TECHNIQUE FOR QUANTITATIVELY DETECTING ALKALINE PHOSPHATASE ACTIVITY IN SEAWATER BASED ON SURFACE-ENHANCED RAMAN SPECTROSCOPY

Abstract

The present disclosure provides a technique for quantitatively detecting alkaline phosphatase (ALP) activities in seawater and other aquatic environments, based on surface-enhanced Raman spectroscopy by taking 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as a substrate and dimethyl sulfoxide (DMSO) as an internal standard. Results show that ALP activity has a good linear correlation with the intensity ratio of a characteristic Raman peak to that of the internal standard peak (600 cm.sup.−1/677 cm.sup.−1) (R.sup.2=0.977). The technique was successfully applied to detect ALP activity of a seawater sample. By extension this technique can also be used in detecting the activity of other microbial extracellular enzymes (e.g., aminopeptidase) in seawater and thus, lays a solid scientific foundation for in-situ detection of the activities of other extracellular enzymes in seawater and other aquatic environments.

Claims

1. A technique for quantitatively detecting alkaline phosphatase in seawater based on surface-enhanced Raman spectroscopy, comprising the following steps: a. preparing a variety of alkaline phosphatase samples with different activities in advance, separately mixing and incubating the alkaline phosphatase samples with a BCIP solution for a period of time, adding a DMSO solution as a standard solution; b. dropping a plurality of standard solutions with different activities onto a surface-enhanced Raman scattering substrate separately, conducting an SERS detection separately, and drawing a standard curve representing relations between intensity ratios of obtained SERS signals of the standard solutions with different activities to obtained SERS signal of the DMSO, and the logarithm values of the activities of the standard solutions; c. dropping a solution of a sample to be tested containing DMSO onto a surface-enhanced Raman scattering substrate to directly detect SERS signals of the sample to be tested and the DMSO; and d. comparing the SERS signals obtained in step c with the standard curve to obtain the activity of the sample to be tested.

2. The technique for quantitatively detecting an alkaline phosphatase based on surface-enhanced Raman spectroscopy according to claim 1, wherein in step b and step c, the SERS signal of the DMSO is obtained by selecting a peak intensity of the DMSO at a Raman shift of 677 cm.sup.−1.

3. The technique for quantitatively detecting an alkaline phosphatase based on surface-enhanced Raman spectroscopy according to claim 1, wherein in step b and step c, the SERS signal of the alkaline phosphatase is obtained by selecting a peak intensity of the alkaline phosphatase at a Raman shift of 600 cm.sup.−1.

4. The technique for quantitatively detecting an alkaline phosphatase based on surface-enhanced Raman spectroscopy according to claim 1, wherein in step b, the standard curve has a fitted equation of y=0.454*x+0.513 with a correlation coefficient R.sub.2 of 0.977.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is a reaction equation of ALP hydrolyzing BCIP;

[0019] FIG. 2 shows surface-enhanced Raman spectroscopy (SERS) of an experimental solvent and a substrate;

[0020] FIG. 3a shows surface-enhanced Raman spectroscopy corresponding to different activities of ALP;

[0021] FIG. 3b is surface-enhanced Raman spectroscopy of selected wave numbers ranging from 400 cm.sup.−1 to 800 cm.sup.−1;

[0022] FIG. 4 shows fitting a standard curve by a linear equation; and

[0023] FIG. 5 is an SERS image after a seawater sample reacts with BCIP.

DETAILED DESCRIPTION

[0024] The present disclosure will be further described in detail with reference to the accompanying drawings and specific examples.

Example 1 Quantitative Model

[0025] Eleven 900 μL of ALP solutions with different activities (10 U/mL, 5 U/mL, 1 U/mL, 0.5 U/mL, 0.1 U/mL, 50 mU/mL, 10 mU/mL, 5 mU/mL, 1 mU/mL, 0.5 mU/mL and 0.1 mU/mL) were separately mixed with 100 μL of 1 mg/mL of a BCIP solution for incubation for 2 h. After a DMSO solution with 20% volume was added, respective SERS signals were detected and obtained spectra were as shown in FIG. 3.

[0026] As can be seen from FIG. 3, there was no direct linear correlation between SERS characteristic peak intensity of a product BCI oxidized dimer and the concentration of the ALP. Therefore, it is difficult to directly use the intensity of the characteristic Raman peak to conduct quantitative analysis due to interference by factors of stability of laser power, uniformity of an enhancing reagent, background noise of a solvent, etc. Thus the characteristic Raman peak of the added DMSO solvent near wave number 677 cm.sup.−1 was used as an internal standard peak. An internal standard method was used to establish a quantitative detection model to quantitatively detect the ALP. Table 1 shows intensity of SERS characteristic peaks of a substrate and an internal standard.

[0027] Table 3 Intensity of characteristic peaks of substrate and internal standard.

[0028] It can be seen from Table 1 that generally the intensity of the characteristic peak of the product (600 cm.sup.−1) and the intensity of the internal standard peak (677 cm.sup.−1) gradually decreased with decrease of the activity of the ALP, but there was no good functional relationship. RSD of a corresponding intensity ratio of each enzyme activity was less than 15%, indicating that the SERS data were highly reliable. A linear fitting was conducted on the concentration of the ALP and the SERS intensity ratio (600 cm.sup.−1/677 cm.sup.−1) by using a least squares method. The result is as shown in FIG. 4.

[0029] The DMSO was introduced as the internal standard, the characteristic peak at the wave number of 677 cm.sup.−1 was used as the internal standard peak. Logarithm value of a total of 10 ALP activities of 10 U/mL, 5 U/mL, 1 U/mL, 0.5 U/mL, 0.1 U/mL, 50 mU/mL, 10 mU/mL, 5 mU/mL, 1 mU/mL and 0.5 mU/mL was taken as the x-coordinate, and the ratio of the product peak (600 cm.sup.−1) to the internal standard peak (677 cm.sup.−1) was taken as the y-coordinate. A standard equation was fitted: y=0.454*x+0.513 with a correlation coefficient R.sup.2 of 0.977, indicating a strong linear correlation between ALP activity and the ratio of the intensity of the characteristic Raman peaks (600 cm.sup.−1/677 cm.sup.−1). The model was capable of quantitatively detecting ALP activity.

Example 2 Verification Test of a Seawater Sample

[0030] A fresh seawater sample was collected from the East China Sea (30° 39′48″N, 122° 29′48″E) in December 2020. The sample was surface seawater and obtained by using a fishing boat. 900 μL of the fresh seawater sample was mixed with 100 μL of 1 mg/mL of a BCIP solution for incubation for 2 h, after a DMSO solution with 20% volume was added, a SERS signal was detected and an obtained spectrum was as shown in FIG. 5.

[0031] As shown in FIG. 5, there was an obvious Raman peak at 600 cm.sup.−1, indicating that the technique successfully and qualitatively detected ALP in the seawater sample. A Raman intensity at wave number 677 cm.sup.−1 reached 6389.6, which indicated a Raman peak caused by C—S—C symmetrical stretching vibration in the DMSO. The ratio of the two peaks (600 cm.sup.−1/677 cm.sup.−1) is 0.377, and the value was substituted into the above model, and ALP activity of the seawater sample was quantitatively detected, and the obtained ALP activity of the water sample was equivalent to activity of 0.5 mU/mL of ALP in Escherichia coli.

[0032] The technique for quantitatively detecting the activity of the ALP based on SERS by taking the BCIP as the substrate and the DMSO as the internal standard was provided. Results showed that ALP activity and the intensity ratio of the product characteristic peak to the internal standard peak (600 cm.sup.−1/677 cm.sup.−1) had a good linear correlation with a correlation coefficient of 0.977. The activity of ALP in the seawater sample was successfully and quantitatively detected by using the model, thus ALP activity of the seawater sample can be rapidly detected. Meanwhile, the technique could also be used for detecting the activity of other extracellular enzymes of microorganisms in seawater and lays a solid scientific foundation for in-situ detection of the activity of microbial extracellular enzymes in seawater.

[0033] The above description is only preferred embodiments of the present disclosure and not limited in the present disclosure. All equivalent modifications, equivalents, improvements, etc. made within the spirit and the principle of the present disclosure should be included in the scope of the present disclosure.