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High-throughput enzyme sensor and method for detecting urea in human urine

The technology of an enzyme sensor and detection method is applied in the direction of material analysis by observing the influence on chemical indicators, and analysis by causing the material to undergo chemical reaction. and other problems, to achieve the effect of repeated use, qualitative and quantitative detection, and reduction of experimental costs

Inactive Publication Date: 2017-03-15
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

Moreover, there are a large number of clinical samples to be tested in hospitals every day. How to achieve high-throughput detection of urea has become another challenge for urea biosensors.
[0006] The traditional 96-microwell plate method can achieve high-throughput detection, but the operation of controlling the enzymatic reaction is cumbersome, the amount of reagents is large, and it cannot be reused

Method used

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  • High-throughput enzyme sensor and method for detecting urea in human urine
  • High-throughput enzyme sensor and method for detecting urea in human urine
  • High-throughput enzyme sensor and method for detecting urea in human urine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Sources of reagents and equipment used:

[0023] 1 Robolids microplate and 96 microplate: Sigma-Aldrich;

[0024] 2 Urease, chitosan: Sigma Aldrich (Shanghai);

[0025] 3 Sodium alginate: Tianjin Guangfu Fine Chemical Research Institute;

[0026] 4 Nessler's reagent, urea: Aladdin;

[0027] 4 High-throughput pipette: Decong Scientific Instrument Co., Ltd.;

[0028] 5 Epoch2 Microplate Reader: Burton, USA

[0029] 6 K5600 miniature ultraviolet-visible spectrophotometer: Beijing Kaiao Technology Development Co., Ltd.

[0030] Preparation of a novel three-dimensional high-throughput enzyme reactor:

[0031] 1) Robolids plate pretreatment

[0032] Seal the commercial Robolid sealing plate (96 silica gel microcolumns, diameter 5mm / column, height 5mm / column) in a 96-microliter microcolumn containing 50uL 0.01% (w / v) poly-lanine (PLL) solution in each well. On the orifice plate, invert the surface of each microcolumn of Robolid to be covered with PLL solution, and inc...

Embodiment 2

[0038] Construction of Urea Standard Curve and Monitoring of Urease Kinetics

[0039] 1) Use 50 mM phosphate buffer solution (pH 7.0) to prepare a series of urea standard substances with different concentrations, add them to the microwell plate one by one, seal the microwell plate with the prepared urease immobilized robolid plate, and invert it to make the sample to be tested contact with the enzyme Start the enzyme reaction at 37°C for 10 min, then invert to separate the sample to be tested from the enzyme to stop the enzyme reaction.

[0040] 2) 24 uL of Nessler's reagent was distributed to the enzyme wells of the microplate in 1) above, and the color reaction was carried out at room temperature for 10 min.

[0041] 3) Use an Epoch2 microplate reader to detect the chromogenic product in 2), and output the absorbance value at a wavelength of 405 nm.

[0042] 4) Draw the standard curve with the absorbance at 405 nm wavelength as the ordinate and the urea concentration as the...

Embodiment 3

[0045] Actual sample testing

[0046] A urine sample was diluted 100 times, and urea standard samples of different known concentrations were added, and the recovery rate of the calculated standard addition was between 95.3% and 104%, as shown in Table 2.

[0047]28 actual urine samples with unknown concentrations were diluted 100 times, 150 times and 200 times respectively for detection with the high-throughput enzyme sensor prepared by the present invention. Substitute the absorbance into the standard curve, and compare the obtained result with the result obtained by the standard urease method, such as Figure 8 It shows that the method has high accuracy.

[0048] Table 1: Comparison of the method of the present invention with other urea detection methods

[0049]

[0050] Table 2: Recovery of standard spiked urea in human urine and ultrapure water

[0051]

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Abstract

The invention relates to a novel high-throughput enzyme sensor based urea detecting method. A robolid sealing plate of a 96-hole microplate is taken as a carrier for enzyme immobilization; enzyme immobilization adopts an embedding method taking sodium alga acid-chitosan as a matrix and barium-alginate gels with the 'egg-box' structure formed by coating pretreatment of poly-l-lysine (PLL)-Ba<2+>, and the three-dimensional high-throughput enzyme sensor is prepared. High-throughput detection (as shown in figure 1) of multiple samples with each micropore sealed is realized through combination of the robolid plate and microplate for enzyme immobilization. The prepared enzyme sensor and a sample solution in the microplate form a 'robolid plate-urease-urea samples' sandwich structure, start and stop of enzyme reaction are realized by simply flipping the sandwich structure, and the urea is detected in an easy, convenient, high-throughput and quantitative manner. Urea concentration is in positive correlation to absorbancy under 405nm wavelength, and the urea can be detected qualitatively and quantitatively. In addition, commercial robolid and robolid for enzyme immobilization can be repeatedly used, and clinical practical and promotion is facilitated.

Description

technical field [0001] The invention belongs to the technical field of enzyme sensor analysis. Specifically relates to a urea detection method based on a high-throughput immobilized enzyme reaction method. Background technique [0002] Urea is the main end product of human protein metabolism, and its normal content in normal human urine and blood is 55–388 mM (9.3–23.3 g / L) and 2.5–7.5 mM (0.15–0.4 g / L) respectively . Abnormal urea levels are indicative of kidney failure, urinary tract obstruction, dehydration, congestive heart failure, shock, severe burns, and liver disease, among others. Therefore, the detection of urea content has become the most effective and direct way of clinical diagnosis and medical treatment to check renal function. [0003] So far, various analytical methods have been reported for the detection of urea in the laboratory, such as electrochemical sensors, spectrophotometry, flow injection spectrophotometry, gas chromatography, high performance l...

Claims

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Application Information

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IPC IPC(8): G01N21/78
CPCG01N21/78
Inventor 杨丽杨继庆
Owner NORTHEAST NORMAL UNIVERSITY
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