Localized surface plasmon resonance effect of 3-mercaptopropionic acid capped on silver nanoparticles for sensing probe application

Metal nanoparticles such as gold and silver are often used in various nanotechnology applications due to the localized plasmon resonance effect that occurs when incident photons interact with metal nanoparticles. 3-mercaptopropionic acid (3-MPA) is one of the capping molecules that can be provided for surface functionalization on gold or silver core because it has thiol and carboxylic group as a linking biomolecule, and this allows the use of nanoparticles for biosensor applications. In this study, we successfully synthesized silver nanoparticles capped by 3-mercaptopropionic acid (AgMPA) by use of two kinds of methods i.e ligand exchange and direct reduction, which reveal the unique characteristic plasmonic peaks of AgNP around 430 nm and 436 nm, respectively with spherical shape of AgNP and diameter size about 6-20 nm. The transmittance spectra from Fourier Transform Infrared (FTIR) measurement shows that S-H coordination from 3-MPA is invisible after 3-MPA attach on Ag core due to the strong coordination between Ag and the sulfur (S) ion, while the broaden peaks at 1589 and 1385 cm-1 for AgMPA are assigned to asymmetric and symmetric vibrations of carboxylate groups which overlap between carboxylate from citrate and 3-MPA. Finally, the colorimetric detection using AgCA and AgMPA as sensing probe was carried out by use of Biocytin-Avidin system molecule as analyte, which resulted in the colour changed rapidly from yellow to transparent-yellow and the red-shifted of absorbance spectra indicating the attachment of analyte to functionalized molecule of our synthesized AgCA and AgMPA.


Introduction
Metal nanoparticles are materials with dimensions of less than 100 nm, which have significantly contributed to the development of nanotechnology in various fields.Noble metal nanoparticles such as gold (AuNPs) and silver (AgNPs) have a unique optical response when interacting with electromagnetic waves in the UV and visible light spectrum.Light incident on metal nanoparticles induces collective oscillations of free electrons at specific resonance wavelengths, and it can increase the localized electromagnetic field intensity.The localized surface plasmon resonance (LSPR) phenomenon arises because light waves are trapped by materials with smaller dimensions in comparison to the wavelength of the incident light.LSPR is one of the methods used in optical transduction because it gives high accuracy due to highly sensitive.The resonant frequency of the plasmon oscillation depends on the material composition, geometry, dielectric, and the distance between the particles [1,2].
Among the metal nanoparticles, silver nanoparticles (AgNPs) show a significant extinction coefficient compared to other metals of the same size.In the application of biosensor, it has a great opportunity to be used as a sensor probe with rapid detection and high sensitivity.So far, the use of AgNPs as sensors has not been widely studied compared to the use of AuNPs because the synthesis of AgNPs is quite complicated and the selection of suitable organic capping materials is limited.In addition, AgNPs also show better antimicrobial characteristics than AuNPs [3].The silver ion can bind to tissue proteins and cause structural changes in the cell wall and nuclear membrane.This activity is related to antimicrobial characteristics in AgNPs [4].As a sensor, colloidal AgNPs can be utilized in colorimetry.In 2021, Hoang and co-workers researched a suitable capping agent for AgNP-based colorimetric platform to detect thiram pesticide in the aqueous environment [5].In 2021, Mahjub and co-workers studied the application of AgNP-based colorimetric bioassay to detect the amount of tobramycin in milk [3].
In usual, Ag has low resistance to oxidation, which occasionally results in instability.Capping ligands on metal NPs is needed for surface passivation.AgNPs need strongly adsorbed ligand molecules, preventing them from oxidation and providing long-term stability [5].A 3-mercaptopropionic acid (3-MPA) is an interesting capping material on metal nanoparticles because it has bifunctional groups i.e thiol and carboxylic end groups.Thiol can be firmly adsorbed on the metal surface via M-S bonds, which provides stability and makes them easy to functionalize.At the same time, carboxylate preserve electrostatic interactions and contribute to surface functionalization between metal nanoparticle and analyte [6].In our previous study, we used 3-MPA as capping for gold nanoparticles (AuMPA).Experimental and simulation results show strong adsorption between 3-MPA and the Au surface.The AuMPA can coordinate with biomolecules such as biocytin and streptavidin to be used as a biosensor probe [7].
In this research, we present a modified synthesis method of AgNP capped by 3-MPA which dissolved in water.We also optimize the concentration ratio of core Ag and capping 3-MPA to obtain stable AgNP colloid which can be further used for probe sensor applications.The optical characterization of AgNP solution was done by use UV-Vis spectroscopy while the chemical characterization was performed by FTIR spectroscopy.

Synthesis of silver nanoparticles
Direct reduction method.Firstly 2.5 mM AgNO3 in aqueous solvent was prepared in a three-neck roundbottomed flask and heated inside the heating mantle under reflux and stirring.When the temperature reached 85 o C, trisodium citrate mixed 3-MPA solution (1 wt%) was added dropwise as shown in Figure 1.The colour of the final solution is changed from clear to brownish yellow, then immediately transferred the product solution into an ice bath to prevent agglomeration.The AgMPA is then stored at 4 o C for further used.Ligand exchange method.The synthesis of AgNPs through the ligand exchange followed the method done by Pambudi et al [7].Firstly, Citrate-capped AgNP (AgCA) was prepared through the reduction method synthesis as describe in the previous method.In our experiment, the formation of AgCA was indicated by a change in colour from clear to brownish yellow.Next, 3-MPA with various concentrations was injected and stirred for 4 hours to make sure the exchange capping from citrate into 3-MPA on Ag surface as shown in Figure 2.

Characterization
The optical characteristic of AgNPs was obtained through UV-Vis spectrometer, while the morphology and size of AgNPs were obtained through TEM image.FTIR spectrometer in transmission mode were used along the experiment to conform citrate and 3-MPA on the surface of Ag core and the samples were evaluated over the wavelength range of 700-3500 cm -1 .

Result
In our experiment, AgCA is synthesized using the chemical reduction method by use of trisodium citrate as reducing and stabilizing agents.The reaction between AgNO & and C ( H * Na & O , • 2H / O can be expressed as follows: In this reaction, some sodium citrate reduces Ag ions from Ag + to Ag 0 , and the rest is physically adsorbed on AgNPs surface.The adsorption of citrate has a role in stabilizing AgNPs by providing a particle surface charge [8,9].AgNPs are known to have maximum absorption peaks in the 390-500 nm range [10].Figure 3  In the case of ligand exchange method, the addition of 3-MPA solution into AgCA solution is intended to replace the capping ligand from citrate to 3-MPA because the sulphur atom has relatively higher binding affinities to the Ag surface than citrate.The addition of 3-MPA solution into AgCA with varied concentration of 4-16µM show a significant red-shifted in the absorbance peaks which indicate the formation of AgMPA after ligand exchange as shown in Figure 4. Addition of 3-MPA solution at low concentration reveals a slight shifted to long wavelength around 12 nm.Meanwhile, at a high concentration of 3-MPA, about 50 µM, the optical characteristic of AgMPA cannot be detected from the visual colour change and from absorbance spectra.The main reason of LSPR peaks shifted caused by the slightly change of refractive index of the organic molecules on surrounding medium of Ag core.Since the experiment was done in room temperature without the involvement of catalysis, the result of exchange capping molecule is not perfectly covering all the area of Ag core, which resulted in quite small peak shifted.The TEM image of AgMPA by use of ligand exchange method in Figure 4 shows nanoparticles have a spherical shape with a 6-10 nm diameter size.In direct reduction method, we used the simultaneous addition of stabilizer (3-MPA) and reductant (trisodium citrate) to reduce Ag completely and to obtain monodisperse AgMPA.We followed the previous method from Yonezawa and Kunitake et.al and we developed the modified method in our laboratory [12].Figure 5 shows the absorbance spectra of AgMPA using the direct reduction method with varied concentrations of 3-MPA.The red-shifted of the plasmonic peaks are clearly shown along with the addition of 3-MPA concentration from the wavelength of 418 nm (AgCA) to 420-436 nm.Absorbance spectrum at 18µM concentration of 3-MPA has a broad shape which indicate the aggregation of AgMPA while the absorbance spectra of 10µM, 13µM, and 15µM of 3-MPA show sharp plasmonic peaks of AgNP characterisitic.The TEM image of AgMPA by direct reduction method has a spherical shape with a 10-20 nm of diameter size.In the direct method the starting material of capping are citrate and 3-MPA which may cause the competition in rapid nucleation and growth process of AgNP formation.The LSPR peak shifted is more clearly shown in comparison to the exchange ligand method.The coordination of citrate and 3-MPA on Ag can be evaluated from FTIR spectra as shown in Figure 6.The peaks of carboxylate asymmetric stretching (vas (COO-)) and symmetric stretching (vs (COO-)) from citrate appeared at 1591 cm -1 and 1395 cm -1 , respectively.In the case of citrate on AgCA and AgMPA the vas (COO-) is slightly shifted to low-frequency (1589 cm -1 ) than the peak in citrate with the same tendency also for vs (COO-), where the peak is found to have a low-frequency shifted (1385 cm -1 ) but it is looked sharper than the peak in reference's citrate.The peak intensity of vs on AgNP is stronger than vas can be explained by ionic bond formation between carboxylate and Ag (anchoring by two oxygens) [11].In the low-frequency region, the peak intensity of C-O stretching from 1279 -1080 cm -1 and C-H stretching from 949 -756 cm -1 are very strong in citrate.In contrast to previous research, it was identified that the bond between AuNP and citrate was covalent.In AgCA, the bonding between AgNP and citrate is an ionic bond with a symmetric stretching direction COO-perpendicular to the surface of the metal nanoparticle [12].On the other hand, for the AgMPA, the chemical coordination between Ag and 3-MPA reveals a strong bonding between the sulphur and Ag core, similar to Au.The S-H coordination at 2569 cm -1 in 3-MPA is invisible in the FTIR spectrum of AgMPA due to the binding of thiol group on Ag.It has been known that the peak at 1713 cm -1 for 3-MPA belongs to the carbonyl group of carboxylic acids.The broaden peaks at 1589 and 1385 cm -1 for AgMPA are assigned to asymmetric and symmetric vibrations of carboxylate groups which overlap between carboxylate from citrate and 3-MPA.In this case, the carboxylate from citrate is attached directly on Ag while carboxylate from 3-MPA is located opposite side from S-Ag.Since we use the methods which both involving the trisodium citrate in the synthesis process, we cannot determine exactly the full coverage of 3-MPA on Ag.The detail band assignment of appearance transmittance peaks for AgNP in comparison to the free citrates and 3-MPA is shown in Table 1.The application of AgNPs as a colorimetric sensing probe was carried out by introducing Biocytin-Avidin as analyte.Avidin has four subunits with high affinity toward Biotin derivative molecules, so it can produce cross-link aggregation and it produces colour change in the solution since the refractive index of medium on organic capping is slightly different.The detection is done by incubating AgCA solution with Biocytin at room temperature and then purification was carried out using the centrifugation method.Next, the analyte (Avidin) were captured on Biotin side with varying concentration.In our case, the treatment of EDC/NHS is should be done previously into AgMPA solution to release the H ion from carboxylic acid part and then it will form carboxylate ion and ready to capture the Biocytin-Avidin system on the surface of AgMPA.The final solution shows a slight colour change from yellow to transparent yellow as shown in Figure 7.In contrast, the addition of Biocytin-Avidin to AgCA with the same concentration does not show a significant colour change even though the Avidin concentration is increased as shown in Figure 8.This situation can be explained by the formation of AgCA's aggregate during the centrifugation process.In addition, the insignificant colour changes in the colorimetric sensor based on AgCA may come from the low concentration (the small number of particles) in final product of AgCA solution.

Conclusion
The synthesis of AgMPA by use of ligand exchange and direct synthesis methods have been successfully done which show the plasmonic peaks at 430-436 nm together with spherical shape and diameter size about 6-20 nm.The coordination of the capping materials.i.e citrate and 3-MPA on Ag core have been evaluated by FTIR characterization which indicating by disappearance of S-H bond in Ag-MPA when 3-MPA adsorb on Ag surface, while the broaden peaks at 1589 and 1385 cm -1 for AgMPA are assigned to asymmetric and symmetric vibrations of caboxylate groups which overlap between carboxylate from citrate and 3-MPA.On the other hand, AgCA and AgMPA as a colorimetric sensing probe were done by introducing Biocytin-Avidin as analyte.The addition of Biocytin-Avidin to AgMPA shows a slight colour change from yellow to transparent-yellow and the slight change of refractive index of medium on capping area of Ag indicated by redshifted of plasmonic peaks which is strengthening the reason of adsorption of biocytin-avidin on carboxylate capping on both AgCA and AgMPA.

Figure 2 .
Figure 2. Mechanism of ligand exchange from AgCA to AgMPA.
(a)  shows the formation of AgCA solution with yellow colour and it has plasmonic peak at 418 nm.TEM images, ini Figure3(b), shows the spherical shape of AgCA with diameter size distribution about 8-14 nm.

Figure 3 .
Figure 3. (a) UV-Vis spectrum of AgCA (insert: solution of AgCA) and (b) TEM image of AgCA with scale bar 50 nm.

Figure 6 .
Figure 6.FTIR spectra of AgCA and AgMPA in comparison to the free capping molecules (citrates and 3-MPA).

Table 1 .
Band assignment AgCA and AgMPA from FTIR measurement.