The HRP-luminol-H2O2 reaction system emits chemiluminescence in the initial steady state of the reaction most brightly, and decays quickly. The optimization experiment condition is important for users to measure chemiluminescence.
3.4.1 Optimization of enzymatic assay condition 3.4.1.1 HRP activity standard curve
According to the data of HRP activity standard curve, the linear range of HRP sits between 0.008~ 0.72 unit. To select an appropriate temperature to manipulate our experiments, we observed the temperature dependent curve which and it indicates that the HRP-luminol-H2O2 system gives similar luminescence around 15℃~40℃ (Table 3). Nevertheless, data shows that change of pH significantly affects the activity of HRP-luminol-H2O2 system (49). The standard condition for HRP-luminol-H2O2
system described in experiments is determined based on these results.
3.4.1.2 Optimal concentration of reagents
Luminol concentration variation vs. reaction initial rate
The optimized concentration of luminol is near 8 mM. By observing the pattern of the graph, it shows that the reaction initial velocity gradually decays after arriving the Vmax, and tends toward saturate state. The initial rate value of it is similar to that when the luminal concentration is 5 mM.
3.4.2 HRP-luminol-H2O2 kinetic Graph
After the adequate concentration of reagents in HRP-luminol-H2O2 reaction are found out, we fix the enzyme amount as well and then drive the coupled enzyme reaction in the uniform condition. By varying the concentration of H2O2, the initial rates differ and lead to a kinetics curve fluctuated by H2O2 variation.
Under uniform reaction condition, we operate the coupled enzyme reactions and detect it with the three different setups, traditional chemiluminescence detector, F4500,
present here. The experiment results are compared to each other and the data of the
early stage CMOS chip is also discussed in our report later.
3.4.3 Frequency Adjusted in the Luminescence Reaction
Frequency is one of the instrument parameters experimenters need to set properly before we start our optical reaction. According to data of the
above-mentioned experiments, different frequency setting may lead to different detected output voltage and that may lead to distinct data analysis.
The following figures display the experiments under the standard condition with different frequency parameters. From these figures, we know that diverse frequency of 35, 45, 50, 65, and 80 Hz, results in different kinetic curves and in lower frequency the initial rate achieves saturation easier than in higher one. The conclusion can be told from the km values of these results with different frequency. In another hand, the Vmax of the initial rate altered as well while we set different frequency parameters.
The results of experiments with different frequency setting are displayed in Figure 24.
Seeing Figure 40(a.), the chip with different frequency parameters would result in different sensitivities even in the same substrate concentration range. Figure 40(a.) showed the different slopes from different frequency parameters. When frequency parameter was set in 45Hz, the slope (∆Initial Rate/ ∆mM) of the recognizable range of H2O2 is 1.6984. The slopes of 50, 65 and 80 Hz are 1.077, 0.307 and 0.0303. The
slope decreased when frequency parameter increases. In another hand, seeing table the saturation concentration shows up earlier when the frequency parameter is lower.
The comparison of the slope and saturation concentration of different frequency parameters is shown in Table 9. That is, the lower frequency parameter leads to a higher slope and sensitivity and there comes with a lower limit of detection (seeing Table 9) but saturates earlier than the higher frequency parameter. The chip detection ability changes with the variation of frequency parameter. This characteristic helps users to find the optimal condition for specific target detection. For example, when the chip measures a wider light signal variation, the higher frequency parameter is necessary to detect the lighter signal where the output voltage may achieves saturation in lower frequency parameter. Else, if there is a dim light signal interval produced by our interested target variation, a smaller frequency parameter could be chosen for detection with its high sensitivity.
3.4.4 Enzyme amount Adjusted in Luminescence Reaction (HRP : 0.16 unit)
The enzymatic reactions designed for PMT measurement system (by Hitachi F-4500) are to be compared with those obtained with CMOS detection system described later. For this purposes, the parameters of the PMT measurement system as described in experimental section are adjusted so that comparable data can be obtained from both instrumentation in similar reaction conditions.
Comparing the luminescence reaction measured by the CMOS chip with the one by the F-4500 fluorescence detector, seeing figure 25, the Km value of the former is 0.889 and the latter is 1.8. The difference between them is notable and we need to return back to discuss our condition setting to correct and lessen the error.
By observing the figure of the kinetics graph, the initial rate inclines to approach saturation rate value as the H2O2 nears 1 mM. Analyzing this graph, the point where the curve bends to near saturation appears earlier. The phenomenon might occur because the luminescence intensity has already over the CMOS chip voltage output range. The overmuch luminescene will not be reflected through the voltage output and that will lead to the errors. To diminish the possibility of overrun, the adequate
amount of enzyme is adjusted to produce the optimal luminescence for detection.
3.4.5 Enzyme amount Adjusted in Luminescence Reaction (HRP :0. 02 unit)
To control the luminescence intensity, the HRP amount is decreased to 0.02 units in figure 26. Under the standard condition as before expects enzyme amount, we experiment the luminescence again and work out the enzyme kinetics graph.
Observing the curve in figure, the point which tends to saturation shows up while H2O2 nears 5 mM and the outcome is more similar to the one detected by F4500 luminescence detector. The Km value resulted from CMOS sensor is 1.4 mM which approaches the Km value of the first generation CMOS chip study published in 2004
(56).
The variation of H2O2 can be identified by the CMOS chip reliably and it indicates that CMOS chip is able to detect luminescence and with a capacity for multi-function biological sensing while the H2O2 oxidation equation is coupled with other biological molecules oxidation reactions capable of H2O2 production.
3.4.6 Comparison of Luminescence Reaction Detection Limitation
Figure 27 displays the HRP-luminol-H2O2 luminescence assay kinetic graph detected with other instruments: F-4500 luminescence spectrometer, integrated commercial photodiode system and the early stage CMOS chip(49). They are all with the similar Km to show the reliability but with the different sensitivity.
The organization of these devices characteristics is shown in table5. It shows the detection limits of luminescence reaction of four detectors, F-4500 luminescence spectrometer, commercial photodiode, early stage CMOS chip and the novel CMOS chip. The data of their sensing area and sensitivity is listed in table5.
Detecting the chemiluminescence by the traditional luminescence detector (F4500), the calibration curve required and the recognizable range starts from 10 μ M and ends in 10 mM. The minimum detection limitation is10 μM so the variation of H2O2 between the interval can be detected unerringly. By the integrated
semiconductor photodiode, the recognizable range appears between 0.1 mM to 10
mM and the detection limitation approaches 100 μM which performs in the same level as the early stage CMOS chip. Comparatively, the new generation CMOS chip has the sensitivity of 50 μM, and it is double sensitive than the early stage CMOS chip and the commercial photodiode system.
Concerning the amount of the enzyme in luminescence method, the quantity required in tests by novel CMOS chip is 0.02 U/ml. The requirement of HRP amount is much less than the early stage chip (49) and also with the better sensitivity than
report published in 2007 (50). The comparison of HRP quantity is listed in Table 6.
The HRP-luminol-H2O2 system used as a platform system can be served to quantify enzymatic reactions that produce H2O. Many biochemical measurements can be done by the HRP-luminol-H2O2 system with suitable design of biochemical
reactions.
3.4.7 Results of the optimized method for HRP-CHOD coupled enzyme reaction Comparing these graphs got from two methods, incubation test and real time test, the graph of incubation test outputs the higher initial rate than real time test ( Vmax
=884.6 CL/ sec in incubation test and the one is 104.3 CL/ sec in the real time test.) The two graphs of tests are displayed in figure 22.
It is thus evident that the result under incubation test presents more sufficient signals for analysis. Because the reaction conditions of both reactions coupled
together is hard to coordinate, the optimization is not easy to carry out. To solve the problem, pretreatment is proceeded to incubate more products for the next reaction going on later.
3.4.8.1 Real time test analysis
Real time test is the most direct way to work our reaction with. The possible variability occurred during incubation can be removed but we need to considerate the rate chemicals emits luminescence with coupled enzyme reaction is not as fast as with single HRP reaction. Thus, we need to adjust ratio of the second reaction enzyme amount to first one. While the initial rate of the second equation is faster than that of the first, the real amount of H2O2 can be detected actually right away and that can be reflected. (The enzyme amount of the second equation needs not to be excess, but the reaction rate of the second one need to be verified to be faster.)
3.4.8.2 Incubation test analysis
The principle of the incubation method is based on the transference of products from the first equation that passes to the next reaction actually to complete the coupled enzyme reaction. Thus, the condition during incubation needs to be identical to acquire the products with equal rate each time. The trusty data output under same
condition hence could be compared with each other exactly.
After samples are incubated, further, the enzyme amount is fixed to carry out the coupled enzyme reactions ( Incubation test). The reaction curve obtained with
incubation test is displayed in figure 23.
Being effected by the low Km value of the first equation(cholesterol oxidation)
（cholesterol oxidation）and substrate inhibition , the Km value of coupled enzyme reaction nears 1.4 mM and the initial rate tends to maximum while cholesterol concentration nears 0.2 mM. Therefore, the recognizable cholesterol range falls between 0.01 mM and 0.2 mM.
Human desired cholesterol concentration is below 3.85 mM, and that means we can test human blood with few sample then dilute it to the recognizable range for analyze. Contrasting the human desired cholesterol concentration to the detectable range of the chip.（The recognizable range of H2O2 concentration falls between 0.1mM to 0.2mM）
It is believed that the cholesterol recognizable range is at least between 0.1 to 0.2mM. The transformation percentage between cholesterol and H2O2 does not reach 100 %, thus, if cholesterol transfers into luminescence more efficiently, the detectable cholesterol range could be amplified and larger than 0.2mM with a wider recognizable range. That would be more flexible for users to quantify the sample concentration
with a wider recognizable range (0.1 to 0.2 mM). Furthermore, following the pathway before, the coupled enzyme reactions can be realized with the photodiode system and the CMOS chip system to verify the better condition for cholesterol detection. Hence, the next step is to enlarge the detectable range and widen it to except a more sensitive and realizable interval for cholesterol analysis.
From the experimental results required by F-4500 luminescence spectrometer detection, we know that cholesterol oxidation could be coupled with the
HRP-luminol-H2O2 system to quantify the amount of cholesterol by luminescence detection. Following the conditions got from the former experiments, the coupled enzyme reaction for cholesterol measurement detected by CMOS chip requires excess enzyme amount, 0.64 U/ml, to produce the detectable light source for CMOS chip detection. The enzyme amount does not fit the bill and is cost consuming. Thus, the HRP-luminol-H2O2 system is established but the method of coupled to cholesterol quantification still needs to be improved to fit the practical application. The co-ordination of the two reactions, cholesterol oxidation and HRP-luminol-H2O2
reaction, is not easy to be achieved. To test the feasibility of cholesterol diagnosis, we have tried to detect the cholesterol signals with the chromogenic method in addition.
The chromogenic assay will be discussed later and it shows that CMOS chip can be integrated with available devices to detect different optical signals,
chemiluminescence or absorbance.
3.4.9 Results of the method for HRP-GOD coupled enzyme reaction
To prove the ability for new generation CMOS chip for target detection with luminescence method. We use glucose and GOD to demonstrate that CMOS
photodiode and HRP-luminol-H2O2 system can be used as diagnostic kit. Figure 20 (from Ude LU’s master thesis) shows the activity of HRP-luminol-H2O2 system is dependent on the concentration of glucose. The Km of glucose is determined to be 3.4 mM + 0.28 mM with PMT instrument (Hitachi F-4500). The same reactions observed by CMOS photodiodes give similar result as shown in Figure 21. Comparing the results with early stage CMOS chip(49), the sensitivity achieves 0.04mM which is 5 folds higher than the sensitivity of CMOS chip, 0.2mM, published in 2004.
Comparing the HRP enzyme amount used in both devices, the quantity required in tests by novel CMOS chip is 0.32U/ml. The requirement of HRP amount is much less than the early stage CMOS chip (49). The data of the comparisons of enzyme
amount and sensitivity is listed in Table 7.