| Chemiluminescence (CL) analysis has been widely applied to environmental sciences, clinical medicine, pharmacy, life science and materials science due to its advantages including simple instrumentation, very low detection limits and wide dynamic ranges. Along with the development of CL analysis, significant progress has been made in techniques to measure CL CL signal could be detected by both conventional photomultiplier (PMT)-based luminometers and high resolution imaging detectors. Light emission down to the single-photon level could be localized and quantified by CL imaging techniques. Imaging techniques are advantageously used when the spatial distribution of the luminescence signal represents crucial analytical information.The research work of the dissertation is made up of three sections of development of CL micro array based on imaging assay, CL immunoassay based on imaging detectors and galvanic cell generated electrochemiluminescence (ECL) imaging assay. 1. The development of CL microarray based on imaging assay.CL imaging assay has been applied to in vitro and in vivo assays, including: quantitative assays performed in various analytical formats, such as microtiter plates, microarrays and miniaturized analytical devices; luminescence imaging microscopy based on enzymatic, immunohistochemical and in situ hybridization reaction; whole-body luminescence imaging in live animals. However, CL imaging is different from fluorescence imaging. In fluorescence imaging assay, an invariance signal could be obtained under certain excitation wavelength, the fluorescence intensity doesn't vary with time, and we can combines the pixel intensities of fluorescence at any time. The emission intensity from a CL reaction varies with time. Most CL reactions have a short-lived signal, complete within a few seconds. Flash-type CL reactions are unsuitable for sensitive and reproducible determinations in CL imaging assay because of lag time between initiation of the reaction and data collection. Therefore, all the well established CL imaging methods use relatively few glow type CL reactions. Fast emitting (flash-type) CL reactions is tuned to furnish a slower-emitting (glow-type) process that is suitable for simple CL imaging. Typically, the CL reaction of luminol-H2O2-horseradish peroxidase (HRP) system is a fast emitting reaction and is unsuitable for simple CL imaging assay. The reaction can be tuned to slower-emitting process by using p-iodophenol (p-IP) as enhancer. However, it is difficult to tune most flash-type reactions to glow-type reactions. The maximum CL signal cannot be monitored even some glow-type reactions are used. For example, the kinetics of the enhanced luminol-H2O2-HRP system usually shows a rapid increase within 1 min until the maximum value is reached, followed by a slow decrease to the background level. When CL reagents are manually added to 96- (384-) well plates or microarray systems by a pipette, it is a great challenge for the operator to add CL reagents to 96- or 384-wells within 1.0 minute; therefore, the total CL signal cannot be collected and the maximum value will be missed. Moreover, a poor reproducibility of CL signal will be induced because the CL reaction cannot be initiated at the same time when CL reagents are manually added to 96-(384- ) well plates or microarray systems. Automated pipetting systems have been applied to CL imaging assay, however, the lag time between initiation of the reaction and data collection still exists. This was the major problem of current CL imaging assay.A luminol/hemoglobin/p-iodophenol CL system was chosen as model, and a novel on line reaction-controlled CL array based on imaging analysis was designed by our group to solve above problem. It is well known that luminol emits strong CL signal in alkaline medium, no CL signal was observed under the acidic conditions. We assumed that CL reactions could be initiated and controlled by controlling pH. Experimental results showed that high sensitivity could be obtained by using reaction-controlled design. The most important thing is the reaction-controlled CL imaging method was expected to monitor fast-emitting CL reaction directly. (1) A design of reaction controlled chemiluminescence imaging and its applicationA reaction-controlled CL imaging analysis was designed. The design was based on controlling pH to initiate CL reaction and obtain high sensitivity. The pH value of CL reaction was controlled by ammonia, which was produced by injecting NaOH solution to NH4Cl solution, and the amount of ammonia could be adjusted by varying concentration, pumping time, and flow rate of NaOH solution or varying concentration of NH4Cl solution. The pH of CL reagents in 96 well microtiter plates increased continuously from the same initial value due to the absorbance of ammonia, and the relative CL intensity increased with the increasing pH. Based on above reaction-controlled design, the CL reaction in 96 wells could be initiated at the same time, and the total CL signal of each well could be monitored. As results of above operation, a high sensitivity and better reproducibility could be obtained. A luminol, H2O2 CL system for determination of hemoglobin (Hb) was selected to validate the presented design. The CL intensity was proportional with the concentration of Hb in the range of 1.0×10-9 to 1.0×10-7 mol/L and the detection limit was 3.0×10-10 mol/L (3s), the relative standard deviation (R.S.D.) for 11 parallel measurements of 1.0×10-8 mol/L Hb was 2.7%.2. CL immunoassay based on imaging detectorsIn the second part of the dissertation, the research work was made up of two sections of immunoassay based on luminol enhanced chemiluminescence (ECL) imaging system, and immunoassay based on peroxyoxalate CL imaging system. These two different CL systems have been successfully applied to determination of recombinant human tumor necrosis factor-a (rh TNF-a), staphylococcal enterotoxin C1 (SEC1), recombinant human interleukin 6 (rHu IL-6) andβ-human chorionic gonadotropin (β- HCG).The major contents in second part are described as follows: (1) Determination of recombinant human tumor necrosis factor-a in serum by CL imagingA simple, sensitive and high throughput CL imaging method was described for the determination of recombinant human tumor necrosis factor-a (rh TNF-a). The proposed method has the advantage of showing the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL), and high throughput of CL imaging method. In this system, 96 well transparent microtiter plates were used as solid phase materials. The method was based on the use of two monoclonal antibodies against rh TNF-a, one "capture" antibody and one labeled with horseradish peroxidase (HRP), in a "sandwich" ELISA format. A cooled CCD camera has been applied to image the weak chemiluminescence from the ECL The CL intensity was proportional with the concentration of rh TNF-a in the range of 9.0 to 312.0 pg/mL and the detection limit was 1 pg/mL The proposed method has been successfully applied to the determination of rh TNF-a in human serum, the reliability of the assay method was established by parallel determination and by standard-addition method. (R.S.D. = 4.7%, recoveries=94.0 -108.2%).(2) Chemiluminescent imaging detection of staphylococcal enterotoxin C1 in milk and water samplesA sensitive, simple and rapid technique for high throughput simultaneous detection of staphylococcal enterotoxin C1 (SEC1) has been developed. The proposed method has the advantage of showing the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL), and high throughput of CL imaging. It was based on a standard sandwich immunoassay format; 96-well ELISA plates were used as solid phase material. A commercial high-sensitivity cooled CCD camera has been applied to image the weak CL from the ECL Under the optimum conditions, the increased CL intensity was proportional with the concentration of SEC1 in the range of 8.0 to 125.0 ng/mL and the detection limit was 0.5 ng/mL(3s). The R.S.D. for eight parallel measurements of 25.0 ng/mL SEC1 was 6.0%. The proposed method has been successfully applied to the determination of SEC1 in milk and water samples. The results obtained compared well with those by ELISA.(3) Development of a gold nanoparticles based chemiluminescence imaging assay and its applicationThe enzyme-linked immunosorbent assay (ELISA) typically carried out in microtiter plates have been widely used in immunoassay with respect to its specificity and sensitivity. The most commonly utilized solid support is polystyrene because of its optical clarity and range of surface properties. As a long chain hydrocarbon, non-modified polystyrene tends to repel water and hydrophilic molecules and attract hydrophobic molecules. Large bimolecular considered hydrophilic will inevitably have stretches of hydrophobic regions that allow the molecule to adsorb to the polystyrene surface. But to enable stable binding of hydrophilic molecules, assay conditions such as high molecule concentration, long incubation time and critical temperature conditions might be required to prevent the molecule from washing off the polystyrene surface. Therefore, the use of alternative methods of immobilization is attractive. Gold nanoparticles have been intensively studied in bio-reagents immobilization via the large specific interface area, desirable biocompatibility and high surface free energy of nanosized particles, and have been widely used in immunoassay. in this paper, a novel gold nanoparticles based protein immobilization method was designed. Bio-composites of gold nanoparticles and proteins were successfully coated on poly (methyl methacrylate) (PMMA) plates and polystyrene microtiter plates. The proteins could be immobilized on solid materials with high density and better bioactivity. Based on above design, CL imaging assay for determination of H2O2 and rHu IL-6 was developed. The linear range and the loading capability were greatly improved when compared with imaging assay performed with direct proteins immobilization. Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of H2O2 in the range of 1.0×10-6 to 1.0×10-4 mol/L, and rHu IL-6 in the range of 2.0 to 312.0 pg/mL. The detection limits were 2×10-1 mol/L (3s) for H2O2 and 0.5 pg/mL for rHu IL-6 with R.S.D. of 3.8% for 3.0×10-5 mol/L H2O2, and 4.4% for 39.0 pg/mL rHu IL-6. This method has been applied to the determination of rHu IL-6 in human serum with satisfactory results.(4) Chemiluminescent imaging detection of recombinant human interleukin 6 (rHu IL-6) in human serumIt is well known that the quantum yield of luminol does not exceed 5%. The TCPO-H2O2-fluorophore chemiluminescent reaction has a quantum yield higher than that observed for most of the chemiluminescent reaction discovered to date. This reaction requires the presence of a suitable fluorophore and has a quantum yield of 30%. The paper presented a novel CL immunoassay method, the proposed method combines the advantages of traditional enzyme-linked immunosorbent assays (ELISA) and bis (2, 4, 6-trichlorophenyl) oxalate (TCPO) - H2O2 CL detection system. A fluorescent product 2, 3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1, 2-diaminobenzene) and H2O2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H2O2 chemiluminescent reaction, and led to CL The dependence of the CL intensity on the concentrations of antigen was studied. As analytical application, the proposed method was used for determination of rHu IL-6. Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of rHu IL-6 in the range of 4.0 to 625.0 pg/mL. The detection limit was 0.5 pg/mL for rHu IL-6 with R.S.D. of 2.3% for 78.0 pg/mL rHu IL-6. This method has been applied to the determination of rHu IL-6 in human serum.(5) Chemiluminescent imaging detection ofβ- human chorionic gonadotropin(β-HCG)A sensitive and simple method has been used for high throughput detection of B human horionic gonadotropin (β-HCG). The proposed method has the advantage of showing the specificity of enzyme linked immunosorbent assays (ELISA), sensitivity of TCPO-H2O2CL reactions, and high throughput of chemiluminescence (CL) imaging. A fluorescent product 2, 3-diaminophenazine PAPN) was produced by reaction between o-phenylenediamine (OPDA, 1,2-diaminobenzene) and H2O2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H2O2 chemiluminescent reaction, and led to CL Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration ofβ-HCG in the range of 12.5 to 400.0 mIU/mL. The detection limit was 3 mIU/mL forβ-HCG with R.S.D. of 3.9% for 50.0 mlU/mLβ-HCG. This method has been applied to the determination ofβ-HCG in urine with satisfactory results.3. Galvanic cell generated electrochemiluminescence imaging assay.Electrogenerated chemiluminescence (ECL) also known as electrochemiluminescence is the luminescence generated by relaxation of exited state molecules that are produced during an electrochemically-initiated reaction. ECL has become an important and valuable detection method in analytical chemistry in recent years. Up to now, most of the reference techniques for ECL generation were carried out by employing an external potential supplier. The potential supplier normally used in research work limited the detection instrumentation miniaturization and hampered further application of ECL detection on a microanalysis system. We assumed that ECL could be generated without employing an external potential supplier. A mini galvanic cell has been designed by our group to generate ECL of luminol and calcein blue; we made an important step towards miniaturization of the equipment. Candidate metals such as aluminum, zinc, chromium and cadmium were used as pure metallic anode, copper, silver, gold, platinum, and graphite for cathode of galvanic cell were investigated. At last, aluminum was chosen as anode and silver as cathode, a mini galvanic cell was formed. The potential of the galvamc cell could be adjusted by varying the components of flow reagent or by using different metals to substitute for aluminum or silver. In our further studies, Cu/Zn alloy galvanic cell was studied. The galvanic cell could supply stable potential for ECL generation of luminol in suitable electrolyte. A galvanic cell sensor array was developed by putting Cu/Zn alloy in 96 well microtiter plates separately. The intrinsic performances of this sensor array were evaluated through luminol/H2O2 CLsystem.The galvanic cell sensor array has several advantages. Firstly, an external potential supplier was not needed for ECL generation, which is benefit for simplification of instrument and application of ECL detection on micro array. Secondly, such devices could lead to the realization of low cost sensors, since the sensors are easily produced using inexpensive instruments. Thirdly, the Cu/Zn alloy sensors can be conveniently stored, and the life time (efficient using time) of described sensors was more than 100 h. Furthermore, in order to form disposable sensor arrays, miao titer plate (MTP) was used.(1) Sensors based on galvanic cell generated dectrochemiluminescence and its applicationIn this paper, a novel electrochemiluminescence (ECL) imaging sensor array was developed for determination of hydrogen peroxide (H2O2), which was based on Cu/Zn alloy galvanic cell generated ECL In alkaline solution, Cu/Zn galvanic cell was formed because of corrosion effect, the galvanic cell could supply stable potential for ECL generation of luminol, and the weak ECL emission could be enhanced by H2O2. The galvanic cell sensor array was designed by putting Cu/Zn alloy in 96 well microtiter plates separately. The relative ECL intensity was proportional with the concentration of H2O2 in the range of 1.0×10-6 to 1.0×10-4 mol/L and the detection limit was 3.0×10-7 mol/L (3s), the R.S.D. for 11 parallel measurements of 1.0×10-5 mol/L H2O-2 was 4.0%.
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