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Bionics-based nano microstructure chip, endotoxin SERS quantitative detection system and method, and application

A biomimetic nano- and micro-structure technology, applied in the fields of biochemical detection and spectral analysis, can solve the problem of near-blank rapid quantitative detection of endotoxin, achieve good uniformity and detection reproducibility, improve detection throughput, and shorten binding time. Effect

Active Publication Date: 2020-06-09
CHONGQING UNIV
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Problems solved by technology

At present, there are very few studies on the identification of endotoxins by SERS spectroscopy, especially the rapid quantitative detection of endotoxins in various biochemical systems is almost blank

Method used

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  • Bionics-based nano microstructure chip, endotoxin SERS quantitative detection system and method, and application
  • Bionics-based nano microstructure chip, endotoxin SERS quantitative detection system and method, and application
  • Bionics-based nano microstructure chip, endotoxin SERS quantitative detection system and method, and application

Examples

Experimental program
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Effect test

Embodiment 1

[0054] Embodiment 1: see figure 1 , Fabrication of arrayed three-dimensional SERS nano-bionic microstructure chips Use a laser engraving machine to engrave a uniformly distributed 4×7 array of small holes on an acrylic plate 1 with a length of 38 mm, a width of 26 mm, and a thickness of 1 mm. , one of which is used as a blank control experiment, and the rest can be used to investigate the release of endotoxin at different time points.

[0055] Put the cicada wings into a DC ion sputtering apparatus, use high-purity argon as the protective gas, and high-purity gold as the target material, the sputtering pressure is 10 Pa, the sputtering current is 4 mA, and the sputtering rate is 4 nm / min. Sputtering for 16 cycles by intermittent sputtering can obtain a SERS substrate with a gold-plated layer thickness of about 50 nm, and then prepare a circular cicada wing 2 with a radius of 1.5 mm, using the prepared acrylic plate 1 as a template , using double-sided tape to stick on the gla...

Embodiment 2

[0058] Example 2 Preparation of endotoxin SERS label

[0059] To prepare silver nanoparticle dimers, add 36 mg AgNO to 200 mL deionized water 3 , heated to boiling, adding 4 mL of trisodium citrate solution with a mass fraction of 1%, and continued boiling for 90 minutes to form a silver nano-sol. After 2 mL of silver nano-sol was centrifuged at 1800 G, the precipitated part was dispersed in deionized water. Take 100 μL of silver nano-sol dispersed after centrifugation, add 3 μL of PBS buffer and 6 μL of HMD (0.4 mg / mL, pH = 4.0), react for two minutes, add 100 μL of deionized water to obtain silver nanodimer Sol, such as figure 2 shown.

[0060] Using PTAP as the Raman reporter molecule, it was mixed with the dimer of silver nanoparticles for 30 min. Add mPEG-SH 7.6×10 -8 mol reaction for 10min, add 1×10 -8 mol of HS-PEG-COOH reacted for 20 min, with 1.5×10 -5 mol mPEG-SH for secondary blocking. Add 100 μL of 10 nmol / L endotoxin nucleic acid aptamer, react at 37 °C...

Embodiment 3

[0061] Example 3 Real-time in-situ detection of endotoxin content during antibacterial process

[0062] In the arrayed three-dimensional biomimetic SERS microstructure, a gradient concentration of endotoxin standard (3.0625-100.0000 ng / mL) was mixed with the SERS label, and DMSO was used as the internal standard for detection, and the detection reaction was 10, 30, 60, 100, 200 s After the SERS spectrum, determine the binding time of the SERS tag and endotoxin as 100 s, and establish the peak intensity ratio I 636 / I 671 A standard curve versus concentration, such as Figure 4 and Figure 5 .

[0063] The Pseudomonas aeruginosa cultured overnight was diluted to 10 with LB medium 5 CFU / mL, with 2 μg / mL ceftazidime (CAZ) as the antibacterial agent, 6 μL of SERS labeling solution was added after 1, 3, 6, 12, 24, 48, and 72 h respectively, according to the established standard curve Calculate the change of endotoxin content in the antibacterial system, and verify its accuracy...

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Abstract

The invention provides a bionics-based nano microstructure chip, an endotoxin SERS quantitative detection system and method, and application. According to the invention, a cicada wing is used as a natural SERS template and is integrated into an array type three-dimensional SERS chip; furthermore, a composite SERS label modified by metal nanoparticles and an aptamer is combined, the defect that theRaman signal of endotoxin is weak is overcome, the rapid and efficient endotoxin detection system and method are established, the rapid and quantitative detection of endotoxin in biological agents such as injections can be achieved, and the rapid and efficient endotoxin detection system and method can also be applied to the monitoring of the change of the endotoxin content after bacteria and antibacterial agents act.

Description

technical field [0001] The invention belongs to the fields of biochemical detection and spectral analysis, and in particular relates to the technology of endotoxin content based on nano-microstructure and SERS spectral analysis. [0002] technical background [0003] Endotoxin poses a huge threat to the safety of drug therapy, drug production and food processing. Efficient inspection and real-time monitoring of endotoxin has become an indispensable link in food, drug production and clinical drug use. At present, the common detection methods of bacterial endotoxin mainly include traditional methods such as the rabbit pyrogen test (RPT) and limulus amoebocyte lysate assays (LAL). The rabbit pyrogen test has individual differences, low sensitivity, and detection time. However, although the LAL detection method has the advantages of rapid detection, simplicity, and high sensitivity, it requires harsh test conditions, and the results are easily affected by enzymes and impuritie...

Claims

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Application Information

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IPC IPC(8): G01N21/65B01L3/00B82Y30/00B82Y5/00B82Y15/00
CPCG01N21/658B01L3/5027B82Y30/00B82Y5/00B82Y15/00
Inventor 徐溢项松涛陈李
Owner CHONGQING UNIV
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