A peptide aptamer for tryptophan detection and a colorimetric tryptophan detection method based on the peptide aptamer

Abstract

The application discloses a peptide aptamer for tryptophan detection and a colorimetric tryptophan detection method based on the peptide aptamer, wherein the peptide aptamer sequence is shown as SEQ ID NO. 1, and the N terminal of the peptide aptamer is modified by a space unit composed of 1 hemi-cystine. The application combines the advantages of the peptide aptamer, such as high affinity, high selectivity, simple structure and easy operation, with the advantages of the colorimetric detection method, such as rapidness, simple operation and no need of labeling, so that the rapid and sensitive detection of tryptophan can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and monitoring applications, specifically relating to a peptide aptamer for tryptophan detection and a colorimetric tryptophan detection method based on the peptide aptamer. Background Technology

[0002] Tryptophan is the most widely distributed indole derivative in nature and was the first amino acid recognized as essential for many animals. Besides being partially incorporated into proteins, tryptophan is metabolized through various pathways to produce important biological compounds such as melanin, 5-hydroxytryptophan, serotonin, and melatonin. Tryptophan is one of the 18 essential amino acids for humans and is a major precursor in the formation of neurotransmitters such as niacin and serotonin, and neurohormones such as melatonin. The neurotransmitter 5-hydroxytryptophan is involved in regulating mood, sleep, appetite and digestion, memory, and sexual behavior. Simultaneously, tryptophan catabolism is a key regulator of host-gut microbiota crosstalk; any disturbance affecting this interaction can lead to disease development. Recent studies have shown that inappropriate Trp levels in the human body may contribute to Alzheimer's and Parkinson's diseases. Therefore, developing a rapid and accurate method to measure tryptophan levels is particularly important. Traditional analytical methods for detecting and measuring tryptophan include X-ray diffraction (XRD), mass spectrometry (MS), nuclear magnetic resonance (NMR), and liquid or gas chromatography (LC / GC). However, the equipment associated with these methods requires significant investment, highly trained personnel to operate, and often dedicated laboratory space. In addition to the high cost, comprehensive analysis also takes a considerable amount of time. Among various sensing strategies, colorimetry is highly regarded for its low cost, fast feedback, simplicity, and ease of readability.

[0003] Aptamers, typically single-stranded DNA, RNA, or peptides discovered from oligonucleotide libraries through exponentially enriched ligand systems, serve as receptors. Furthermore, aptamer sensors can achieve picomolar sensitivity, which is more sensitive than other electrochemical sensors using micro to nanomolar sensitivities. Another advantage of aptamers is that they can be chemically modified according to the detection criteria of the target molecule. Aptamers exhibit high affinity and specificity for homologous ligands and some ligands that antibodies cannot recognize, such as ions or small molecules. Once selected, aptamers can be synthesized in large quantities through chemical processes, at a higher cost than antibody production. Gold (Au)-based nanomaterials are a widely studied topic in nanoscience and nanotechnology. Due to their attractive electronic, optical, magnetic, and thermal properties, as well as catalytic properties, gold nanomaterials have found wide applications in physics, chemistry, catalysis, technology, biomedicine, materials science, and electrochemical sensors. More interestingly, the intrinsic properties of gold nanomaterials can be controlled by adjusting their shape, size, and surrounding chemical environment. Gold nanomaterials are widely used to prepare modified electrode surfaces for electrochemical sensors. Furthermore, gold nanomaterials can be readily multifunctionalized with various organic or biological ligands, and a variety of gold-based nanocomposites have been developed for electrochemical applications. Over the past few decades, the application of gold and gold-based nanomaterials in electrochemical sensors has been extensively reported. Summary of the Invention

[0004] The present invention aims to address the shortcomings of existing technologies by providing a peptide aptamer for tryptophan detection and a colorimetric tryptophan detection method based on the peptide aptamer, which can achieve rapid detection of tryptophan with high sensitivity and specificity.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0006] The peptide aptamer sequence for tryptophan detection is shown in SEQ ID NO.1: N-terminus-CEGNSSSC-C-terminus, wherein the N-terminus of the peptide aptamer is modified by a spatial unit consisting of one cysteine ​​residue. This peptide aptamer can be used to prepare a formulation for the detection of tryptophan.

[0007] The colorimetric tryptophan detection method based on the above-mentioned peptide aptamers includes the following steps:

[0008] (1) Preparation of detection solution: Add chloroauric acid solution dropwise to ultrapure water, then add the above-mentioned peptide aptamer solution dropwise, stir, then add sodium borohydride solution dropwise, and continue stirring to obtain the detection solution; the chloroauric acid solution is a chloroauric acid solution with a mass concentration of 8-15%, the peptide aptamer solution is a peptide aptamer solution with a concentration of 1.5-2.5 mM, and the sodium borohydride solution is a sodium borohydride solution with a concentration of 0.2-0.4 M; the addition ratio of the chloroauric acid solution, peptide aptamer solution, and sodium borohydride solution is: 10-20 μL of chloroauric acid solution, 10-20 μL of peptide aptamer solution, and 15-25 μL of sodium borohydride solution are added per 10 mL of ultrapure water;

[0009] (2) The solution containing tryptophan is dropped into the test solution and the color change can be observed directly with the naked eye. Then potassium chloride solution is added and the absorbance is scanned while the UV-Vis spectrophotometer is running. The volume ratio of the solution containing tryptophan to the test solution is 1:6 to 1:12. The potassium chloride solution is a potassium chloride solution with a potassium chloride concentration of 0.8-1.2 M and the volume ratio of the potassium chloride solution to the test solution is 1:4 to 1:8.

[0010] Preferably, the stirring time after adding the peptide aptamer solution in step (1) is 0.5 h, and the stirring time after adding the sodium borohydride solution is 1.5 h.

[0011] Preferably, the chloroauric acid solution in step (1) is a chloroauric acid solution with a mass concentration of 10%, the peptide aptamer solution is a peptide aptamer solution with a concentration of 2.0 mM, and the sodium borohydride solution is a sodium borohydride solution with a concentration of 0.3 M; the addition ratio of the chloroauric acid solution, peptide aptamer solution, and sodium borohydride solution is: 15 μL of chloroauric acid solution, 15 μL of peptide aptamer solution, and 20 μL of sodium borohydride solution are added per 10 mL of ultrapure water.

[0012] Preferably, the ultrapure water in step (1) is ultrapure water with a resistivity greater than 18 MΩ*cm.

[0013] Preferably, the scanning range of the UV-Vis spectrophotometer in step (2) is 400-800 nm.

[0014] Preferably, the potassium chloride solution in step (2) is a potassium chloride solution with a potassium chloride concentration of 1.0 M.

[0015] Preferably, in step (2), the volume ratio of the solution containing tryptophan to the detection solution is 1:9; and the volume ratio of the potassium chloride solution to the detection solution is 1:6.

[0016] Preferably, the tryptophan colorimetric detection method based on peptide aptamers exhibits good linearity in the range of 10.0 μM–1.0 mM; its linear equation is as follows:

[0017] y = 0.0006812 x +0.2620 (R 2 = 0.9845)

[0018] in, y It is the absorbance of the solution at 589 nm. x It is the tryptophan concentration (μM).

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention combines the advantages of peptide aptamers, such as high affinity, high selectivity, simple structure, and ease of operation, with the advantages of colorimetric biosensors, such as speed, simple operation, and label-free operation, to achieve rapid and ultrasensitive detection of tryptophan. Attached Figure Description

[0021] Figure 1 This is a physical diagram of the tryptophan biocolorimetric detection process based on peptide aptamers according to the present invention;

[0022] Figure 2 The absorbance of tryptophan at different concentration gradients is shown in the colorimetric detection method for tryptophan based on peptide aptamers of the present invention.

[0023] Figure 3 The results demonstrate the specificity of the tryptophan biocolorimetric detection method based on peptide aptamers of the present invention.

[0024] Figure 4 These are the experimental results from the present invention for addressing the problem of cysteine ​​interference. Detailed Implementation

[0025] Reagents not specifically mentioned in this invention are commercially available reagents. Room temperature refers to approximately 25°C. Example 1

[0026] The peptide aptamer sequence for tryptophan detection is shown in SEQ ID NO.1: N-terminus-CEGNSSSC-C-terminus, wherein the N-terminus of the peptide aptamer is modified by a spatial unit consisting of one cysteine ​​residue. Example 2

[0027] The tryptophan colorimetric detection method based on the peptide aptamers described in Example 1 includes the following steps:

[0028] (1) Preparation of detection solution: Chloroauric acid solution is added dropwise to ultrapure water, then the peptide aptamer solution described in Example 1 is added dropwise, stirred for 0.5 h, then sodium borohydride solution is added dropwise, and stirring is continued for 1.5 h to obtain the detection solution; the chloroauric acid solution is a chloroauric acid solution with a mass concentration of 10%, the peptide aptamer solution is a peptide aptamer solution with a concentration of 2.0 mM, and the sodium borohydride solution is a sodium borohydride solution with a concentration of 0.3 M; the addition ratio of the chloroauric acid solution, peptide aptamer solution, and sodium borohydride solution is: 15 μL of chloroauric acid solution, 15 μL of peptide aptamer solution, and 20 μL of sodium borohydride solution are added per 10 mL of ultrapure water; the ultrapure water is ultrapure water with a resistivity greater than 18 MΩ*cm;

[0029] (2) Add a solution containing tryptophan to the test solution. The color change can be observed directly with the naked eye. Then add potassium chloride solution. Under the condition of running a UV-Vis spectrophotometer, scan the absorbance in the range of 400~800 nm (e.g., ...). Figure 1 The volume ratio of the tryptophan-containing solution to the detection solution is 1:9; the potassium chloride solution is a 1.0 M potassium chloride solution; and the volume ratio of the potassium chloride solution to the detection solution is 1:6.

[0030] It exhibits good linearity in the range of 10 μM–1 mM (e.g. Figure 2 Its linear equation is:

[0031] y = 0.0006812 x +0.2620 (R 2 = 0.9845)

[0032] in, y It is the absorbance of the solution at 589 nm. x It is the tryptophan concentration (μM).

[0033] The specificity test results of the tryptophan colorimetric detection method are as follows: Figure 3 :

[0034] The peptide aptamer solution described in this invention was mixed with a 1.0 mM tryptophan or other non-specific amino acid solution at a volume ratio of 9:1. Then, a 1.0 M potassium chloride solution was added at a volume ratio of 6:1. It was found that the absorbance of the other non-specific amino acids (phenylalanine, lysine, alanine, methionine, glycine, glutamic acid, threonine, leucine, histidine, serine, arginine, isoleucine, tyrosine, valine, and aspartic acid) differed significantly from the absorbance of the solution with added tryptophan. Furthermore, the absorbance of the mixed solution of all the above amino acids was similar to that of tryptophan. Meanwhile, because the cysteine ​​side chain -SH readily couples with colloidal gold, and cysteine ​​molecules easily aggregate due to hydrogen bonding, this leads to the aggregation of colloidal gold. To address this, this invention proposes a solution: adjusting the pH of the cysteine ​​solution to 7.50, and then measuring its absorbance according to the above experimental steps. Compared with before pH adjustment, the absorbance decreased significantly, and the interference effect was reduced to an acceptable range (e.g., ...). Figure 4 This demonstrates that the tryptophan biosensor based on peptide aptamers described in this invention has excellent specificity.

Claims

1. A peptide aptamer for tryptophan detection, said peptide aptamer being a sequence in which the N-terminal cysteine ​​of the sequence shown in SEQ ID NO.1 is modified by a spatial unit consisting of an additional cysteine.

2. The application of the peptide aptamer as described in claim 1 in the preparation of a tryptophan detection agent.

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