dual antigen analytes. Thecation-exchange membrane coated nanoparticles as matrix support successfully performedthe duplex function of entrapping antibody and labeling probes in the multianalysis strategy,foreseeing a prospect of fast, effective and specific non-covalent label mode in the serologic multianalytes assay to assist the diagnosis of early-state lung cancer.