METHODS FOR DETECTION, DETERMINATION, AND ACTIVITY MEASUREMENT OF PEROXIDASE BASED ON CHEMILUMINESCENCE

Abstract

A further high-sensitive method for detection, determination, and activity measurement of peroxidase with no special enhancer argent. The substance, for example, high-concentration ammonium sulfate, is dissolved in the reaction solution to give rise to the micro-hydrophobic property, for detection, determination, and activity measurement of peroxidase using luminol and hydrogen peroxide as substrates.

Claims

1. The method for detection, determination, and activity measurement of peroxidase using luminol as its substrate comprising: dissolving the substance to produce hydrophobic property.

2. The method for detection, determination, and activity measurement of peroxidase using luminol as its substrate virus concentration method according to claim 1, wherein the substance to produce hydrophobic property is ammonium sulfate.

3. The method for detection, determination, and activity measurement of peroxidase using luminol as its substrate virus concentration method according to claim 2, wherein the ammonium sulfate has a concentration of 3 M to 3.5 M.

4. The method for detection, determination, and activity measurement of peroxidase using luminol as its substrate virus concentration method according to claim 2, further comprising: adding ethylenediamine-tetraacetate together with ammonium sulfate.

5. The method for detection, determination, and activity measurement of peroxidase using luminol as its substrate virus concentration method according to claim 3, further comprising: adding ethylenediamine-tetraacetate together with ammonium sulfate.

6. The solution for detection, determination, and activity measurement of peroxidase comprising: containing of luminol, hydrogen peroxide, peroxidase, and ammonium sulfate; wherein the ammonium sulfate has a concentration of 3 M to 3.5 M.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIGS. 1A-AH Luminol chemiluminescence spectra recorded as a function of time in the presence of various concentrations of ammonium sulfate.

[0034] FIG. 2 Plots of the integrated intensity (area under each spectrum) of luminol chemiluminescence recorded in the presence of various concentrations of ammonium sulfate against time elapsed after the initiation of the reaction.

[0035] FIGS. 3A-3D Detection and determination of HRP based on the present chemiluminescence under the optimal conditions.

[0036] FIG. 4 Effect of EDTA on the removal of background chemiluminescence. EDTA concentration (ppm) is indicated on each graph.

[0037] FIG. 5 Reaction mechanism for the Luminol-H.sub.2O.sub.2-HRP reaction.

DESCRIPTION OF THE EMBODIMENTS

[0038] The present invention is explained in detail, as below.

[0039] General experimental conditions in the present invention are described as follows.

[0040] First, 1 μL of HRP standard solution ranging in concentration between 1×10.sup.−6M and 1×10.sup.−9 M is placed in an empty 1-mL cuvette, subsequently 1000 μL mixture of equal parts of luminol solution and hydrogen peroxide solution is added to the cuvette to initiate the chemiluminescence reaction. Particularly, the concentrations of HRP, subjected to the evaluation of the present invention, is in the range of 5×10.sup.−9 M to 1×10.sup.−7M. In this case, the concentration of HRP in the reaction mixture is in the range of 5×10.sup.−12 M to 1×10.sup.−1° M. Luminol solution used for the above-mentioned evaluation is the mixture of 1 vol of 30 mM luminol prepared in 0.75 M NaOH and 5 vol of pH8.5 tris(hydroxymethyl)aminomethane (0.1 M) buffer solution containing various concentrations of ammonium sulfate. The feature of this luminol solution is favorably prepared so as to make the final reaction mixture pH optimal. On the other hand, hydrogen peroxide solution used for the evaluation is various concentrations of ammonium sulfate solution containing 100 mM hydrogen peroxide: concentration of ammonium sulfate is corresponding to that used for luminol solution. The solution pH is in the range of 8.3 to 8.8. However, in the absence of ammonium sulfate, pH of the reaction mixture increases to approximately 12, because of the absence of buffer action by ammonium sulfate.

[0041] In the evaluation of the effect of ammonium sulfate (AS), luminol chemiluminescence was characterized at various molar concentrations of AS using the reaction system described above. Specifically, the effect was evaluated by using the light intensity calculated from the chemiluminescence spectra recorded as a function of time. Chemiluminescence spectra was repeatedly recorded 5 times at one minute interval. First spectral measurement was carried out at 10 s after the initiation of the chemiluminescence reaction, i.e., five spectra were obtained at 0.17 min (corresponding to a00 designated in FIGS. 1, 2 and 4), 1.17 min (a01), 2.17 min (a02), 3.17 min (a03), and 4.17 min (a04); the third digit is expressed as “a”. For example, in FIGS. 1A-1H, correspondence between legend for each spectrum and time is as follows; 0420! 900.fwdarw.0.17 min, 901.fwdarw.1.17 min, 902.fwdarw.2.17 min, 903.fwdarw.3.17 min, 904.fwdarw.4.17 min.

[0042] FIGS. 1A-1H show chemiluminescence spectra recorded at various concentrations of ammonium sulfate used to prepare pH8.5 tris (0.1 M) buffer solution and hydrogen peroxide solutions: (a) AS=0.0 M, (b) AS=0.9 M, (c), AS=1.8 M, (d) AS=2.3 M, (e) AS=2.5 M, (f) AS=2.8 M, (g) AS=3.0 M, and (h) AS=3.2 M. As shown in FIGS. 1A-1H, the intensity of the chemiluminescence spectra is intensified with an increase in the ammonium sulfate concentration. The intensity obtained at 3.2 M ammonium sulfate is intensified by 100 times as compared to that obtained in the ammonium sulfate free system. In FIGS. 1A-1H, it is shown that the weak chemiluminescence is observed in the absence of ammonium sulfate. This is possibly because iron (III) ion liberated from HRP in the relatively high pH solution catalyzes the luminol chemiluminescence but not from the HRP catalyzed luminol reaction. It is possible to achieve 200 times intensification of chemiluminescence by adjusting the concentrations of luminol and hydrogen peroxide in the solution. FIGS. 1A-1H shows changes in chemiluminescence spectra recorded at 1-min interval in the presence of various concentrations of ammonium sulfate (AS). First spectral measurement was carried out at 10 s after the initiation of the chemiluminescence reaction, i.e., five spectra were obtained at 0.17 min (corresponding to a00 designated on the graphs in FIGS. 1A-1H, 2 and 4), 1.17 min (a01), 2.17 min (a02), 3.17 min (a03), and 4.17 min (a04); the third digit is expressed as “a”. For example, in FIGS. 1A-1H, correspondence between legend for each spectrum and time is as follows; 0420!900.fwdarw.0.17 min, 901.fwdarw.1.17 min, 902.fwdarw.2.17 min, 903.fwdarw.3.17 min, 904.fwdarw.4.17 min.

[0043] The Integrated intensity (area under each spectrum) vs. Time elapsed after the initiation of the reaction.

317 #800; [HRP]=0

[0044] #800 (10 s after), #801 (1.17 min after), #802 (2.17 min after), #803 (3.17 min after), #804 (4.17 min after),

317 #600; [HRP]=5×10.SUP.−12.M

[0045] #600 (10 s after), #601 (1.17 min after), #602 (2.17 min after), #603 (3.17 min after), #604 (4.17 min after),

317 #100; [HRP]=1×10.SUP.−1.° M

[0046] #100 (10 s after), #101 (1.17 min after), #102 (2.17 min after), #103 (3.17 min after), #104 (4.17 min after)

[0047] FIG. 2 exhibits the relationship between the luminol chemiluminescence intensity recorded at various concentrations of ammonium sulfate and time elapsed after the initiation of the reaction. In FIG. 2, each integrated intensity (=area under each spectrum) is plotted as a function of time elapsed. It is shown that the intensity is evidently increased, especially when the AS concentration is greater than 3.0 M. FIG. 2 shows plots of the integrated intensity (area under each spectrum) of the luminol chemiluminescence generated in the presence of various concentrations of ammonium sulfate (AS) against time elapsed after the initiation of the reaction.

[0048] It is clearly shown in FIG. 2 that the effect of AS on the chemiluminescence becomes remarkable when the AS concentration is greater than approximately 2.8 M. This means that the threshold value is present to express the effect of AS. This result also suggests that AS markedly affects the reaction mechanism. That is, it can be concluded that high concentration AS contributes to accelerate two reactions in the reaction mechanism, resulting in the increase in the overall reaction rate, followed by the increase in the chemiluminescence intensity.

[0049] FIGS. 3A-3D exhibit that the results obtained under the optimal conditions (concentration in the reaction mixture; AS=3.2 M, luminol=2.5 M, and H.sub.2O.sub.2=50 mM). Based on the present method, detection, and determination of pM level of HRP become feasible.

[0050] FIGS. 3A-3D show a detection and determination of HRP. It is possible to detect and determine pM level of HRP at high SN ratio. The chemiluminescence spectra, are repeatedly recorded 5 times at 1-min interval. First spectral measurement was carried out at 10 s after the initiation of the chemiluminescence reaction.

[0051] 317 #100, 317 #600, 317 #800—about 10 s (0.17 min) after the initiations of the reaction

[0052] 317 #101, 317 #601, 317 #801—1.17 min

[0053] 317 #102, 317 #602, 317 #802—2.17 min

[0054] 317 #103, 317 #603, 317 #803—3.17 min

[0055] The Integrated intensity (area under each spectrum vs. Time elapsed after the initiation of the reaction is shown.

[0056] 317 #800; [HRP]=0

[0057] #800 (10 s after), #801 (1.17 min after), #802 (2.17 min after), #803 (3.17 min after), #804 (4.17 min after),

[0058] 317 #600; [HRP]=5×10-12 M

[0059] #600 (10 s after), #601 (1.17 min after), #602 (2.17 min after), #603 (3.17 min after), #604 (4.17 min after),

[0060] 317 #100; [HRP]=1×10-10 M

[0061] #100 (10 s after), #101 (1.17 min after), #102 (2.17 min after), #103 (3.17 min after), #104 (4.17 min after)

[0062] Subsequently, the effect of ethylenediamine-tetraacetic acid (EDTA) added to the present reaction solution on the chemiluminescence was examined. The results obtained are shown in FIG. 4. The concentrations of ammonium sulfate, luminol, and hydrogen peroxide are 3.2 M, 2.5 mM, and 50 mM, respectively. These concentrations are common to all experiments in this examination. FIG. 4 shown the effect of EDTA on the removal of background chemiluminescence. EDTA concentration (ppm) in the reaction mixture is indicated on each panel.

[0063] The concentration of each component is indicated in FIG. 4. Graphs shown in the left column (column (a)) are obtained in the presence of 1×10.sup.−10 M HRP, and on the other hand, graphs in the right column (column (b)) are obtained in the absence of HRP. It is clearly shown that background chemiluminescence is almost completely removed in the systems to which EDTA is added. Therefore, it can be regarded that the chemiluminescence observed in the presence of EDTA (FIG. 4, column (a)) is arising from the HRP catalyzed luminol reaction. The bottom graph in column (b) is obtained in the absence of not only EDTA but also HRP. Despite being that HRP is not present, chemiluminescence is observed. This is possibly due to background chemiluminescence attributed to the trace amount of metal ions contained in ammonium sulfate as impurity. This background can be fully removed by masking effect for contaminant metal ions with EDTA, originally added to stabilize hydrogen peroxide for the long-term storage. Furthermore, it is evident that EDTA does not exert any harmful effect on the luminol-H.sub.2O.sub.2-HRP chemiluminescence intensified in the presence of ammonium sulfate.

[0064] FIG. 5 shows a reaction mechanism for the luminol-H2O2-peroxidase chemiluminescence reaction.

INDUSTRIAL APPLICABILITY

[0065] According to the present study, the present chemiluminescence system is available to detect and determine against all types of analytes. For example, the present method is available for the chemiluminescent detection, determination, and activity measurement of peroxidase, which is useful in the detection of biological macromolecules, organic substances and so on.