化学发光成像分析的研究
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摘要
化学发光(Chemiluminescence，CL)分析因其具有仪器设备简单、检出限低和线性范围宽等优点，已被应用于环境科学、临床医学、药学、生命科学、材料科学等领域。伴随着CL分析的发展，CL信号的检测技术取得明显进步，既可以利用传统的光电倍增管(Photomultiplier，PMT)测CL信号，也可以利用具有高灵敏度、高分辨率的CL成像装置检测发光信号，利用CL成像技术可对低至单光子水平的光进行定位及定量检测。当发光信号的空间分布体现了重要的分析信息时，成像检测技术更有优势。
本论文的研究工作主要由化学发光阵列成像平台的建立；化学发光免疫成像分析和自发电池激发的电化学发光成像分析三大部分构成。
1化学发光阵列成像平台的建立
CL成像分析主要包括微孔版、微阵列及微型化装置中的定量检测；基于酶、免疫组织化学和原位杂交反应的显微成像分析；生物体的发光成像分析等。然而，CL成像不同于荧光成像，在进行荧光成像时，在一定波长光的激发下，产生稳定的光信号，光信号不随时间而变化，我们可在任一段时间内积分，获得荧光强度，然而，CL信号是时间的函数，CL强度随时间而改变。大多数CL反应是快反应，发光在数秒内便完成，因为反应的引发与数据采集间存在延迟时间，利用传统的的CL成像方法不能检测这样的快反应。因而现有的CL成像分析只能够检测一些慢CL反应，或者将快发光反应转化为慢发光反应才能进行成像。例如辣根过氧化物酶(horseradish peroxidase，HRP)催化的鲁米诺与H_2O_2间的CL反应是快发光反应，不适合成像分析，通常要加入对碘苯酚等增强剂增强发光强度并延长发光时间。然而，很难将多数快CL反应转变为慢反应。即使将一些慢发光反应用于成像分析，最大发光强度仍然不能被CCD捕获到。例如，加入增强剂的鲁米诺／H_2O_2／HRP体系，在几十秒内，发光强度迅速增加并达到最大值，随后缓慢的降到与背景值相当的数值。然而，在几十秒内，操作者很难将所有CL试剂手动地加入到96孔、384孔酶标板或微阵列系统中，CL反应过程的信号不能全部被记录下来，在已开始反应的孔中，最大发光信号将被错过。这将导致检测发光信号的灵敏度相对较差。并且，如果采用手动加样方式将CL试剂加到微孔板或微阵列中，由于CL反不能在同一时间被引发，将导致CL信号的重现性较差。即使采用自动加样系统，反应的引发及信号采集之间的延迟时间仍然存在，这是传统CL成像方法存在的问题。
我们利用鲁米诺／血红蛋白／对碘苯酚CL反应体系作为模型，建立了一种新的在线调控的CL阵列成像平台，解决上述问题。众所周知，在碱性介质中，鲁米诺能产生很强的CL信号，而在酸性条件下，不能产生CL信号。所以，我们设想可以通过控制反应的pH值引发并控制CL反应。最终的实验结果表明利用本方法提高了检测的灵敏度。这种反应可控的CL成像分析平台的建立，将使快CL反应的成像检测成为可能。
(1)反应可控的化学发光成像分析的设计及应用
设计了反应可控的CL成像分析方法。本方法通过控制反应的pH值引发CL反应，并获得了很高的灵敏度。反应的pH值由在线产生的氨气调节，氨气由NaOH溶液与NH_4Cl溶液反应产生，氨气的量可以通过改变NaOH溶液的浓度，进样时间及流速来调节，也可以通过改变NH_4Cl溶液的浓度来控制。96孔酶标板各孔中的CL试剂吸收氨气后，pH值从相同的起始值持续增加，相对CL强度随pH值增加而增加。基于以上的设计，96孔酶标板上的CL反应在同一时间被引发，每孔中整个CL反应过程的信号可以被记录下来。从而获得高的灵敏度及良好的重现性。试验了鲁米诺／H_2O_2CL体系，用于测定血红蛋白(Hb)。CL强度与Hb浓度在1.0×10~(-9)～1.0×10~(-7)mol／L范围内呈线性，检出限为3.0×10~(-10)mol／L(3s)，对1.0×10~(-8) mol／L的Hb进行11次平行测定，相对标准偏差(R．S．D．)为2.7％。
2化学发光免疫成像分析
本论文的第二部分的研究工作主要由两部分内容构成：基于鲁米诺增强化学发光(ECL)体系的免疫成像分析和基于草酰酯CL反应的免疫成像分析。并应用到重组人肿瘤坏死因子a(rhTNF-a)，金黄色葡萄球菌肠毒素C_1(SEC_1)，白介素6(rHuIL-6)和β-人绒毛膜促性腺激素(β-HCG)的检测。
(1)化学发光成像分析法检测人血清中的肿瘤坏死因子a
设计了一种简单、灵敏、高通量的分析方法用于重组人肿瘤坏死因子(recombinant human tumor necrosis factor-a，rh TNF-a)的检测。本法具有酶联免疫吸附(enzyme-linked immunosorbent assays，ELISA)法的特异性、增强化学发光法(ECL)的高灵敏度及CL成像分析高样品通量等优点。透明的96孔板被用做固相载体。基于ELISA分析法中双抗体夹心法的原理，将对rh TNF-a抗原具有特异结合能力的一种抗体作为包被抗体，另一被辣根过氧化物酶(HRP)标记的抗体作为酶标抗体。一台带有低温冷却装置的CCD成像系统被用于检测CL信号。CL强度值与rh TNF-a浓度在9.0～312.0 pg／mL范围内呈良好线性关系，检出限为1 pg／mL(3s)。本法已被成功地用于人血清中的rh TNF-a的检测，对78.0 pg／mL rh TNF-a 8次平行测定结果的R．S．D．为4.7％，利用标准加入法所得回收率结果为94.0～108.2％，实验结果表明本方法具有实际应用的可行性。
(2)化学发光免疫成像分析法测定牛奶和水中的金黄色葡萄球菌肠毒素C_1
设计了一种灵敏、简单、快速及高通量的方法测定金黄色葡萄球菌肠毒素C_1(staphylococcalenterotoxin C_1，SEC_1)，本方法具有ELIsA法的特异性，增强化学发光分析法(ECL)的高灵敏度，及成像分析法高通量检测的优势。基于双抗体夹心法的免疫反应原理，以96孔酶标板作为固定化载体。用一台商品化的带有低温冷却装置的CCD成像系统检测CL信号。在最佳实验条件下，发光强度值与SEC_1浓度在8.0～125.0 ng／mL范围内呈良好线性关系(r~2=0.9976)，检出限为0.5 ng／mL(3s)，对25.0 ng／ml SEC_18次测定结果的R．S．D．为6.0％。将本方法用于检测牛奶和水中SEC_1含量，测定结果与ELISA法相吻合。
(3)以纳米金为载体的化学发光免疫成像分析的研究及应用
以微孔板为载体的ELISA分析因其具有的高灵敏度和好的特异性被广泛应用于免疫分析。聚苯乙烯因其具有光学透明性并且表面性质可改变，成为应用最为广泛的固定相载体。未修饰的聚苯乙烯表面带有长的碳氢链，会排斥水及亲水性分子，吸引疏水性分子。大的亲水性分子一般都带有疏水链，会使分子吸附在聚苯乙烯表面。但需要很长的孵育时间、较高的物质分子浓度和严格的控制温度等条件，以防止物质分子从聚苯乙烯表面被洗脱下来。因此，亟需建立更好的的固定化方法以满足免疫分析的需求。纳米金因其比表面积大、生物兼容性好及表面自由能高，已在生物试剂的固定化及免疫分析等领域得到广泛应用。
本文设计了一种以纳米金为载体固定蛋白质的新方法。纳米金和蛋白质形成的生物复合材料被成功地固定在聚甲基丙烯酸甲酯(PMMA)片和聚苯乙烯微孔板上。蛋白质可被高密度地固定在固相载体上并较好地保留生物活性。基于以上设计，建立了利用CL成像检测H_2O_2及重组人白介素-6(rHu IL-6)的分析方法。本方法的线性范围和包被效率与直接固定蛋白的CL成像分析相比，都有显著提高。在选定的实验条件下，CL强度与H_2O_2浓度在1.0×10~(-6)～1.0×10~(-4)mol／L呈线性关系，与rHu IL-6浓度在2.0～312.0 pg／mL呈线性关系，检测H_2O_2的检出限为2.0×10~(-7)mol／L(3s)，rHu IL-6的检出限为0.5pg／mL，对3.0×10~(-5) mol／L的H_2O_26次测定结果的R．S．D．为3.8％，对39.0 pg／mLrHu IL-6 6次测定结果的R．S．D．为4.4％。将本方法用于人血清中rHuIL-6的检测，结果令人满意。
(4)化学发光成像分析法检测人血清中的自介素6(rHuIL-6)
众所周知，鲁米诺CL体系的发光量子产率不超过5％，而双[2，4，6-三氯苯基】草酰酯(TCPO)=H_2O_2-荧光剂CL体系具有较高的发光量子产率，通常可达30％。本文建立了一种新的化学发光免疫分析(CUA)方法，结合了传统ELISA方法和双[2，4，6-三氯苯基]草酰酯(TCPO)-H_2O_2CL体系的优点。邻苯二胺(OPDA)与H_2O_2在HRP催化下反应生成荧光物质2，3-二氨基吩嗪(DAPN)。DAPN被TCPO和H_2O_2反应的中间产物激发产生CL信号，本文研究了所产生的CL信号强度与抗原浓度间的关系。作为方法的分析应用，将本方法用于检测rHuIL-6．在最优化的实验条件下，CL强度与rHu IL-6浓度在4.0～625.0 pg／mL呈线性关系，检测rHu IL-6的检出限为0.5 pg／mL，检测78.0 pr／mL rHu IL-6的R．S．D．为2.3％，将本方法用于测定人血清中rHu IL-6，结果令人满意。
(5)化学发光成像分析法检测β-人绒毛膜促性腺激素(β-HCG)
本文采用一种灵敏、简单的方法，可实现对β-HCG的高通量检测。本法具有ELISA方法的特异性、双[2，4，6-三氯苯基]草酰酯(TCPO)-H_2O_2 CL体系的高灵敏度及CL成像分析高样品通量等优点。邻苯二胺(OPDA)与H_2O_2在HRP催化下反应生成荧光物质2，3-二氨基吩嗪(DAPN)。DAPN被TCPO和H_2O_2反应的中间产物激发产生CL信号，本文研究了所产生的CL信号强度与抗原浓度间的关系。作为方法的分析应用，将本方法用于检测β-HCG。在最优化的实验条件下，CL强度与β-HCG浓度在12.5～400.0 mIU／mL呈线性关系，检测β-HCG的检出限为3 mIU／mL，对50.0 mIU／mLβ-HCG 9次平行测定结果的R．S．D．为3.9％。将本方法用于测定尿液中β-HCG的含量，结果令人满意。
3自发电池激发的电化学发光成像分析
电化学发光(Electrogenerated chemiluminescence，ECL)也称电致化学发光，是电化学反应过程中产生的激发态分子返回基态产生的光辐射，已成为分析化学中重要且有价值的检测方法。目前，文献报道的ECL分析都是使用外加的电源来实现ECL的激发，这就限制了ECL检测系统的微型化。及其在微全分析系统中的进一步应用。我们设想能否不使用任何外加电源便可实现ECL的激发?我们曾经设计了微型自发原电池为鲁米诺体系及钙黄绿素的电化学发光提供激发电位。大大的简化了ECL的设备，在ECL系统的微型化方面，迈出了新的步伐。金属Al，Zn，Cr，Cd等可作为阳极，Cu，Ag，Au，Pt及石墨等可作为电池的阴极。最后选择Al作为自发原电池的阳极，Ag作为阴极。可通过改变流动试剂的组成或用其他金属替换Al和Ag电极对调节电池的电位。在后续的工作中，进一步研究了Cu／zn合金形成的自发原电池。Cu／Zn合金自发原电池可以为鲁米诺电化学发光提供稳定的电位，将Cu／zn合金颗粒单独置于96孔板中形成自发电池传感器阵列，并利用鲁米诺／H_2O_2CL，体系验证传感器的性能。
自发电池传感器阵列具备以下几个优点：首先，不需要为ECL的产生提供外加电源，这有利于仪器的简单化及ECL检测在微阵列中的应用。其次，因为这些传感器利用廉价的仪器很容易制备，可实现传感器的低成本制作。再次，Cu／Zn合金传感器贮备方便，有效使用寿命大于100h。为了形成可任意使用的传感器阵列，使用了酶标板。
(1)自发电池激发的电化学发光传感器的研究及应用
研究了一种新型的电化学发光(ECL)成像阵列传感器并将其用于H_2O_2的测定。此传感器基于Cu／zn合金自发电池产生的ECL。在碱性溶液中，Cu／Zn合金由于金属腐蚀效应形成自发电池，此电池可为鲁米诺的ECL的产生提供稳定的电位，产生的弱的ECL信号可被H_2O_2增敏。将Cu／Zn合金的颗粒置于96孔板的微孔中形成自发电池传感器阵列。相对ECL强度与H_2O_2浓度在1.0×10~(-6)～1.0×10~(-4)mol／L范围内呈线性关系，检出限为3.0×10~(-7)mol／L(3s)，对1.0×10~(-5)mol／L的H_2O_2进行11次平行测定，相对标准偏差(R．S．D．)为4.0％。
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-H_2O_2-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-H_2O_2-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 application
A 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 NH_4Cl solution, and the amount of ammonia could be adjusted by varying concentration, pumping time, and flow rate of NaOH solution or varying concentration of NH_4Cl 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, H_2O_2 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 detectors
In 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 C_1 (SEC_1), 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 imaging
A 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 C_1 in milk and water samples
A sensitive, simple and rapid technique for high throughput simultaneous detection of staphylococcal enterotoxin C_1 (SEC_1) 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 SEC_1 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 SEC_1 was 6.0%. The proposed method has been successfully applied to the determination of SEC_1 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 application
The 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 H_2O_2 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 H_2O_2 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 H_2O_2 and 0.5 pg/mL for rHu IL-6 with R.S.D. of 3.8% for 3.0×10~(-5) mol/L H_2O_2, 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 serum
It is well known that the quantum yield of luminol does not exceed 5%. The TCPO-H_2O_2-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) - H_2O_2 CL detection system. A fluorescent product 2, 3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1, 2-diaminobenzene) and H_2O_2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H_2O_2 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-H_2O_2CL 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 H_2O_2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H_2O_2 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/H_2O_2 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 application
In this paper, a novel electrochemiluminescence (ECL) imaging sensor array was developed for determination of hydrogen peroxide (H_2O_2), 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 H_2O_2. 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 H_2O_2 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 H_2O-2 was 4.0%.
本论文的研究工作主要由化学发光阵列成像平台的建立；化学发光免疫成像分析和自发电池激发的电化学发光成像分析三大部分构成。
1化学发光阵列成像平台的建立
CL成像分析主要包括微孔版、微阵列及微型化装置中的定量检测；基于酶、免疫组织化学和原位杂交反应的显微成像分析；生物体的发光成像分析等。然而，CL成像不同于荧光成像，在进行荧光成像时，在一定波长光的激发下，产生稳定的光信号，光信号不随时间而变化，我们可在任一段时间内积分，获得荧光强度，然而，CL信号是时间的函数，CL强度随时间而改变。大多数CL反应是快反应，发光在数秒内便完成，因为反应的引发与数据采集间存在延迟时间，利用传统的的CL成像方法不能检测这样的快反应。因而现有的CL成像分析只能够检测一些慢CL反应，或者将快发光反应转化为慢发光反应才能进行成像。例如辣根过氧化物酶(horseradish peroxidase，HRP)催化的鲁米诺与H_2O_2间的CL反应是快发光反应，不适合成像分析，通常要加入对碘苯酚等增强剂增强发光强度并延长发光时间。然而，很难将多数快CL反应转变为慢反应。即使将一些慢发光反应用于成像分析，最大发光强度仍然不能被CCD捕获到。例如，加入增强剂的鲁米诺／H_2O_2／HRP体系，在几十秒内，发光强度迅速增加并达到最大值，随后缓慢的降到与背景值相当的数值。然而，在几十秒内，操作者很难将所有CL试剂手动地加入到96孔、384孔酶标板或微阵列系统中，CL反应过程的信号不能全部被记录下来，在已开始反应的孔中，最大发光信号将被错过。这将导致检测发光信号的灵敏度相对较差。并且，如果采用手动加样方式将CL试剂加到微孔板或微阵列中，由于CL反不能在同一时间被引发，将导致CL信号的重现性较差。即使采用自动加样系统，反应的引发及信号采集之间的延迟时间仍然存在，这是传统CL成像方法存在的问题。
我们利用鲁米诺／血红蛋白／对碘苯酚CL反应体系作为模型，建立了一种新的在线调控的CL阵列成像平台，解决上述问题。众所周知，在碱性介质中，鲁米诺能产生很强的CL信号，而在酸性条件下，不能产生CL信号。所以，我们设想可以通过控制反应的pH值引发并控制CL反应。最终的实验结果表明利用本方法提高了检测的灵敏度。这种反应可控的CL成像分析平台的建立，将使快CL反应的成像检测成为可能。
(1)反应可控的化学发光成像分析的设计及应用
设计了反应可控的CL成像分析方法。本方法通过控制反应的pH值引发CL反应，并获得了很高的灵敏度。反应的pH值由在线产生的氨气调节，氨气由NaOH溶液与NH_4Cl溶液反应产生，氨气的量可以通过改变NaOH溶液的浓度，进样时间及流速来调节，也可以通过改变NH_4Cl溶液的浓度来控制。96孔酶标板各孔中的CL试剂吸收氨气后，pH值从相同的起始值持续增加，相对CL强度随pH值增加而增加。基于以上的设计，96孔酶标板上的CL反应在同一时间被引发，每孔中整个CL反应过程的信号可以被记录下来。从而获得高的灵敏度及良好的重现性。试验了鲁米诺／H_2O_2CL体系，用于测定血红蛋白(Hb)。CL强度与Hb浓度在1.0×10~(-9)～1.0×10~(-7)mol／L范围内呈线性，检出限为3.0×10~(-10)mol／L(3s)，对1.0×10~(-8) mol／L的Hb进行11次平行测定，相对标准偏差(R．S．D．)为2.7％。
2化学发光免疫成像分析
本论文的第二部分的研究工作主要由两部分内容构成：基于鲁米诺增强化学发光(ECL)体系的免疫成像分析和基于草酰酯CL反应的免疫成像分析。并应用到重组人肿瘤坏死因子a(rhTNF-a)，金黄色葡萄球菌肠毒素C_1(SEC_1)，白介素6(rHuIL-6)和β-人绒毛膜促性腺激素(β-HCG)的检测。
(1)化学发光成像分析法检测人血清中的肿瘤坏死因子a
设计了一种简单、灵敏、高通量的分析方法用于重组人肿瘤坏死因子(recombinant human tumor necrosis factor-a，rh TNF-a)的检测。本法具有酶联免疫吸附(enzyme-linked immunosorbent assays，ELISA)法的特异性、增强化学发光法(ECL)的高灵敏度及CL成像分析高样品通量等优点。透明的96孔板被用做固相载体。基于ELISA分析法中双抗体夹心法的原理，将对rh TNF-a抗原具有特异结合能力的一种抗体作为包被抗体，另一被辣根过氧化物酶(HRP)标记的抗体作为酶标抗体。一台带有低温冷却装置的CCD成像系统被用于检测CL信号。CL强度值与rh TNF-a浓度在9.0～312.0 pg／mL范围内呈良好线性关系，检出限为1 pg／mL(3s)。本法已被成功地用于人血清中的rh TNF-a的检测，对78.0 pg／mL rh TNF-a 8次平行测定结果的R．S．D．为4.7％，利用标准加入法所得回收率结果为94.0～108.2％，实验结果表明本方法具有实际应用的可行性。
(2)化学发光免疫成像分析法测定牛奶和水中的金黄色葡萄球菌肠毒素C_1
设计了一种灵敏、简单、快速及高通量的方法测定金黄色葡萄球菌肠毒素C_1(staphylococcalenterotoxin C_1，SEC_1)，本方法具有ELIsA法的特异性，增强化学发光分析法(ECL)的高灵敏度，及成像分析法高通量检测的优势。基于双抗体夹心法的免疫反应原理，以96孔酶标板作为固定化载体。用一台商品化的带有低温冷却装置的CCD成像系统检测CL信号。在最佳实验条件下，发光强度值与SEC_1浓度在8.0～125.0 ng／mL范围内呈良好线性关系(r~2=0.9976)，检出限为0.5 ng／mL(3s)，对25.0 ng／ml SEC_18次测定结果的R．S．D．为6.0％。将本方法用于检测牛奶和水中SEC_1含量，测定结果与ELISA法相吻合。
(3)以纳米金为载体的化学发光免疫成像分析的研究及应用
以微孔板为载体的ELISA分析因其具有的高灵敏度和好的特异性被广泛应用于免疫分析。聚苯乙烯因其具有光学透明性并且表面性质可改变，成为应用最为广泛的固定相载体。未修饰的聚苯乙烯表面带有长的碳氢链，会排斥水及亲水性分子，吸引疏水性分子。大的亲水性分子一般都带有疏水链，会使分子吸附在聚苯乙烯表面。但需要很长的孵育时间、较高的物质分子浓度和严格的控制温度等条件，以防止物质分子从聚苯乙烯表面被洗脱下来。因此，亟需建立更好的的固定化方法以满足免疫分析的需求。纳米金因其比表面积大、生物兼容性好及表面自由能高，已在生物试剂的固定化及免疫分析等领域得到广泛应用。
本文设计了一种以纳米金为载体固定蛋白质的新方法。纳米金和蛋白质形成的生物复合材料被成功地固定在聚甲基丙烯酸甲酯(PMMA)片和聚苯乙烯微孔板上。蛋白质可被高密度地固定在固相载体上并较好地保留生物活性。基于以上设计，建立了利用CL成像检测H_2O_2及重组人白介素-6(rHu IL-6)的分析方法。本方法的线性范围和包被效率与直接固定蛋白的CL成像分析相比，都有显著提高。在选定的实验条件下，CL强度与H_2O_2浓度在1.0×10~(-6)～1.0×10~(-4)mol／L呈线性关系，与rHu IL-6浓度在2.0～312.0 pg／mL呈线性关系，检测H_2O_2的检出限为2.0×10~(-7)mol／L(3s)，rHu IL-6的检出限为0.5pg／mL，对3.0×10~(-5) mol／L的H_2O_26次测定结果的R．S．D．为3.8％，对39.0 pg／mLrHu IL-6 6次测定结果的R．S．D．为4.4％。将本方法用于人血清中rHuIL-6的检测，结果令人满意。
(4)化学发光成像分析法检测人血清中的自介素6(rHuIL-6)
众所周知，鲁米诺CL体系的发光量子产率不超过5％，而双[2，4，6-三氯苯基】草酰酯(TCPO)=H_2O_2-荧光剂CL体系具有较高的发光量子产率，通常可达30％。本文建立了一种新的化学发光免疫分析(CUA)方法，结合了传统ELISA方法和双[2，4，6-三氯苯基]草酰酯(TCPO)-H_2O_2CL体系的优点。邻苯二胺(OPDA)与H_2O_2在HRP催化下反应生成荧光物质2，3-二氨基吩嗪(DAPN)。DAPN被TCPO和H_2O_2反应的中间产物激发产生CL信号，本文研究了所产生的CL信号强度与抗原浓度间的关系。作为方法的分析应用，将本方法用于检测rHuIL-6．在最优化的实验条件下，CL强度与rHu IL-6浓度在4.0～625.0 pg／mL呈线性关系，检测rHu IL-6的检出限为0.5 pg／mL，检测78.0 pr／mL rHu IL-6的R．S．D．为2.3％，将本方法用于测定人血清中rHu IL-6，结果令人满意。
(5)化学发光成像分析法检测β-人绒毛膜促性腺激素(β-HCG)
本文采用一种灵敏、简单的方法，可实现对β-HCG的高通量检测。本法具有ELISA方法的特异性、双[2，4，6-三氯苯基]草酰酯(TCPO)-H_2O_2 CL体系的高灵敏度及CL成像分析高样品通量等优点。邻苯二胺(OPDA)与H_2O_2在HRP催化下反应生成荧光物质2，3-二氨基吩嗪(DAPN)。DAPN被TCPO和H_2O_2反应的中间产物激发产生CL信号，本文研究了所产生的CL信号强度与抗原浓度间的关系。作为方法的分析应用，将本方法用于检测β-HCG。在最优化的实验条件下，CL强度与β-HCG浓度在12.5～400.0 mIU／mL呈线性关系，检测β-HCG的检出限为3 mIU／mL，对50.0 mIU／mLβ-HCG 9次平行测定结果的R．S．D．为3.9％。将本方法用于测定尿液中β-HCG的含量，结果令人满意。
3自发电池激发的电化学发光成像分析
电化学发光(Electrogenerated chemiluminescence，ECL)也称电致化学发光，是电化学反应过程中产生的激发态分子返回基态产生的光辐射，已成为分析化学中重要且有价值的检测方法。目前，文献报道的ECL分析都是使用外加的电源来实现ECL的激发，这就限制了ECL检测系统的微型化。及其在微全分析系统中的进一步应用。我们设想能否不使用任何外加电源便可实现ECL的激发?我们曾经设计了微型自发原电池为鲁米诺体系及钙黄绿素的电化学发光提供激发电位。大大的简化了ECL的设备，在ECL系统的微型化方面，迈出了新的步伐。金属Al，Zn，Cr，Cd等可作为阳极，Cu，Ag，Au，Pt及石墨等可作为电池的阴极。最后选择Al作为自发原电池的阳极，Ag作为阴极。可通过改变流动试剂的组成或用其他金属替换Al和Ag电极对调节电池的电位。在后续的工作中，进一步研究了Cu／zn合金形成的自发原电池。Cu／Zn合金自发原电池可以为鲁米诺电化学发光提供稳定的电位，将Cu／zn合金颗粒单独置于96孔板中形成自发电池传感器阵列，并利用鲁米诺／H_2O_2CL，体系验证传感器的性能。
自发电池传感器阵列具备以下几个优点：首先，不需要为ECL的产生提供外加电源，这有利于仪器的简单化及ECL检测在微阵列中的应用。其次，因为这些传感器利用廉价的仪器很容易制备，可实现传感器的低成本制作。再次，Cu／Zn合金传感器贮备方便，有效使用寿命大于100h。为了形成可任意使用的传感器阵列，使用了酶标板。
(1)自发电池激发的电化学发光传感器的研究及应用
研究了一种新型的电化学发光(ECL)成像阵列传感器并将其用于H_2O_2的测定。此传感器基于Cu／zn合金自发电池产生的ECL。在碱性溶液中，Cu／Zn合金由于金属腐蚀效应形成自发电池，此电池可为鲁米诺的ECL的产生提供稳定的电位，产生的弱的ECL信号可被H_2O_2增敏。将Cu／Zn合金的颗粒置于96孔板的微孔中形成自发电池传感器阵列。相对ECL强度与H_2O_2浓度在1.0×10~(-6)～1.0×10~(-4)mol／L范围内呈线性关系，检出限为3.0×10~(-7)mol／L(3s)，对1.0×10~(-5)mol／L的H_2O_2进行11次平行测定，相对标准偏差(R．S．D．)为4.0％。
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-H_2O_2-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-H_2O_2-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 application
A 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 NH_4Cl solution, and the amount of ammonia could be adjusted by varying concentration, pumping time, and flow rate of NaOH solution or varying concentration of NH_4Cl 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, H_2O_2 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 detectors
In 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 C_1 (SEC_1), 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 imaging
A 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 C_1 in milk and water samples
A sensitive, simple and rapid technique for high throughput simultaneous detection of staphylococcal enterotoxin C_1 (SEC_1) 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 SEC_1 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 SEC_1 was 6.0%. The proposed method has been successfully applied to the determination of SEC_1 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 application
The 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 H_2O_2 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 H_2O_2 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 H_2O_2 and 0.5 pg/mL for rHu IL-6 with R.S.D. of 3.8% for 3.0×10~(-5) mol/L H_2O_2, 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 serum
It is well known that the quantum yield of luminol does not exceed 5%. The TCPO-H_2O_2-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) - H_2O_2 CL detection system. A fluorescent product 2, 3-diaminophenazine (DAPN) was produced by reaction between o-phenylenediamine (OPDA, 1, 2-diaminobenzene) and H_2O_2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H_2O_2 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-H_2O_2CL 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 H_2O_2 catalyzed by horseradish peroxidase (HRP). DAPN was excited by the reactive intermediate of TCPO-H_2O_2 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/H_2O_2 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 application
In this paper, a novel electrochemiluminescence (ECL) imaging sensor array was developed for determination of hydrogen peroxide (H_2O_2), 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 H_2O_2. 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 H_2O_2 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 H_2O-2 was 4.0%.
引文
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