Colorimetric Sensor for Cr(VI) Ion Detection in Tap Water Using a Combination of AuNPs and AgNPs

Colorimetric sensors are a promising technique for the simple screening of water, food, and environmental samples contaminated with interferents, allowing for color changes to be observed with the naked eye or a spectrophotometer. In this study, a colorimetric sensor for the selective detection of hexavalent chromium ion (Cr(VI)) contamination in water was developed. A combination of sodium borohydride-coated gold and citrate-capped silver nanoparticles (Na-AuNPs/cit-AgNPs) was employed as a colorimetric probe. Upon the addition of Cr(VI)-contaminated tap water in the colorimetric probe solution, the color sequentially transitioned from its initial orange to dark reddish-purple, dark purplish-red, dark blue-violet, and gray. This colorimetric strategy relies on AgNP dissolution and AuNP aggregation in the presence of the Cr(VI) ions. The dissolution of AgNPs is evidenced by the reduction of the characteristic peak of AgNPs at 400 nm, while the aggregation of AuNPs leads to a red shift in the absorption band at 514 nm, accompanied by broad absorption in the 500–700 nm range. The limits of detections were found to be 22.9 and 50 ppb using a spectrometer and by visual observation, respectively. The synthesized AuNPs and AgNPs are very stable in the presence of media containing complicated ions. We further demonstrated the practical applicability of the developed system for detecting Cr(VI) in real samples, including natural water and artificial urine, highlighting its potential for addressing Cr(VI) contamination in practical scenarios.


Details of AuNPs synthesis
In this study, AuNPs stabilized with two different capping agents (NaBH 4 and TSC) separately prepared.The NaBH 4 capped-and TSC capped-AuNPs were denoted as Na-AuNPs and cit-AuNPs, respectively.Na-AuNPs were synthesized via reduction reaction method using NaBH 4 as a reducing agent.Briefly, freshly prepared NaBH 4 solution in ice-cold water (0.1 M) was rapidly added in 0.25 mM HAuCl 4 aqueous solution (20 mL).The solution was stirring for 10 min to complete the reduction reaction.Moreover, The AuNPs capped with TSC were synthesized with a similar protocol to previous method with additional of TSC.To do this, 5mg TSC was added to HAuCl 4 aqueous solution followed by adding NaBH 4 solution.Both Au colloids were left for overnight at room temperature before use.
Particle size of Na-AuNPs can be altered by varying volume of gold precursor.In this study, four different sizes of AuNPs were prepared and the volume and concentration of each reagent were listed in Table S1.

Details of cit-AgNPs synthesis
In this study, AgNPs stabilized with two different capping agents (NaBH4 and TSC) were prepared.
For Na-AgNPs synthesis, as-prepared NaBH4 aqueous solution (0.1 M, 0.3 mL) was rapidly injected in 0.3 mM AgNO3 aqueous solution (10 mL).To complete the reaction, the mixture was continuously stirred at 700 rpm for 10 min.The solution color changed from brown to light yellow.In case of cit-AgNPs synthesis, cit-AgNPs were prepared by reducing silver salt with TSC, which acts as both reducing and capping agents.Briefly, 0.368 mL of 10 mM AgNO3 aqueous solution was mixed with 23.75 mL DI water.Then, the solution was boiled (100 C) and stirred at 700 rpm for 10 min.Freshly prepared 1.25 mL of 1% TSC aqueous solution was quickly injected.The color was developed from clear to light yellow after continuously boiling for 30 min, confirming the formation of AgNPs.The colloidal solution of both Na-AgNPs and cit-AgNPs was stored in refrigerator before used.
Moreover, the particle size of Na-AgNPs can be tuned by varying volume of silver precursor.The volume and concentration of each reagent were listed in Table S2.

Synthesis of Na-AuNPs at five different concentrations
As the 5.93 nm-Na-AuNPs was selected as optimum particle, this size was quite small for centrifugation for variation of AuNPs concentration.The concentration of AuNPs was varied by varying the volume of all precursors as shown in Table S3.The concentration of Na-AuNPs colloid was observed by measuring optical density using spectrophotometer.Fig. S2 exhibits the extinction spectra and inset (on the right) shows the corresponding photographic images of all Na-AuNPs at five different concentrations.These five concentrations of Na-AuNPs gave similar LSPR at the wavelength of 514 nm.The optical density (OD) varied from 0.30, 0.58, 0.87, 1.37, and 1.76.From photograph image, color intensity of probe colloid increased as increased concentration of Na-AuNPs.

Optimization Na-AuNPs:cit-AgNPs combination for Cr(VI) ion detection in TW
To achieve a suitable probe colloid for highly sensitive detection of Cr(VI) ion contaminated TW, the optimization of Na-AuNPs:cit-AgNPs combination including particle size of Na-AuNPs and cit-AgNPs, optical density of Na-AuNPs, ratio between Au:Ag, and volume of Cr(VI) ion in tap water was studied.
First, it is well known that particle size of metal NPs strongly influence the aggregation behavior of NPs in the solution, which directly affected the sensitivity and LOD of colorimetric probe.
In this study, different particle sizes of Na-AuNPs  Consequently, the 40.43-nm cit-AgNPs were selected as the optimal size for further investigation.In case of small volume of Cr (VI) addition (Figure S6a), the probe color developed from orange to dark red, purple, blue, an grey color when reacted with high Cr(VI) concentration (0.5 to 100 ppm).This indicates that the small amount of analyte may not be sufficient to elicit a detectable color difference by naked eyes.For large volumes of Cr(VI) solution (50, 75 µL) (Figure S6c and d), the color of probe colloid changed to dark purple and grey color when mixed with Cr at concentrations ranging from 0 to 5 ppm and 10 to 100 ppm, respectively.This higher Cr(VI) amount can suddenly induce the aggregation of the probe for entire detection range.Therefore, this excess Cr content was not suitable for this developed colorimetric probe.When 35 µL of Cr(VI) solution was added to the probe colloid (Figure S6b), multicolor transformation from orange to dark red, purple, blue and finally greygreen color was found for Cr (VI) detection in the range of 0.05 to 100 ppm.Additionally, the LSPR responses among each Cr(VI) content in the detection probe were also investigated.Broadening of the Na-AuNPs and reduction of the cit-AgNPs were observed under all detection conditions.With increasing Cr(VI) volume, the absorption peak at longer wavelengths (> 600 nm) also increased.This is caused by the aggregated AuNPs induced by high content of Cr(VI).This result is in good agreement with the result of color development from photograph.According to the result, the suitable Cr(VI) volume of 35 µL showed a favorable system to visual detection process, which was chosen as an optimum Cr(VI) content for further investigation.Upon the addition of Cr(VI) solution, a wide absorption band with a shoulder peak at longer wavelengths was observed for Na-AuNPs (Figure S7a).The presence of LSPR band at higher wavelength confirmed the formation of Na-AuNPs aggregation in the solution.The sensitivity of this probe colloid could not be enhanced by increasing the amount of Na-AuNPs.In the case of probe colloid with Au:Ag volume ratio of 1:1, the color of the probe obviously developed from orange to dark purple and grey when reacted with high Cr(VI) concentration of 5 to 100 ppm.While the color of probe in the control TW was comparable to probe reacted with Cr(VI) at concentration of 0 to 1 ppm.Moreover, two characteristic LSPR bands of both cit-AgNPs and Na-AuNPs were clearly observed at 400 and 514 nm, respectively (Figure S7c).The absorption band of both cit-AgNPs and Na-AuNPs gradually decreased with increasing Cr(VI) content (0 to 1 ppm).Subsequently, the LSPR peak vanished when exposed to high concentrations of Cr(VI) (5 to 100 ppm).This optical property gave a good agreement with the corresponding color transformation.This probe colloid gave a narrow visual detection range at high Cr(VI) content.For probe colloid prepared by mixing Na-AuNPs and cit-AgNPs at 3:1 volume ratio (FigureS7b), the multicolor evolution (original orange to reddish purple, dark purplish red, dark blue violet, and grey color) with wide visual detection range from 0 to 100 ppm Cr(VI) concentration was observed.The LSPR peak of Na-AuNPs gradually decreased with wide absorption wavelength as increased the Cr(VI) concentration (0 to 10 ppm).Subsequently, the characteristic LSPR bands of both AuNPs and AgNPs disappeared and addition absorption band at about 345 nm was found when reacted with high Cr(VI) concentration at 50 to 100 ppm.This probe system exhibited a correlation between the spectral changes in AuNPs bands upon the addition of Cr(VI), potentially enabling quantitative determination of Cr(VI).Furthermore, it achieved high sensitivity for Cr(VI) detection by visual observation.Considering all optimization results, the suitable detection system comprises (1) a probe colloid consisting of a mixture of 5.93-nm Na-AuNPs and 40.43-nm cit-AgNPs at a fixed volume ratio of AuNPs:AgNPs of 3:1, and (2) an optimum Cr(VI) volume of 35 µL.These parameters were employed for further analytical performance studies.

Electrochemical measurement.
Electrochemical measurement was carried out using screen-printed carbon electrode consisting of carbon based working and auxiliary electrodes and Ag/AgCl reference electrode (SPCE, 100TE, Zensor) was selected as a disposable electrode.A 50 µL colloidal solution of the probe was applied directly to completely coat the electrode.Differential pulse voltammetry was recorded with portable potentiostat (electrochemical analyzer (ECAS 100), Zensor) incorporated with ECA100 software at scan rate of 50 mVs -1 .Differential pulse voltammograms were collected under ambient environment at room temperature.Figure S9 depicts the spectral response of the probe colloid for entire measurement range of Cr(VI) detection (0 to 100 ppm).Tap water was used as a solvent in this study.The spectral shape changes by altering the amount of Cr(VI) content.

Figure S9
The corresponding extinction spectra of the probe for entire measurement range of Cr(VI) detection from 5 ppb to 100 ppm performed in tap water.

Extinction spectra of selectivity study.
Figure S10 shows the extinction spectra of probe colloid in the presence of various heavy metal ions (Pb, Cd, Ni Cu, Zn, Fe, Mn, Hg, As, Cr(III)) compared to Cr(VI) ion.A wider absorption band was only observed for probe colloid reacted with Cr(VI).Whereas extinction spectra of probe colloid reacted with other heavy metal ions gave similar result to the control sample of tap water.

Figure S10
The corresponding extinction spectra of selectivity study.The concentration of all interfering ions was fixed at 0.1 ppm.

Extinction spectra of the Na-AuNPs and cit-AgNPs upon storage
The stability of individual Na-AuNPs and cit-AgNPs was separately investigated using UV-Vis measurement.Figure S11a   The feasibility of determining chromium levels in various real samples, such as natural water and synthetic urine, using our developed colorimetric probe colloid was demonstrated.Figure S12a   12. Summary of other previous reports using metallic NPs towards Cr(VI) detection.

3 .
UV-Vis spectra and photographic image of each nanomaterial mixed with DI and TW.The solid and dotted curves in Figure S1 are the extinction spectra of each metal colloid in the presence of DI water and tap water, respectively.The photograph image (inset on the right of Figure S1) displays the color transformation of each colloid after being mixed with DI and TW.

Figure
Figure S1 UV-Vis spectra of each nanomaterial mixed with DI and TW.Inset reveals the photographic image of resultant colloid upon DI and TW addition.

Figure S2
Figure S2 UV-Vis spectra of five different concentrations of Na-AuNPs (5.93 nm in diameter).Inset (on the right) shows the corresponding photograph taken under visible light.

( 3 .
91, 5.93, 10.81, 35.92 and 52.84 nm) were synthesized by altering the amount of gold precursor in the synthesis reaction.The particle size of cit-AgNPs was fixed at about 40 nm.The volume ratio between AuNPs:AgNPs was fixed at 3:1 (final volume = 100 µL) and the volume of Cr(VI) was fixed at 50 µL.The concentration of Cr(VI) ranged from 0.1 to 100 ppm.UV-Vis spectra and photographs depicting the AuNPs:AgNPs combination upon addition of Cr(VI) are displayed in Figure S3.All AuNPs:AgNPs combinations with various sizes of Na-AuNPs show no aggregation upon addition of Cr(VI) ion.The efficacy of Cr(VI) detection varied with the particle sizes of Na-AuNPs.The probe combination of Na-AuNPs (3.91 and 5.93 nm):cit-AgNPs exhibited a highly sensitive response towards Cr(VI) determination as observed multicolor transformation from orange to purple, blue and finally green color.Nonetheless, the probe containing the smallest Na-AuNPs showed a pale color, which was hardly distinguished the correct color by naked eyes.The corresponding UV-Vis spectra of both systems (Figure S3a and S3b) reveal distinct spectral changes of Na-AuNPs:cit-AgNPs combination in the presence of Cr(VI) over entire measurement range.The pristine absorption bands of both cit-AgNPs and Na-AuNPs were observed at approximately 400 and 514 nm, respectively.The absorption band of AuNPs was gradually broadened with increasing the concentration of Cr(VI) ion.The characteristic absorption band of cit-AgNPs (400 nm) disappearedupon reaction with high Cr(VI) (5-100 ppm).The spectral response of LSPR of both Na-AuNPs and cit-AgNPs show the possible relationship for quantitative determination of Cr(VI) ion.In case of probe containing Na-AuNPs (10.81nm) and cit-AgNPs, the probe color changed to grey and dark grey upon mixing with Cr(VI) solution at concentrations of 10-100 and 0.5-5 ppm, respectively (FigureS3c).For larger Na-AuNPs (35.92 and 52.84 nm) mixed with cit-AgNPs, the color of probe solution notably changed at higher Cr(VI) content (10-100 ppm) (FigureS3d and S3e).No difference in color transformation by visual observation and less reduction of absorbance were observed when probe colloids reacted with lower concentration of Cr(VI) (0 to 5 ppm).These results indicate that different particle sizes of Na-AuNPs exhibit varying sensing behaviors in response to changes in Cr(VI) concentration.The mixed Na-AuNPs (5.93 nm) and cit-AgNPs (40 nm) showed the best response toward Cr(VI) detection.Consequently, Na-AuNPs (5.93 nm) was selected for further investigation.

Figure S5
Figure S5 Extinction spectra and inset (on the top) shows the corresponding photograph image of probe colloid (Na-AuNPs:cit-AgNPs) upon Cr(VI) addition.Cit-AgNPs were separately mixed with five

Figure S7
Figure S7 Extinction spectra and inset (on the top) shows the corresponding photograph image of probe colloid (Na-AuNPs:cit-AgNPs) at three different Au:Ag volume ratios upon Cr(VI) addition.The volume ration of Au:Ag was varied from (a) 9:1, (b) 3:1, and (c) 1:1.

7 .
The relationship between particle size of both Na-AuNPs and cit-AgNPs upon addition of Cr(VI) solutions.The possible mechanism of our developed probe colloid was investigated by observing the shape evolution of both AuNPs and AgNPs upon addition of Cr(VI) ion in the solution.The correlation between the particle sizes of Na-AuNPs and cit-AgNPs upon the introduction of Cr(VI) solutions might provide insight into predicting the detection mechanism.FigureS8shows the double y-axes plot, showing diameter of Na-AuNPs (on the left) and diameter of cit-AgNPs (on the right) versus the probe colloid reacted with different concentrations of Cr(VI) in TW.

Figure S8 8 .
Figure S8 Plot with double y-axes: diameter of Na-AuNPs (on the left) and diameter of cit-AgNPs (on the right) versus the probe colloid reacted with different concentrations of Cr(VI) in TW.
and b indicate the extinction spectra of Na-AuNPs and cit-AgNPs upon 4 months storage, respectively, with photographic insets on the right side showing the resulting colloids.Both nanomaterials are stable upon storage time as observed no change of UV-Vis spectra and no change of solution color.

Figure
Figure S11 UV-Vis spectra of (a) Na-AuNPs and (b) cit-AgNPs upon 4 months storage.Inset reveals the photographic image of resultant colloid.
and b depict the spectral response of the probe colloid for entire measurement range of Cr(VI) detection (0 to 100 ppm) in natural water and synthetic urine, respectively.

Figure S12
Figure S12The corresponding extinction spectra of the probe for entire measurement range (5 ppb to 100 ppm) of Cr(VI) detection in (a) natural water and (b) synthetic urine.

Table S1
Volume of each reagent for AuNPs synthesis

Table S2
Volume of each reagent for cit-AgNPs synthesis

Table S3
Summary of other previous reports using different types and morphologies of metallic NPs towards Cr(VI) detection.