Abstract
Smart biocompatible materials that respond to a variety of external stimuli have a lot of potential in the creation of low-cost diagnostic biosensors. The present work describes the creation of core–shell nanoparticles as a biosensor for smart enzyme detection of salivary alpha-amylase (sAA). A chitosan-tripolyphosphate core was generated via ionic gelation and was coated with a starch–iodine shell to create biocompatible core–shell nanoparticles. The starch–iodine shell was ruptured in the presence of certain amounts of amylase, exposing the core. This application explains a noticeable color change from blue to white that can be used to identify sAA at the point of care. Synthesized nanoparticles were examined for scanning electron microscopy analysis and energy-dispersive X-ray (EDX). An EDX report reveals that the nanoparticles have higher carbon content at 55% followed by an oxygen atom of 35%. Fourier-transform infrared spectroscopic analysis revealed that the core–shell nanoparticles have carbonyl (C═O) functional groups present. A confirmatory test of amylase reaction on nanoparticle-impregnated paper turns blue to white indicating that the nanoparticle reacts with amylase as an indicator. This paper-based method can be used in future applications in forensic and medical applications.
1 Introduction
Sensor devising has been widely applied in various platforms in recent years, which are mostly stimulus-responsive smart materials. These sensors are made for detecting pH, enzymes, DNA, temperature, electric impulses, and other biomolecules in trace amounts (1). Based on the existence of certain chemicals, the smartness of the materials involved can be employed in smart nanoparticles for diagnostics (2,3). Smart materials are being extensively investigated in the theme of biosensors and detectors to leverage these changes in characteristics caused by changes in stimulus, for use as actuators and sensors, or self-assembly methods (3). Fluorescence-based magnetic (Fe/Fe3O4) core–shell nanoparticles, for example, have been shown to work as nano biosensors in cancer diagnostics to detect protease activity in biospecimens (4). In other experiments, biocompatible nanoparticles such as liposomes were mixed with standard labels such as gold nanoparticles. The use of liposome-gold nanodot hybrids for phospholipase-C biosensing was described, which led liposomes to hydrolyze membrane phospholipids, generating byproducts that boosted fluorescence quenching in the oxygen-rich environment as well to enhance the stability of gold nanodots (5). In addition, a recent example described the application of core–shell nanoparticles as biopolymer assembly to construct a device for detecting antimicrobial resistance by observing changes in starch–iodine chemistry in the presence of beta-lactamases (6).
Salivary alpha-amylase (sAA) (EC 3.2.1.1), also known as 1,4-alpha-d-glucan glucanohydrolase, is an enzyme in the oral cavity that aids in the breakdown of starch into simple sugars (7). sAA has a pH value of 7, which coincides with the normal pH of saliva which ranges from 6.4 to 7.0. sAA is secreted by the salivary gland an exocrine gland which is secreted based on autonomic nervous stimuli. sAA has a moderate role in glucose regulation in blood next to that of glucagon hormone through the digestion of carbohydrates in food. Apart from its involvement in carbohydrate digestion, sAA is also used as a biomarker to identify changes in sympathetic nervous system (SNS) activity caused by stress (7). The SNS responds very instantly in the presence of a stressor, while the hypothalamus pituitary adrenal system axis is activated after a time lag (8,9). In addition to forensic biology, point-of-care amylase detection is being used in foods and drinks (honey, malt, eggs), as well as detergents and textiles (10).
Salivary amylase is an enzyme that is putative evidence for many forensic investigations at crime sites since amylase enzyme is very sensitive and easy to detect by biosensors or any other electronic detection system even in trace amounts (11). The current study describes the creation of smart core–shell nanoparticles that can detect the presence of amylase in saliva for stress diagnosis and forensic applications. The use of smart materials on paper-based point-of-care devices to detect amylase for the aforementioned applications is discussed in this study. The goal of this research is to create enzyme-responsive core–shell biocompatible nanoparticles which can be an alternate method to detect amylase enzyme in saliva rather than implementing electronic and other biochemical tests.
2 Materials and methods
2.1 Materials
All the necessary chemicals and reagents used in the present study are purchased from Sigma-Aldrich, Hi-Media Labs. All the chemicals used are of analytical grade (AR). The low molecular weight chitosan (7,000 kDa) was used in the present study. Soluble starch, potassium iodide, iodine, glacial acetic acid, sodium hydroxide, and sodium tripolyphosphate (TPP) were procured from Sigma-Aldrich. Food-grade dye was obtained from an essence shop (Chennai). Whatman qualitative chromatography paper grade 1 was purchased from Whatman, India. MilliQ water was used for the experimental reactions. All the tissue culture plates and flasks used were sterile and clean.
2.2 Preparation and characterization of core–shell nanoparticles
Chitosan core–shell nanoparticles were synthesized following the earlier methodology (12) with slight modifications. In brief, 1 mg·mL−1 of chitosan solution in 1% acetic acid was prepared along with a hydrophilic red-colored dye which acts as a color change indicator. About 10 mL of TPP solution was added into 30 mL of chitosan solution dropwise at room temperature for 15 min. This brings an ionic gelation process spontaneously with TPP solution to form chitosan-TPP pellet nanoparticles which can then be separated by centrifugation at 5,000 rpm for 10 min. A resuspension agent of soluble starch solution (5 mg·mL−1 in 1 N NaOH) mixed with Lugol’s iodine (5:1) was used for pellet resuspension after the centrifugation process. The resuspended nanoparticles were then vortexed and sonicated for 2 min. The core–shell nanoparticles synthesized were centrifuged again for 10 min under 6,000 rpm and the pellets were dispersed in 2 mL of deionized water, followed by sonication for 2 min. The resulting pellets were then stored at 4°C till further use. The physicochemical characterization of nanoparticles was done using Fourier-transform infrared (FTIR), Scanning Electron Microscopy (SEM) (JOEL JSM-7600F), and UV-visible (UV-Vis) analysis. The plausible nanoparticle coating was achieved using electrostatic affinity binding of positively charged nanoparticles in red suspension with that of the negatively charged starch–iodine complex which occurs as blue suspension.
2.3 SEM, field emission scanning electron microscope (FE-SEM)-energy-dispersive X-ray analysis (EDAX), FTIR, and UV-Vis spectroscopic analysis of the synthesized core–shell nanoparticles
Suspended core–shell nanoparticles were subjected to UV-Vis spectroscopy absorbance (Shimadzu, Japan) between 350 and 600 nm (13). FTIR spectroscopy (Shimadzu, Japan) was performed on lyophilized samples to determine the functional groups present as a result of chemical reaction in starch–iodine complex in whole nanoparticles. Field emission scanning electron microscopy (FE-SEM) (JOEL JSM-7600F) analysis was performed to assess the surface morphology of the prepared core–shell nanoparticles. It was done in the maximum magnification range from 20× to 30,000× with a spatial resolution of 50–100 nm. Elemental analysis in core–shell nanoparticles was assessed through a (FE-SEM) fitted with EDAX (Hitachi S-4700 FE-SEM).
2.4 Application of the nanoparticles on a paper-based format
Detection of sAA using core–shell nanoparticle-impregnated Whatman filter paper grade 1 was employed based on the methodology of Yetisen et al. (14) with slight modifications. In brief, 5 µL of synthesized core–shell nanoparticle suspension was placed on Whatman filter paper and made dry. Following this, 5 µL of the sample was added, which was then sealed and blocked by suitable membranes to avoid the iodine loss. The adhesiveness was enhanced by a scotch tape backing membrane of 200 GSM (grams per square meter) of glossy paper. Whatman filter paper (1 cm × 6.5 cm) with an air-dried nanoparticle suspension was placed on blotting paper for saliva absorption. The change in color due to an enzyme reaction was observed after 15–20 min as a confirmatory test.
2.5 Application of alpha-amylase for the detection of saliva on the paper-based test
Nanoparticles were put to widely used Whatman Qualitative Grade 1 filter paper to create an economical point-of-care device, and the same was evaluated using different amylase concentrations on paper (15). In brief, 5 mL of the nanoparticle suspension was applied to the filter paper and left to completely dry. After that, 5 mL of the sample was added. The color changes were noticed from 5 min to 1 h later. It was discovered that 10 min was the ideal amount of time for results to be observed, and this time frame was also employed for subsequent investigations on 12 sAA concentrations (for each concentration; total n = 60). The transition of the color from blue to white was regarded as a positive reaction. The paper remains blue in the absence of alpha-amylase.
3 Results and discussions
3.1 Making of nanoparticles, tube-based format, and physicochemical characterization of core–shell nanoparticles
The nanoparticles were obtained and lyophilized. Varying concentrations of alpha-amylase (0.5, 1, 1.5, and 2 mg) in 10 mL of water alongside the core–shell nanoparticles and starch–iodine result in varying color change reactions (Figure 1). It was noticed that the color change is observed and the adsorption of core–shell nanoparticles with alpha-amylase increases with an increase in the concentration of alpha-amylase. The different absorption and color change samples are taken for SEM, EDAX, and UV spectroscopic analysis.
(a) Core–shell nanoparticles and (b) tube-based adsorption study.
The nanoparticles obtained, consisting of a core–shell, were spherical and size range of 90 μm as seen through an electron microscope (Figure 2a). Core–shell nanoparticles imparted unevenness to the surface (Figure 2b). This could be due to the addition of alpha-amylase. In a similar study, the size range of nanoparticles was found to be 200–250 nm. The core–shell prepared as chitosan-TPP nanoparticles by ionic gelation was seen as uneven to the surface (16).
SEM showing core–shell nanoparticles: (a) aggregated nanoparticles and (b) enlarged nanoparticles.
3.2 FESM-EDAX analysis
Core–shell nanoparticles subjected to FESM-EDAX reveal the surface scan and its overall elemental composition at various energy dispersion levels (Figures 3 and 4). FESM-EDX analysis of the synthesized core–shell nanoparticles reveals that the major elements present are carbon, oxygen, and iodine at major peak and abundance values (Table 1). The sum of the atomic percentages should add up to 100%. The table shows that the sample contains primarily carbon (55.01%) and oxygen (35.27%), with smaller amounts of potassium (4.17%), iodine (4.95%), phosphorus (0.35%), aluminum (0.18%), and chlorine (0.07%). EDX results of the present study correlate with prior alginate-coupled chitosan nanoparticles with the same elemental composition through EDX analysis, which is efficiently tested as nano drug delivery against an in vivo antidiabetic model (17).
(a) Electron microscopic image of FESEM with EDX showing the chitosan core and the core–shell nanoparticle. (b) Enlarged portion of the nanoparticle in the EDX region for elemental analysis.
The FESM-EDX pattern of the sample with O as its highest peak and I as its lowest peak.
Elemental concentration in the core–shell nanoparticle by EDAX dispersive analysis
| Element | Line type | Weight (%) | Weight (% sigma) | Atomic (%) |
|---|---|---|---|---|
| C | K series | 32.48 | 0.44 | 55.01 |
| O | K series | 27.73 | 0.28 | 35.27 |
| Al | K series | 0.23 | 0.04 | 0.18 |
| P | K series | 0.54 | 0.04 | 0.35 |
| Cl | K series | 0.12 | 0.03 | 0.07 |
| K | K series | 8.02 | 0.09 | 4.17 |
| I | L series | 30.88 | 0.26 | 4.95 |
| Total | 100.00 | 100.00 |
3.3 FTIR spectroscopic analysis
FTIR was performed on the lyophilized sample. In the above FTIR graph (Figure 5), we observe peaks or bands corresponding to the different functional groups of the sample. For instance, a peak near 3,257 cm−1 indicates the presence of C–H bonds in the sample, while a peak near 1,638 cm−1 indicates the presence of a carbonyl group (C═O) in the sample. The spectral peak values of 2,924 cm−1 indicate the C–H stretching plane of the alkane group, those of 1,638 cm−1 indicate C═C stretching, and those of 1,543 cm−1 indicate the presence of strong N–O stretching which are indicators of the chitosan functional group present in the core–shell nanoparticles. Out-of-plane bending moments are those which are caused by out-of-plane forces (18). The shape and position of the peaks provide information about the molecular structure and chemical composition of the sample. A sharp peak indicates a strong absorption, while a broad peak indicates a weaker absorption due to multiple overlapping peaks.
FTIR analysis of the core–shell nanoparticles.
3.4 Spectroscopic analysis of nanoparticles in a tube-based format
The UV-Vis analysis was carried out from 200 to 700 nm (Figure 6). The above UV-Vis analysis shows results similar to the results found by Adhikary and Banerjee (16), where the distinct peaks appear at 375 and 549 nm for the core–shell nanoparticles while the red food dye displayed a peak at 507 nm. The 375 nm peak was attributed to the shell of the nanoparticles containing Lugol’s iodine, which shows an absorption peak at 355 nm due to triiodide ions. The interaction with other components of the nanoparticles led to a red shift of the peak to 375 nm as has been described for other polymers as well. The addition of amylase led to an increase in the amount of free triiodide resulting in an increase in the absorbance peak at 375 nm. The corresponding release of red dye from the core showed an increase in the absorbance at 507 nm. Thus, in addition to amylase, the nanoparticles obtained had characteristic absorbance peaks at 375 and 507 nm. The transition of blue to red on exposure to sAA was thereby confirmed through spectroscopic analysis. As explained previously, the iodine-bound shell of starch in the core–shell nanoparticle leads to the formation of tri-iodide anion complexes at the helices of starch producing an intense blue/purple color. This color disappears with the degradation of starch by sAA. This color change can be further observed by the naked eye or through image analysis. Thus, the addition of amylase led to a gradual reduction in the blue color and an increase in the red color. The above UV-Vis analysis shows clearly that the absorption value increases with an increase in the amylase concentration.
UV-Vis analysis of (a) blank solution, (b) chitosan-TPP nanoparticles, (c) starch–iodine complex, and (d) core–shell nanoparticles.
3.5 Development of the paper-based device
In the present study, the nanoparticle-coated paper method is developed to detect the presence of alpha-amylase. As shown in Figure 7, we observe two images: Two nanoparticle-coated SI papers were placed, in which, the first paper shows the negative result where we do not observe any color change (Figure 7a). In the second image (Figure 7b), we observe a color change of white color from blue color as indicated by an arrow. In brief, 0.5 mL of saliva was added to the paper. The starch iodine complex broke and led to the white color. The shell ruptures revealing the core, indicating the color from blue to white. A closer look can be seen in Figure 7b. This confirms the presence of alpha-amylase in the paper.
Paper-based device for detection of alpha-amylase in saliva samples: (a) SI-coated paper strip and (b) nanoparticle-coated paper strip showing reduction of color change indicated by an arrow.
Paper-based techniques are ready-to-use and portable techniques to detect biological samples since the twenty-first century. This technique is cheap and effective rather than conventional instrument-based detection (19,20). These paper-based nanoparticle-impregnated detection kits are easily monitored and quantified through mobile phone applications in modern days, especially in monitoring environmental trace hazards and aerosol pollution (21). This sAA paper-based detection kit is further ensured for its stability to avoid leaching and contamination due to the presence of Lugol’s iodine in the starch shell in the detection of sAA.
3.6 Advantages and limitations of the smart core–shell nanoparticle sensors
The core–shell nanoparticle sensors are an advanced replacement rapid technology instead of using antibodies and immune fluorescent dye markers in detecting biomolecules in unknown samples. The most intriguing nanoparticles available in use for such biosensors are chitosan, TPP, and poly-vinyl alcohol, which are widely used in the development of smart core nanoparticles in the detection of food spoilage, DNA, and other body fluids (22). Apart from that core–shell nanoparticles like titanium dioxide, aluminum oxide, and zinc oxide are successfully trailed as nano pesticide formulations (23). Trace elements like copper, zinc, manganese, and iron nonmaterial are used as nano fertilizers for agriculture, especially in micro irrigation (24). Nanomaterials are also employed to ensure agricultural parameters like soil quality and water quality checking (25). In the present study, these smart core–shell nanoparticles are stable and robust and can be modified to detect various concentrations of sAA and this technology can be made ready to use in the form of a paper-based kit and swab (24). This technology will be a non-invasive method of detecting sAA in any biological samples by visualizing instant color change from blue to white as a result of the enzymatic reaction and nanoparticle detection in a few minutes. This technique replaces the earlier salivary detection through some instruments like desktop scanners and other expensive chemical tests. As a further development, this technology is formulated to detect several biological fluids like serum and DNA for better prototype product development which can be the best alternative source for routine usage of biochemical and instrument-based methods in the detection of biological samples.
4 Conclusion
The present work envisaged the core–shell nanoparticles based on stimulus oriented, which is feasible to be applied as biosensor agents in the form of sAA detection in the form of a paper-based device. This is a ready-to-apply technology to detect saliva samples in the forensic and medical fields. As a further outstretch, this technology will be updated with further advancements to be applied on several biological samples in terms of blood, pH, DNA, and other enzymes as well as much more in agricultural applications to make it as easy to use as a rapid technology for multiple biological, medical, and forensic applications instead of using instrument and antibody-mediated techniques which is very expensive.
Acknowledgment
Authors thankful to the Princess Nourah Bint Abdulrahman University Researchers Supporting Program (PNURSP2024R356) Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia for providing financial grant.
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Funding information: This research was funded by Princess Nourah Bint Abdulrahman University Researchers Supporting Program (number: PNURSP2024R356), Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia.
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Author contributions: Kumaravel Kaliaperumal, Kumaran Subramanian: writing – original draft, writing – review and editing, formal analysis; Akshara Seenivasan, Renitta David: experimental work up, results validation; Nahaa Miqad Alotaibi, Modhi Obaidan Alotaibi, Nawaf Alshammari, Mohd Saeed: data interpretation, project administration, funding support.
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Conflict of interest: The corresponding author (Kumaran Subramanian) is a Guest Editor of the e-Polymers’ Special Issue “Biodegradable and bio-based polymers: Green approaches” in which this article is published.
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Data availability statement: All data generated or analyzed during this study are included in this published article.
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