In this study described a green approach for synthesizing silver nanoparticles (AgNPs) with corncob acting as a reducing and stabilizing agent to rapidly detect of L-Cysteine (Cys). To obtain an appropriate condition of synthesizing AgNPs variations in the reactant condition such as concentration, pH, and processing speed were altered. The synthesized AgNPs, that have sizes of 50–100 nm were capped by corncob extract (CCB), became virtually hexagonal and re-dispersed well in aqueous solution. AgNPs were explored for their possible structural characterization. Significantly, the CCB/AgNPs combination demonstrated very high sensitivity for Cys detection with a 30 nM limit of detection (LOD) and selective detection of Cys among 12 amino acids. Furthermore, the CCB/AgNPs combination was applied to detect Cysteine human serum with an error rate of less than 5%. As a result of this work, a rapid approach for Cys detection employing green-synthesized AgNPs was developed.
Research Article
Green Synthesis of Silver Nanoparticles from Corn Cob Aqueous Extract for Colorimetric Cysteine Detection in Serum Simulated with Cysteine Samples
https://doi.org/10.21203/rs.3.rs-1512012/v1
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Bio-synthesize
Corncob
AgNPs
Cysteine
Colorimetric
The only amino acid that exists is cysteineamong the 20 non-essential amino acids in the human system that contains the thiol group [1, 2]. Its found in keratin the primary protein that builds up nails, skin, and hair contain it aids in the production of collagen, which keeps skin supple and smooth [3, 4]. Cysteine also protects liver parenchymal cells, improves hematopoietic function, boosts leukocyte production, and speeds up skin cell turnover. A shortage of cysteine can lead to hair loss, psoriasis, swelling, tiredness, liver damage, decreased hematopoietic white blood cell loss, and other problems [5, 6]. As a consequence, it plays a crucial role in protein synthesis.
Congenital metabolic abnormalities and cystinuria may be linked to abnormal cysteine concentrations in the body [7]. The measurement of cysteine is critical for precise pre-diagnosis of a variety of diseases. Capillary electrophoresis [8], high-performance liquid chromatography [9], mass spectrometry [10], fluorescence analysis [11], and electrochemical voltammetry are among the sensitive and selective methods being developed for the detection and measurement of cysteine in environmental, pharmaceutical, and biological samples or precursors [12].These processes depend on redox chemistry or derivative products of chromo pore/fluorescent groups, and they necessitate high temperatures, specialized instruments, and the use of powerful and harmful reagents to improve the detection of trace components and the removal of basic disruptions in a cost-effective manner. Therefore, colorimetric sensing differs from earlier approaches in that it relies on a colour shift in the nanoparticles that can be observed and analyzed with the naked eye and a basic UV-vis spectrometer [13]. Its more convenient to use, but it’s also less expensive.
Because of the advancement of nanotechnology, colorimetric detection based on silver nanoparticles has recently been recognized a promising strategy for detecting cysteine [13, 14]. Due to their high valueabsorption coefficient and range dependent optical features. Silver nanoparticles are substantially less expensive compared to other metal nanoparticles and have a high affinity for nitrogen and sulfur-containing molecules [15]. AgNPs bind strongly to the biomolecules accessible thiol groups.The aim of green nanoparticles synthesize is to reduce waste and establish a long-term procedure. In recent years, the development of nanotechnology has prioritized green processes that use mild reaction conditions and nontoxic precursors to promote environmental sustainability [16]. As biological agents, such as plants or microbiological sources, are used as reducing and capping agents in an environmentally friendly method [17]. Green synthesized silver nanoparticles are a novel and promising alternative to nanoparticles producingchemically produced. Due to its unique antibacterial capabilities [18], larvicidal activity [19], anticancer activity [20], and Metabolites detection [21] green silver nanoparticles have gotten a lot of interest.
As shown in Scheme 1, the purpose of this study was to use CCB extract to synthesize AgNPs for cysteine detection. Initially, CCB extract was extracted from the husk. Then, utilizing Tollens reagent [Ag (NH3)2OH] and CCB, well dispersed Ag-NPs were produced at 80°C under magnetic stirring.CCB extract was used as a reducing and stabilizing agent during the synthesis, rather than some other synthetic reducing or stabilizing chemical, resulting in the development of a synthesize mechanism from metal nanoparticles from agriculture wastes. Finally, employing the naked eye and UV-vis spectra, the generated Ag-NPs were used in a sensitive and selective colorimetric manner. Inaddition, the detection of cystine inhuman serum was also discussed in order to evaluate the potential direct implementation of the generated Ag-NPs.
Materials
Silver Nitrate (AgNO3)was purchased from Merck, India. Cysteine (Cys), Proline (Pro), Tryptophan (Trp), Phenylalanine (Phe), Histidine (His), Serine (Ser), Leucine (Leu), Threonine (Thr), Arginine (Arg), Valine (Valn), Tyrosine (Tyr), N-Acetyl Cysteine (NAc), Glutathione Reduced (GSH). These amino acids were purchased from HiMedia Laboratories Pvt Ltd, Mumbai, India.
Preparation of Corn Cob Extract
A fresh corn was purchased from local market, Salem, Tamil nadu, India. The corn was carefully rinsed and seeds were removed. After that, the CCB was chopped into small pieces and dried in the dark for 15 days. After drying, the CCB was ground into fine powder. A 10 g of CCB powder was taken in a beaker and add 100 mL of distilled water was added and boiled it for 2 hrs. After boiling the solution was filtered using 0.2 mm Whatman filter paper. The filtered solution was used for synthesis of silver nanoparticles.
Green Synthesis of Silver Nanoparticle
Freshly prepared aqueous solution of corn cob extract was used to prepare silver nanoparticles. The stock solution of 10 mL corn cob extract was added dropwise in sequence to the dilute and freshly prepared 1mM AgNO3 in 200 mL distilled water and stirred for 10 min at 60°C in a magnetic stirrer[19]. Brown AgNPs were formed under ambient light settings after the contents of the reaction vessels were mixed with gentle swirling.
Synthesized and Characterization of Ag-NPs
A spectrophotometer was used to obtain the UV-vis spectra of Ag-NPs (UV-1800, Shimadzu, Japan), in the fast-speed mode with a spectral scanning rangeof 300–700 nm [13]and the scan intermissionwas 0.5 nm. A transmission electron microscopy JEM-2100 (TEM; JEOL, Japan) was used to investigate the shape and distribution of the AgNPs, which operated at a 200-kV accelerating voltage. A few drops of the suspending AgNPs were dropped on a copper grid coated with ultrathin materials surface.For each area, selected area electron diffraction (SAED) patterns were also obtained for the particle size distributions of AgNPs (3000 HSA, Malvern, England). Energy dispersive X-ray analysis (EDS) was used to determine the total silver content of the composite (TEM; JEOL, Japan).
Sensitive detection of Cysteine
The prepared CCB/AgNPs colloids were 75 times diluted in the detection experiment, and the pH was set to 5.0. After that, 1mL of the AgNPs solution was added to 2mL of cysteine aqueous solution at various concentrations (0 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 500 mM, and 1000 mM), to observe the color change within 5 min, the mixture was well mixed and homogenized at room temperature. The related surface plasmon resonance (SPR) absorption data was collected using a UV- vis spectrophotometer.
Selective detection of Cysteine
Under the same condition, 12 different amino acids in 2 mL (Pro, Trp, Phe, His, Ser, Leu, Thr, Arg, Valn, Tyr, NAc and GSH) with 100 mM concentration were added to 1mL AgNPs solution. To see if any other amino acids interfered with cysteine detection, 1 mL of each of the other 12 amino acids (1000 mM) were added to a mixture of 1 mL AgNPs and 1 mL cysteine (1000 mM). UV-vis spectroscopy was used to notice the color shift and record the associated data.
Cysteine detection in serum samples
To evaluate cysteine in human serum samples, the traditional addition strategy was applied with minor modifications to the techniques specified [22]. At 4°C, human blood was centrifuged for 30 min at 4000 rpm after being drawn from a healthy volunteer. The plasma was produced from the supernatant, which contained proteins and amino acids among other things. After that, a 2 mL serum sample was mixed with 1.2 mL acetonitrile, which was then combined with various cysteine concentrations. After vortexing for 1 min, the serum protein residue was eliminated by centrifuging the solution for 20 min at 10,000 rpm.The supernatant was collected and dissolved in PBS (pH = 7.0) to reach final cysteine concentrations of 10, 50, 80, 100, and 200 mM for detection, with the serum being 50 times diluted in PBS.The UV-vis spectra at the respective wavelengths were recordedafter adding 0.5 mL of the above cysteine solution to the AgNPs solution (2.5 mL).The following formula was used to obtain the recovery values.
Green Synthesis of AgNPs
UV-vis spectra were used to investigate the synthesis of AgNPs in the presence of CCB. UV-vis spectroscopy is among the most major technique was used to analyze the formation of metal nanoparticles [23, 24]. The production of CCB capped AgNPs visible at 400 nm, and the addition of cysteine to the NPs produced particle aggregation, resulting in a change in solution color from bright yellow to red. Figure 1a shows the plasmon band centers at 400 nm blue shift, generating a second peak at 530 nm.The complex generated as a function of particles aggregation could explain the appearance of a second peak in the spectra.There is no obvious peak in the only CCB aqueous extract solution shown in Fig. 1b. Finally, various conditions were used to synthesize CCB/AgNPs, as indicated in Table 1.
Table 1
Synthesis of AgNPs under various ratio, temperature, and time conditions.
|
Samples |
CCB/AgNO3 (mL/Conc. mM) |
Temperature (°C) |
Time (min) |
|---|---|---|---|
|
AgNPs-1 |
30/0.1 |
90 |
20 |
|
AgNPs-2 |
30/0.5 |
70 |
10 |
|
AgNPs-3 |
30/1.0 |
80 |
15 |
|
AgNPs-4 |
30/1.5 |
60 |
10 |
|
AgNPs-5 |
30/2.0 |
50 |
5 |
| was deep red, leading to a substantial increase in absorbance intensity at 530 nm. b) Aqueous extract of CCB. | |||
Morphology and structural of synthesized AgNPs
The Fig. 2 shows the TEM micrographs of AgNPs before and after cysteine was added. Figure 2a shows polydisperse and hexagonal particles. When cysteine was added to the solution, the color of AgNPs changed from yellow to red, and they began to aggregate, as seen in Fig. 2b.
The AgNPs were capped with CCB,which also served as a reducing and stabilizing agent during the reaction.After adding cysteine, cysteine bonded to the surface of AgNPs via an Ag-S bond that was stronger than the interaction between AgNPs and CCB, and the metal-sulfur connection was strong enough to replace CCB and immobilize the thiol groups on the nanoparticles' surface, establishing a metal-complexing ligand [25, 26]. The NH2 and COOH from cysteine formed hydrogen bonds with AgNPs, causing them to aggregate [27, 28]. Meanwhile, the cysteine made a hydrogen bond with the AgNPs.
As shown in Fig. 2c, four planes of silver might be classed as diffraction circles in the selected area electron diffraction (SEAD) pattern (311, 220, 200, and 111) indicating that the nanoparticles formed were highly crystalline. Figure 2d shown the Malvern particle size distribution of AgNPs size about 90 nm after adding cysteine that was in a very narrow distribution, spanning from 200–400 nm in Fig. 2e. In general, which accords with the UV-vis spectrum analysis. Figure 2f shows the component of the AgNPs composite that was studied using energy-dispersive X-ray spectroscopy (EDS).The residual C and O elements were as from CCB, which capped the AgNPs throughout their action, while the Ag signal came from AgNPs. Because the capping CCB effectively prevented nanoparticle aggregation, its not strange that the particles re-distributed well in water. As a result, this approach is important for the pollution free reuse and recycling of AgNPs [29, 30]. The scheme 2 shows the mechanism of colorimetric cysteine detection with AgNPs.
Optimization of parameter detection of Cysteine
In this study, various parameters were optimized for cysteine determination, including the effect of pH, concentration AgNPs, stirring rate, and reaction time. Figure 3 shows the efficient defection of use at 1.5 µM concentrations of AgNPs and a pH of 7.0, stirring at 400 rpm for 15 min. Figure 3a shows by adding appropriate volumes of diluted HCl or NaOH solution to AgNPs after the addition of cysteine with different pH were obtained. The absorbance increased as the pH increased, as seen in Fig. 3a. As can be observed, poor absorbance at pH < 5.0 may be due to proton competition with AgNPs for cysteine association, whereas higher pH may be due to AgNPs being unstable [31, 32].As a response, pH 7.0 was accepted for further investigation.
Figure 3b shows the effect of different AgNPs concentrations in the range of 0.2–2 µM on cysteine determination using a colorimetric approach. The absorbance of cysteine increased when the concentration of AgNPs was raised up to 1.5 µM. Because of the rapid coagulation process of AgNPs after by settling on the bottom of the quartz cuvette, using larger concentration of AgNPs was not a fine decision [33]. Hence, for further study, an AgNPs concentration of 1.5 µM final concentration was employed to determine cysteine.
Figure 3c shows the using a magnetic stirrer, different stirring rates were investigated for determining cysteine for a chemical reaction surface area of AgNPs. For aggregation using cysteine and AgNPs, a stirring rate of 200–500 rpm was used for 5 min at pH 7.0. After decreased absorbance, raising the stirring rate up to 400 rpm enhanced the absorbance value of the analyte in UV-vis spectrophotometry [31]. For the purposes of the experiment, the stirring rate set to 400 rpm.
For the determination of cysteine from sample solution, the influence of reaction time was also examined in the range of 5–20 min. The reaction time is another important component that affects colorimetric readings. The mean color intensity increased progressively from 4–15 min and stayed constant at 15 min, showing that the AgNPs were etched within 15 min showed in Fig. 3d.
Sensitive Detection of Cysteine
AgNPs were incubated to solution containing varying concentration of cysteine (1000-10 µM). As the concentration of cysteine in a suspension of AgNPs was increased, the color of the solution gradually changed from yellow to deep red, as shown in Fig. 4a. Even the addition of a 100 mM cysteine solution resulted in a color shift that could be identified from the initial suspension.Modification in the absorption spectra of the AgNPs confirmed these results. As shown in Fig. 4b, increasing the cysteine concentration from 0–1 µM caused the AgNPs typical absorbance at 400 nm to gradually decrease (A400). At the same time, the additional absorption band at 530 nm was gradually increasing (A530). The amount of cysteine in the solution was measured using the ratio of 530 nm to 400 nm absorption (A530/A400). There was a good positive relationshipbetween both the absorption ratio as well as the cysteine concentration in the range of 10-1000 µM (Fig. 4c).
Selective detection of Cysteine
The significantsensor systems must be able to selectively identify their target analyte, because they must also be able to detect and transmit specific events that occur when analyte molecules interact [34, 35]. We carried out the same studies with 12 other amino acids the same under the conditions to demonstrate that the current method is suitable for developing a colorimetric detection platform for cysteine, including cysteine, proline, tryptophan, phenylalanine, histidine, serine, leucine, threonine, arginine, valine, and tyrosine, acetyl cysteine (NAc) and glutathione (GSH), two other thiol-containing molecules were also decreased. Only cysteine caused a noticeable color change from yellow to red in the AgNPs solution, as well as a significant increase in the absorption ratio (A530/A400) as shown in Fig. 5a UV-vis spectra and Fig. 5b photographic images. As a result of these findings, the current colorimetric technique based on AgNPs was found to have good selectivity for cysteine detection. The structural diversity of these compounds, as well as difference in their ability to bind with AgNPs, can explain this selectivity. Under the optimum experimental circumstances, amino acids that lack a thiol group are unable to bond AgNPs and hence cannot interfere with the cysteine determination process. Although NAc and GSH both have a thiol moiety capable of forming a silver Sulphur bond with AgNPs, they have a limited ion complexing ability.
Detection of Cysteine in Real Samples
Due to their high sensitivity and selectivity, the probes were also tested for their viability in colorimetric measurements of cysteine in serum. The color of the AgNPs gradually changed from yellow to red when the content of cysteine in the tested samples increased, as seen in Fig. 6. The colorimetric assay allowed for the rapid visualization of cysteine by comparison with a blank sample, allowing for the identification of cysteine at concentrations as low as 100 µM. We performed recovery experiment with serum samples spiked with 200 µM, 100 µM, 80 µM, 50 µM, and 10 µM of cysteine, respectively, to further confirm the efficacy of the proposed approach in terms of its application to real biological samples for cysteine determination. Table 2 summarizes the findings. The detection rate was within an acceptable range of 95.0–105.3%, indicating the methods reliability and applicability.
|
Samples |
Spikes (µM) |
Calculated (µM) |
Detection (%) |
|---|---|---|---|
|
1 |
200 |
204.2 |
102.1 |
|
2 |
100 |
105.3 |
105.3 |
|
3 |
80 |
83.4 |
104.25 |
|
4 |
50 |
51.7 |
103.4 |
|
5 |
10 |
9.5 |
95 |
In conclusion, using AgNPs as a chemical sensor, we produced a colorimetric probe for measuring cysteine that is highly selective and sensitive. CCB was found to have a high value usage method for synthesizing AgNPs for cysteine detection. Different reaction conditions were described when synthesized and well-dispersed AgNPs with aqueous extract of CCB as reducing and stabilizing agent.The results show that the amount of AgNPs was greatly influenced by the ratio of corn cob aqueous extract to AgNO3, but temperature and time had little effect.After centrifugation, the hexagonal AgNPs composite was capped with CCB and re-dispersed thoroughly in water. The AgNPs composite showed sensitive and selective detection of cysteine by watching the color change from yellow to red with naked eye.With a cysteine detection error of less than 5% in human serum, the present sensor has a lot of potential in the medical field. Therefore, this study presents not only a new avenue for the advantageous use of CCB aqueous extract, but also a novel AgNPs synthesis method that can detect cysteine easily, rapidly and selectively.
Author Contribution
This research was planned by C.R and T. P. Numerical simulation was performed by S.C.B.R did the experiments. The authors G.M and P. A discussed the results. C.R. and T. P. wrote the original manuscript.
Funding This work
Not applicable
Availability of Data and Material
The data generated by the simulations and experiments used in this work is not available to the public.
Code Availability
The codes for the current study are not available.
Declarations
Ethics Approval
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Consent to Participate
Not applicable.
Consent for Publication
Not applicable.
Competing Interests
The authors declare no competing interests.
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Scheme 1 and 2 are available in the Supplementary Files section
No competing interests reported.
- scheme1.png
Scheme 1. Representation of the green synthesis pathway leading to corn cob AgNPs for cystine detection.
- scheme2.png
Scheme 2. AgNPs aggregation hypothesis after cysteine addition.
