The Preparation Of Water Soluble CdTe Quantum Dots And Its Study On Immuno-imaging Of Colorectal Cancer Cells

PrefaceIn the field of biomedicine,with the ever developing probe technology of high sensitivity and high specificity,limitations of organic fluorescent dyes application are becoming more and more obvious.In comparision,quantum dots has shown its remarkable superiorities in various fields.Quantum dots(QDs) are nanometer-scale semiconductor crystals composed of groupsâ…¡-â…¥(eg.CdTe,CdSe) orâ…¢-â…¤(eg.InP, InAs) elements,and are defined as particles with physical dimensions smaller than the exciton Bohr radius.As a new fluorescent dye,QDs have many special and excellent optical features—continuous and wide excitation spectra,possible adjustment of fluorescent spectra location by changing the diameter of QDs,thus QDs of different colours fluorescence could be activated through laser of but one wavelength,to carry out multicolor meassurement;narrow emission spectra,thus light of different colours could be shown without overlapping;stokes,preventing the overlapping of emission and excitation spectra,thus permitting the optical measurement under the condition of low signal tensity;high resistance of photobleaching,the high stability of QDs is extremely significant for research requiring long time imaging;high stability of the fluorescence,facilitating to gain fluorescent signal and so fotrh without background disturbance.Therefore,QDs has shown its unique advantages and broad prospect in the application of many fields.Marking QDs on the antibody to create immuno-fluorescent probe can specificly identify antibody on cells,and perform detection of antigen.Because of the unique optical properties of QDs,introducing it to mark antibody and study of immuno-fluorescent imaging can greatly increase the sensibility of specific detection, and it cal also be applied in the multiple-colored imaging and real-time monitoring, which has great significance for the biomedical study,especially for early detection of tomor in high performance.According to the difference of solvents adopted for preparating QDs,methords of preparating high quality QDs mainly comes in the following two ways.One way is to compose by taking advantage of precursor thermolysis in organic solvent of high boiling point.The other way is to compose directly in aqueous solution by stabilizer. QDs prepared in aqueous solution has both water solubility and biocompatibility, without the necessary modification of hydrophilic molecules and group for conjugating biomolecule,which is more suitable for conjugation with biomolecule.Therefore,this research is on creating mercapto-acetic acid coated CdTe QDs through aqueous solution.Colorectal cancer is one of malignant tumors with the highest incidence,ranking the third in our country.Finding an effective device of early detection and letting the patients receive earlier diagnosis and treatment is a vital factor for increasing the patients' survival rate.ND-1 is a kind of monoclonal antibody which is prepared using the colorectal cell line CCL187 as the immunogen.Thousands of clinical pathology assays have shown that monoclonal antibody ND-1 has specific affinity to LEA in middle and high differentiated colorectal cancer tissue,and outweighs monoclonal antibody anti-CEA which is broadly utilized in clinics.The tumor associated antigen LEA which is identified by ND-1 is located on the cancer cell surface,it is a kind of antigen related to cell differentiation and invasive power.In normal tissue, non-colorectal cancer tissue or low differentiated colorectal cancer tissue,it is rarely expressed,if any,the expression rate is quite low,while in middle or high differentiated colorectal cancer tissue,the expression rate is quite high.The check of LEA can help early colorectal cancer diagnosis,and used as a referring index for deciding malignancy of cancer.makes more significant sence in early diagnosis.Thus,this study prepare the CdTe quantum dots in water phase,and conjugate it to monoclonal antibody ND-1, preparing a specific fluorescent probe for colorectal cancer associated antigen LEA. This study realized dectetion of colorectal cancer cells in high sensibility,and provide a new method for early diagnosis and imaging.Materials and Methods1.Preparation,purification and exosydrome of Water soluble CdTe QDs1.Preparation of water soluble CdTe QDsPrepare NaHTe precursor at 0℃under no-oxygen condition,precursor as the core to compose QDs solution,obtain QDs with different particle diameters by heating at different time at 140℃.2.Purification of water soluble CdTe QDsQDs obtained from the above was filtered through 0.22μm micropore film to remove Te which is not sufficiently reacted.3.Exosydrome of water soluble CdTe QDsDetect UV-vis excitation spectra and emission spectra of QDs and get a rough estimate of particle diameter of QDs by spectrum peak;perform TEM measurement of the QD₆₁₃ obtained through 140℃heating 195min.2.Preparation,purification and characterization of the monoclonal antigen ND-11.Preparation of BALB/C's abdominal dropsy for monoclonal antigen ND-1Inject in female BALB/C mice abdominal cavity the hybridoma cell line IC₂ that can help excrete ND-1.every mouse was injected 0.5ml,cell concentration is1-2×l0⁶/ml,and take out abdominal dropsy after 10-20 days.2.Abstraction and purification of BALB/C's abnormal dropsyAdd CaCl₂ to abdominal dropsy and remove protein fiber clot,after dialysis of 24h through HiTrap protein G column affinity chromatography purifying monoclonal antibody.Antibody after purification was conditioned to the approriate concentration and displace buffer to PBS,4℃for sort-term use or -70℃for long-term conservation. SDS-PAGE was used to identify mAb purity.Bradford was used to measure the concentration of the mAb.3.Identification of the immuno-activity of the purified mAb ND-1To use purified mAb ND-1 as the primary antibody,goat anti mouse FITC-IgG as the secondary antibody to incubate CCL187 and HeLa respectively,detect the immuno-activity of mAb by immuno-fluorescent imaging. 3.Preparation and purification of the conjugate of quantum dots and ND-1(QD₆₁₃-ND-1)1.Bioconjugation of quantum dots and mAb ND-1Using different buffer condition of conjugation,to inspect the activating condition of-COOH on the surface of QDs.EDAC connection,covalently and stably connecting -COOH of TGA on the surface of QDs with primary amine on mAb ND-1.2.Purification of bioconjugatesSephacryl S-200 for gel filtration chromatography,purifying conjugates of QDs and mAb ND-1.4.Specific immuno-fluorescent imaging on colorectal cancer cells(1) With the conjugate of water soluble QDs CdTe and mAb ND-1,QD₆₁₃-ND-1 used as the fluorescent probe,study on specific immuno-fluorescent imaging was carried out for the high colorectal cell line of CCL187 whose LEA is high expressed on the surface.CdTe QDs incubating CCL187 of unconjugated mAb ND-1 and QD₆₁₃-ND-1 fluorescent probe incubating LEA negative Hela was performed as a control group.(2) Compatitive inhibition between QD₆₁₃-ND-1 and ND-1.Using mAb ND-1 of different concentration to incubate CCL187 for 1h,and QD₆₁₃-ND-1 to incubate for 1h, observe the fluorescence intensity.5.Study on the ability of anti-photobleaching of CdTe QDsWe stimulated QD₆₁₃ and CCL187 cells fluorescently labeled with FITC with 488nm,We collected one image every 285s and obtained totally twelve images.Randomly choose two ROI on the fluorescent image of QD₆₁₃ and FITC,using software MeanROI attached by Carl Zeiss LSM510 laser confocal microscopy to analyze the mean fluorescence intensity of the two ROI.6.Quantum dot modified with PEG to reduce nonspecific adsorptionTo reduce nonspecific adsorption of QDs on cell surface,we adopt NH₂-PEG to modify the QDs.Using PEG-QD₆₁₃ and QD₆₁₃ to incubate CCL187 and HeLa for 30min,to study the effectiveness of PEG to reduce the nonspecific adsorption.Results 1.Preparation,purification and exosydrome of Water soluble CdTe QDs1.The stability of mercaptoacetic acid synthesis of water-soluble CdTe quantum dotsWe obtained the CdTe quantum dots from the green to yellow,orange and gradient under fluorescence arouse after heated for 75min 95min,115min,135min,155min, 175min and 195min at 140℃.Quantum dot constantly increased and launch peaks redshifted With the increase of the heating time,that showed obvious quantum size.2.The detection of the uv-visible spectra of CdTe quantum dotsAbsorption spectrum happen red shift and the CdTe quantum dots of different size and different color fluorescent were obtained,along with the increase in the preparation of heating time.Each quantum dot absorption peaks was obvious and its granularity was uniform.Absorption spectrum peaks were not only,but in a wide range,that conduced to,launch in single color and emit multiple colors.We can obtain multiple color image with only one excitation wavelength.3.The detection of the fluorescence spectroscopy of CdTe quantum dotsEmission spectral happen redshift along with the increase in the preparation of heating time.Emission peak was symmetrical,the most symmetrical peak width was between 35 and 55nm,that showed a narrow size distribution.4.The detection of the TEMSynthesized quantum dots were spherical.Their particle size distribution was uniform and the average particle size was 3.93±0.14nm,that coincided with the quantum dot size according to formula.Notes:The red quantum dot QD₆₁₃ was the most distinct quantum dot compared its color with cell background in different heating time of quantum synthesis with fluorescent.Its stimulation and emission characteristics were excellent,so it was used in the subsequent research.2.Preparation,purification and identification of monoclonal antibody 1.The purification of the mAb ND-1Purified monoclonal antibody displayed two bands of 55kDa and 25kDa on SDS-PAGE electrophoresis,respectively was the heavy chain and light chain of antibody IgG.Its purity reached at 95%according to gel imaging scans.2,the detection of the immunologic activity of mAb ND-1In immuno-fluorescence assays,monoclonal antibody ND-1 was the direct antibody and a goat anti mouse FITC-IgG antibody was the indirect antibody incubating LEA positive CCL187 cells,which surface showed obvious fluorescent signal.If ND-1 was instead of PBS or LEA positive CCL187 cells were instead of LEA negative HeLa cells,we didn't obtain obvious fluorescent signal.That proved that the purified monoclonal antibody ND-1 have immunologic activity.2.Preparation and purification of quantum dot fluorescence probe QD₆₁₃-ND-11.The preparation of QDs fluorescence probe QD₆₁₃-ND-1Acording to EDAC connection assay,covalent reaction within the mercaptoacetic acid carboxyl groups-COOH of QD₆₁₃ quantum dot and the amine NH₂ at the end of mAb ND-1 happened.pH7.4 PBS was chosen as activation buffer.2.The identification and purification of QDs fluorescence probe QD₆₁₃-ND-1In order to identify the effective coupling within QDs and mAb ND-1,we employed gel chromatography with Sephacryl S-200 gel.Thus,we preliminarily separated and purified the coupling product QD₆₁₃-ND-1.3.The specific immune fluorescence imaging of QD₆₁₃-ND-1 probe for colorectal cancer cells1.The specific immune fluorescence imaging of QD₆₁₃-ND-1 probe for colorectal cancer cellsAccording to fluorescence assays,QD₆₁₃-ND-1 immuning to CCL187 cell surface display specific fluorescent signals.But QD₆₁₃-ND-1 to HeLa cells,or QD₆₁₃ to CCL187 cells,membrane didn't show specific fluorescent signals.That indicated that the binding of QD₆₁₃-ND-1 and LEA positive CCL187 cell surface was specific. 2.The competitive inhibition experiment of QD₆₁₃-ND-1 with ND-1We implemented competitive inhibition experiment with QD₆₁₃-ND-1 and onoclonal antibody ND-1 of different concentration,CCL187 cell surface QD₆₁₃ red fluorescent signal strengthen gradually with the reduction of ND-1 concentration.The specifical binding of QD₆₁₃-ND-1 and CCL187 cell surface can be competitively inhibited by mAb ND-1 which specifically combined with LEA.The results showed that the fluorescent signal of cell surface was active group ND-1 of QD₆₁₃-ND-1 specifically binding cell surface antigen.4.Study on the ability of anti-photobleaching of CdTe QDsWe stimulated QD₆₁₃ and CCL187 cells fluorescently labeled with FITC with 488nm and compared the resistance to photobleaching ability about QD₆₁₃ and FITC. We collected one image every 285s and obtained total twelve images.The FITC signal quenched significantly at 1710th second,but the fluorescence signal of QD₆₁₃ remain the line in all the process.According to the confocal microscopy bringing with MeanROI software,we obtained the time curve for average fluorescence intensity,that FITC average fluorescence intensity dropped significantly,but QD₆₁₃ keep the same level.That indicated that the resistance to light bleaching ability of the quantum dot was evidently superior to the FITC.5.QDs modified with PEG reduced nonspecific adsorptionThe PEG modification of QDs preliminarily attained the aims that it eliminate quantum dot nonspecific adsorption effects.Coupling mAb ND-1 and PEG-QD₆₁₃ is the further research.That will be a kind of immune fluorescent probe which is unable to specific absorption.ConclusionWe obtained mercaptoacetic acid stabilized CdTe quantum dot of with hydrothermal method.It was covalently coupled with monoclonal antibody ND-1.That was used for specificly detection of colorectal cancer cells.We provided a kind of high sensitivity detection method for early diagnosis and imaging studies of colorectal cancer.