Construction Of A Novel Surface Chemistry For SPR Protein Chip And Its Application In The Immune Disorder Diagnosis For CITP

Research backgroundSurface plasmon resonance (SPR) is a free labeled optical biosensor technology, which is more and more widely used in many fields today, such as life sciences, which do not need to lable the analyte and measure the kinetics of molecular biological activity in non-modified conditions, be suitable for the high throughput screening of bioactive molecules, especially for the small molecules, the trace analysis of the unknown materials and the clinical samples online.Chronic idiopathic thrombocytopenic purpura(CITP) is a kind of common bleeding autoimmune disease, the incidence is on the rise in China, which seriously affect the life quality of the CITP patients, but the mechanism is unknown, it is difficult to find really specific and effective method for treatment. Many studies show that the pathogenic CD4+Th cell proliferation rising, apoptosis reducing, activity increasing and the Thl,Th17, Th22cell differentiation imbalance.Recently, a new regulatory B cells has been paid more and more attention and has been actively studied in the field of immunology. So far there are no related reports in CITP patients, we will study the role of Breg in immune disorders of patients with CITP, and to explore the relationship between Breg and CD4+Th cell subsets and platelet antibody, and we will look for one new ideas for the clinical diagnosis and treatment of CITP. Therefore, we will construct a novel SPR chemistry chip, and explore the application of SPR technology in the detection of immune disorders for CITP patients. SPR technology has a very important significance for the choice of treatment strategy effective evaluation and prognosis and platelet compatibility.Research purposeTo construct and optimize the novel chemical surface chip for SPR technology, and to establish the new detection method based on SPR technology, which is simple and rapid, high sensitivity and specificity. As the treatment effect for CITP patients is poor, whose pathogenesis is complex and unknown to the role of new regulatory B cells in autoimmune diseases, we will analyse the characters of the regulatory B cells in the peripheral blood of patients with CITP and its effect on CD4+Th cell immune disorder in this study. We plan to explore its application value in clinical diagnosis and treatment of CITP patients, which will provide a new target and a novel assay for the immune disorder of CITP.Research contentsThis study intends to carry out the following work:1Construct a novel surface chemistry SPR protein chip, which is quick and simple with high sensitivity and specificity, and evaluate the methodological character.2Analyse the changes of regulatory B cells and its correlation with platelet antibody in the peripheral blood of patients with CITP.3Using the novel SPR protein chip, to establish a new assay with high throughput and free lable for platelet antibody screening and match in CITP patients, and to compare the characters with the MAIPA and SPA assays.4Study the correlation between the changes of regulatory B cells and CD4+T cell and related cytokines in the peripheral blood of patients with CITP, to analyse the role of them for CITP immune disorder. 5Using the novel SPR protein chip, to establish a new assay with high throughput and free lable for detecting the special cytokines in CITP patients.Research methods1Preparation for poly (OEGMA-co-HEMA) matrixUsing binary self-assembly system to control the concentration of initiator surface. Specific operation process is as follows:ethanol as solvent, mixed mercaptan initiator and PEG mercaptan, a total concentration of1mM, the SPR chips treated with UV-ZONE are immersed in the mixed solution for15hours, then chips are taken out, washed with ethanol fully, blown dry with nitrogen. According to2,2’pyridine (tendency)12.5mg (0.8mM),2.62g OEGMA526(5mM), HEMA0.65g (5mM), dissolved them in5mL ultrapure water and5mL methanol. Add1mL CuCl2solution (0.04mM), nitrogen gas is piped in, eliminate oxygen for15min. Then slowly inject1mL ascorbic acid(AscA) solution (0.04mM), Cu (II) complexes are gradually turned into Cu (I) complexes, reaction solution is changed from blue to pink. Continue to anaerobic treatment for15min, polymerization reaction is processed in the glove box of nitrogen gas environment on the chip surface. Chip surface agent will trigger polymerization reaction on the chip surface at room temperature for8hours, then remove chips and terminate reaction, using methanol and ultrapure water clean them, blow dry with nitrogen for further analysis, such as elliptical polarization measurement of film thickness, etc. The hydroxyl activity is poor at Poly (OEGMA-co-HEMA) matrix chain end, which is difficult to couple reaction with the protein under mild conditions, so we must have functionalization for hydroxyl groups, such as functional hydroxy. Hydroxy functional experiment operation is as follows:the surface modified poly (OEGMA-co-HEMA) chips are soaked in DMF reaction solution with succinic anhydride (10mg/mL)and DMAP(15mg/mL), react under room tempture for12hours. Remove chips and wash them with DMF and ethanol fully, blow dry by nitrogen.2Preparation for dynamic cyclodextrin surface (1) Using self-assembly monolayer technique, PEG monolayer is assembled on chip surface as the main chain, we use thinner to control the density of PEG main chain, then soake the chips in cyclodextrin solution, when plenty of cyclodextrin went into the PEG main chain, stopping the linear PEG at the other end.(2)By hydroxylation reaction, cyclodextrin hydroxyl groups are turned into active carboxyl group, which is advantageous for the immobilized biological probe. Using system experimental parameters (chain density, chain length and carboxyl numbers) of SPR technology for their influence on biological detection performance, we optimize the best conditions.(3) Under the optimization experimental conditions, we have prepared for the novel sensors based on cyclodextrin molecule matrix, and applied the chip matrix to detect protein model.3Characterization methods for SPR chips(1)AFM characterization:Characterization of matrix morphology structure is analysed by atomic force microscope (AFM,Veeco,Dimension3100). The experiment is done by the contact model.(2) Elliptic polarization characterization:film thickness is measured by M-2000V spectral elliptic polarization measuring instrument (J.A.Woollam Co, inc). Measurement angle is65,70,65degrees, wavelength varies from400nm to800nm. From elliptic polarization meter data, we get the thickness of the film with a specific model, including SAMs and Poly (OEGMA) using Cauchy Model,(An, Bn)=(1.45,0.01) and (1.46,0.01), respectively. Sample thickness are measured as the average values for the three position and reported the results for x±s values.4Performance test(1)Anti-fouling effect test:According to the instrument manuals, carboxyl functional chip will be loaded, set the working temperature at25℃, flow velocity at2μL/s, ran in PBS buffer solution (pH=7.4), after a stable baseline appear, inject specific concentrations protein solution (PBS dilution), finally inject PBS buffer solution.(2)Protein detection ability test:According to the operational requirements, set parameters of instrument for sample and add30μL sample in384well plates, set temperature at25℃,50%humidity, protein sample will be pointed on the SPR chip surface by contacted way. After sample chip was placed in the box of25℃temperature and75%humidity, keep constant temperature and humidity for2hours, then install the protein chip in SPR system, ran in PBS solution (pH=7.4) and at2μ L/s flow velocity, after a stable baseline is seen, ethanol is run into the chip for7min, closed the extra active site, then a specific concentration protein solution(PBS dilution) is injected, finally glycine solution (lOmM, pH=2.0) can wash out the combination of proteins, so as to achieve the goal of regeneration.5Collect specimens and separate PBMC from CITP patientsExtraction of5mL venous blood with heparin sodium anti-coagulation from CITP patients and control group, PBMC is separated by density gradient centrifugation assay, and adjusted in RPMI1640medium containing10%fetal bovine serum,PBMC density is1×109/L.1mL suspension cell is used for detecting CD4+T and Breg subsets with fluorescent antibody staining by FCM,2mL cell suspension is incubated in phorbolester PMA (final concentration50μg/L) and ionomycin (final concentration1μmol/L) at37℃overnight, then collecting the supernatant and saving them at-70℃for detection of cytokines.6Determination of Thl, Th17,Th22subsets and Breg by FCMTake five tubes for detection, respectively take the mononuclear cells using lymphocytes stratified separation, wash twice by PBS,106mononuclear cells for each tube, No.1tube as isotype controls, No.2tube including5μL FITC-CD4antibodies and5μL PE-IFN-y, No.3tube including5μL FITC-CD4antibody and5μL IL-17A antibody, No.4tube including5μL FITC-CD4antibody and5μL PE-IL-22antibody, No.5tube including CD5-FITC, CDld-PE, PerCP-CD19antibodies5u L, respectively. The specimen after thoroughly incorporated with antibody, incubated at room temperature away from light for1h. Take2mL cracking fluid for red blood cells to act for10mins, after the centrifugal supernatant is removed, then wash2times by PBS and detect them by FCM, the related analysis and comparison is done using the software.7Detection of IFN-γ, IL-17, IL-22and IL-10cytokines by ELISATake out the PBMC culture supernatant, we detect IFN-y, IL-17, IL-22and IL-10levels by ELISA method, and do it according to the reagent operation manual, providing three holes for each sample and standard, read the OD value at450nm.8The conditions for platelet immobilized on SPR chip surfaceThe influencing factors mainly are the ionic strength, pH value of solution and the concentration of the protein. In this study, under certain ionic strength, we dilute platelet antigen and point them on the novel SPR chips, respectively. According to not wasting samples, and making sure that the immobilized platelets reach a certain amounts, to determine the platelet antigen dilution degrees. At the same time, we choose the different pH value (4.0,4.0,4.5,5.5,6.0)10mM acetate buffer solution as stationary liquid for platelets, to explore the influence of pH value for platelet antigen, to find the optimum pH value. The principle of chip regeneration is to use a certain concentration of acid, alkali, or high ionic strength of solution to wash away the combination of antigen and antibody, so as to achieve the regeneration of the sensor chip. On the regenerating reagent, we try to adopt the10mM NaOH,1:300diluted phosphoric acid and pH=2.0glycine as regenerative reagent, finally to determine the best regenerative reagent types.9Platelets immobilization on the SPR chip surfacePlatelet antigens are immobilized:According to the explored conditions, amino coupling method is used to immobilize corresponding general-purpose platelet antigen on the SPR sensor chip surface for detecting the corresponding platelet antibody. Method as follows:(1) Activation for the SPR chip:First,0.3939g EDC (0.4M) and0.430g NHS (0.1M) were dissolved at room temperature, then make up10mL EDC/NHS activation fluid mixture, the SPR chip is activated in the activation fluid for30min.(2) Point platelet antigen:10%glycerol fluid is made in10mM acetate (pH=6.0) and glycerin, the universal platelet antigen is diluted for five times with it,0.5μL mixture is pointed on the chip surface, and immobilized at room temperature for1hour.(3) The surface of the chip is enclosed:after chip immobilization, which will be installed in the SPR analyzer, inject PBS solution to choose optimum resonance angle (debugging from minimum angle to maximum angle, then choose the optimum resonance angle between70and100), when baseline is stationary, treated for10min with closed fluid ethanolamine, enclosing unreacted activated surface, removing physical adsorption material and sealing fluid by injecting PBS, then testing the samples.10The performance analysis of SPR technology(1) Using the exploring conditions and the fixed SPR chips, to detect the positive control, negative control and patient samples serum correspondingly, analyse the stability, sensitivity and specificity of SPR technology, data from SPR analyzer are analysed using BIAevaluation software.(2) Comparative study:detect platelet antibody for40CITP patients using SPR technology,SPA assay and MAIPA assay, compare with the differences of three methods, and to analyze their performance.11Application for platelet antibody detection by the novel SPR chipsFor CITP patients, screen platelet antibody by SPR technology, and choose compatible platelets for transfusion. The known type O general platelets are immobilized on a chip (preparation for chip according to the above), to detect whether there are relevant antibody in CITP patients serum. Also the platelets supplied by blood center are washed and diluted, then0.5u L platelet is pointed on the chip, to detect SPR signal change in serum for platelet antibody positive patients, if no signal change, show that they are in matches, or matching statements do not agree. Using the SPR technology for10CITP patients with platelet antibody positive to choose cooperated platelets for infusion, and to analyze the clinical effectiveness of platelet transfusion according to the long-term follow up. 12Application of cytokines detection by the novel SPR chip(1) First method:take out the PBMC culture supernatant, using SPR chip technology to detect IFN-y, IL-17, IL-22and IL-10levels. The ways as follows: after SPR chip is activated, anti-IFN-y, anti-IL-17, anti-IL-22, anti-IL-10mAb are immobilized on the SPR chip for60minutes, respectively. At the same time pointing PBS as blank control (due to performance analysis of the chip has been ae studied), standard cytokines are diluted into different concentrations, detecting their corresponding signal value and make up standard curves, according to the standard curves, the concentrations of the corresponding cytokines can be calculated.(2) Second method:for exploratory research, for detecting the specific cytokines in CITP patient, First the anti-CD4antibody is immobilized on the chip, capturing CD4+T cells, then culture cells on the chip, and have the dynamic detection of cytokines, and comparing with normal control group, this method will help to analyse the cellular immunity disorders for CITP patients more accurately. We will study to detect IFN-gama from CD4+T secretion as an example.13Statistical processingExperimental results show as x±s, analysed by the SPSS16.0software, using two samples t test, muti-samples ANOVA test and spearman correlation analysis for the relation degree between the two variables, the difference was statistically significan with P<0.05.Research results1The construction of novel SPR matrix:For the preliminary master in SIP dynamics, we made full use of the controllable polymerization technology, and adapt the poly matrix related parameters at the nanoscale molecular, built a cyclodextrin matrix platform and Poly(OEGMA-co-HEMA) matrix, and achieved a high unity between the "zero" protein nonspecific adsorption and the high protein fixed ability, solved the common problems of biological sensors.2(1)For the novel chip, protein nonspecific adsorption amount is lower than the detection sensitivity of SPR. The experimental results showed that the objective of the research and development of the chip had good ability to resist protein nonspecific adsorption.(2)This project adapt the model protein IgG/goat anti-human IgG system to verify application effect for two kinds of chips in the detection of protein interactions. The experimental results showed that both chips had the ability of high immobilization protein, and can maintain protein biological activity,but the cyclodextrin matrix platform was more sensitive than the Poly (OEGMA-co-HEMA) matrix.3FCM results showed that the proportion of peripheral blood CD19+B, CD5+CD19+B cells were increased in CITP patients compared with control group, the difference was statistically significant (P<0.05), but the proportion of CD1d (hi)CD5+CD19+B(Breg) was reduced, the difference was statistically significant (P<0.05).4ELISA results showed that, compared with healthy controls, the expression levels of IL-10and TGF-betal from PBMC cultures supernatan were reduced in CITP patients, the difference was statistically significant (P<0.05). The proportions of Breg positively correlated with IL-10levels from PBMC culture supernatant (P<0.05), but the proportions of Breg were of no correlation with TGF-betal level (P>0.05). The changes of regulatory B cells had negative correlation with the changes of platelet related antibody(PAIgG) in CITP patients.5Before glucocorticoid treatment in CITP patients, the platelet counts were (5-37) x109/L, mean count was28x109/L, the percentage of peripheral blood Thl, Th17, Th22subsets increased obviously, the difference was statistically significant compared with normal controls (P<0.05). The proportion of regulatory B cells (Breg) was reduced, the difference was statistically significant (P<0.05). After glucocorticoid treatment for four days, the platelet counts rised, mean counts were71×109/L, compared with normal control group, the percentages of peripheral blood Thl, Thl7, Th22subsets decreased obviously, they had no significant difference (P>0.05). The percentages of Thl, Th17, Th22subsets had negative correlations with the platelet counts (P<0.05), but the proportion of Breg had positive correlation with the platelet counts (P<0.05).6According to the ELISA results, compared with normal control group, before glucocorticoid therapy in CITP patients, IFN-y, IL-17, IL-22levels from PBMC culture supernatant rised, the expression of IL-10level was lower, the difference was statistically significant (P<0.05), but after glucocorticoid treatment, compared with normal control group, there was no significant difference. The ratio of Breg cells in CITP patients showed positive correlation with the change of IL-10level in PBMC culture supernatant(P<0.05), the changes between IL-10and IFN-y, IL-17, IL-22levels showed a negative correlation,respectively (P<0.05).7Detection conditions for platelet anibody by the novel SPR chips in CITP patients:(1) Platelet diluted times:Using acetic acid buffer solution to dilute platelets, respectively for5times,10times,20times,50times,100times, results showed that with the concentration of pointed analyte increasing (including platelet antibody in serum) signal value increased, after20times dilution, SPR detection signal is not obvious, so10times dilution was pointed concentration for sample platelets.(2) Series of pH value (pH=4.0,4.5,5.0,5.5,6.0) acetic acid buffer solution were used as platelets pointing sample dilution, respectively, the results showed that the acetic acid buffer solution at pH=4.5had more strong signal.(3) In NaOH buffer solution, partially hydrolyzed polymer chain, lead to the baseline drift down, but using1:300diluted phosphoric acid, baseline did not return to the original baseline. Using the pH=2.0glycine regeneration, baseline return to the initial value, after regeneration the probe still maintained high biological activity.8Detection limit for platelet antibody:Platelet reagent antigen was pointed on the same chip, and selected the corresponding seven areas, one for blank control point, the other6points for testing, standard controls were taken from positive serum5,10,20,30,40,60uL, the corresponding concentration was50ng/ml,100ng/ml,200 ng/ml,300ng/ml,400ng/ml,600ng/ml, respectively, at2u L/s the velocity of sample testing, processing by the BIAevaluation analysis software, results showed that had good sensitivity and minimum detection limit was about50ng/ml.9Platelet antibody specific detection:reagent platelet antigen and nonspecific NS1antigen were pointed on the same chip, one spot for blank control, the other each point for sample testing, using platelet antibody positive controls and NS1antibody positive serum, respectively, results showed that, when platelet antibody positive controls run into the SPR chips, strong signal was seen at the platelet antigen spot, but had weak signal at nonspecific NS1antigen spot and control spot. This study suggests that the chip specificity is better, the novel SPR chip can detect specific platelet antibody.10Platelet antibody screening was analysed by SPR chip technology,SPA assay and MAIPA method for40CITP patients, test results were shown that, by McNemr test, there was no statistically significant difference between SPR technology and SPA assay (P>0.05). There was also no statistically significant difference between SPR technology and MAIPA assay (P>0.05), the sensitivity, specificity and total consistency for SPR method were91%,97.9%,97.2%, respectively. In the40CITP patients,26of them platelet antibody were positive by SPR assay. Of the10platelet antibody positive CITP patients, by SPR technology, we choose compatible platelets transfusion from a number of donor platelet, after clinical follow-up analysis, of which8cases of patients, the platelet added value1h CCI>7.5,24h CCI>4.5, got effective infusion and they were in good condition, the other2cases were invalid for other basic diseases.11Results for detection of related cytokines by the novel SPR chip in CITP patients:cytokines IL-10, IFN-y, IL-17, IL-22levels form PBMC culture fluid in CITP patients were detected by the novel SPR chip, the results showed that, compared with ELISA, there were no significant difference(P>0.05), but we can assayed all the cytokines in one time test, which was more simple and convenient.12Another application of SPR model for specific cytokines detection, CD4cells were captured on the surface of SPR protein chip, and then we analysed cytokines.The activity and purity of CD4+T cells are more than90%, by the dynamic analysis method, IFN-gama levels from CD4+T cells culture stimulation were detected on the surface of the chip, results showed tha the detected signal time was shorter than those of stimulation in vitro, and had better specificity.Research conclusion1This project has used controllable surface activity by polymerization technology to regulate the related parameters of polymer on nanometer scale, and built a novel chemistry surface SPR biosensor chip based on oligomeric ethylene glycol methyl methacrylate(Poly(OEGMA-co-HEMA)),which implemented the resistant protein nonspecific adsorption effect, and reduced background noise.2The project has built another novel dynamic cyclodextrin SPR chip surface. Through the chemical modification of cyclodextrins in polyethylene glycol (PEG) chain on dextrin free rotation and sliding, freedomly taking advantage of cyclodextrins rotation and sliding ability in polyethylene glycol (PEG) chain, which realized the biosensor surface with the dynamic state, can improve the space adaptation and the space selectivity of the probe for detecting protein molecules.3This project has shown that the proportion of Breg is reduced, the proportion of the Breg positively correlated with IL-10levels from PBMC culture supernatant, the changes of regulatory B cells and platelet antibody show a negative correlation in CITP patients, which may provide a new idea for pathognisis of CITP.4We have established a new type of SPR microarray to analyse platelet antibody and the compatibility between the serum of CITP patients and platelets from donors, and to cross matching test. By clinical follow-up analysis, showing effective infusion and good conditions,which showed that the novel SPR technology can provide preliminary application for platelet antibody screening and matches.5The percentages of peripheral blood Thl, Th17, Th22subsets increased obviously in CITP patients, and showed negative correlation with the platelet counts. The proportion of Breg is reduced, and show positive correlation with the platelet counts, IFN-y, IL-17, IL-22levels are reduced, IL-10levels are rised, but show a negative correlation between IL-10with IFN-y, IL-17, IL-22level, which may provide a new target for clinical diagnosis and treatment of CITP.6This kind of novel SPR protein chip can be used to detect the cytokines from PBMC secretion, to study the CITP immune disorders with high-throughput and rapid analysis. Besides, a new special SPR chip platform was established for capturing CD4+T cells to analyse the related cytokine IFN-gama in dynamic detection, the real situation of immune disorders is more evident, which is better in favor of clinical diagnosis and treatment for CITP.7This study provides a new target and the new detection method for clinical diagnosis and treatment in CITP immune disorders. This novel cyclodextrin chemical surface SPR chip has important significance in the detection of platelet antibody and the cytokines disorder for CITP patients with high-throughput and rapid analysis.