Hydrogen-Bonding Recognition-Induced Colorimetric Determination of Hydrazine Based on the Tryptophan Capped Gold Nanoparticles

A simple, cost-effective, and rapid colorimetric method for hydrazine detection using tryptophan-caped gold nanoparticles (TrpAuNPs) has been developed. Tryptophan (Trp) is a protein with reducibility and amino group which can reduce chloroauric acid (HAuCl4) to AuNPs and modify the surface of AuNPs simultaneously. e Trp-AuNPs could be used to quantitatively detect hydrazine and showed different responses to vary concentration of hydrazine in an aqueous solution based on the aggregationinduced color change of Trp-AuNPs.e real water sample analysis veri�ed the conclusion.e sensitivity of the detection system was in�uenced by the size of AuNPs which is determined by the pH of the detection system, the concentration of Trp, and the react time. We found that higher temperature contributed to more rapidly results. e detection system can detect as low as 1 μμM hydrazine. We expect our approach to have wide-ranging applications in the developing region for monitoring water quality in some areas.


Introduction
Hydrazine, a highly reactive base and a strong reducing agent, has been used as an important reactant in the preparation of pharmaceuticals, pesticides, photography chemicals, emulsi-�ers, and dyes in various chemical industries [1][2][3].However, it is highly toxic and readily absorbed by oral, dermal, or inhalation routes of exposure, and long-term studies with laboratory animals indicate that hydrazine is mutagenic and carcinogenic [4].Exposure to hydrazine at high levels (10 ppb, threshold limit value) can induce irritation of nose, temporary blindness, pulmonary edema, DNA damage, and even severe damage of the central nervous system [5][6][7].In addition, contamination of rural drinking water supplies with hydrazine by livestock waste, organic wastes, and chemical fertilizers continues to be a problem.It has been a major concern throughout the world for several decades.Due to its widespread applications and toxic effects on humans, developing reliable and sensitive analytical methods for the selective detection of hydrazine is highly desirable.
So far, hydrazine can be routinely analyzed by a wide variety of techniques, such as gas chromatography [8], high performance liquid chromatography (HPLC) [9], ion chromatography [10], chemiluminescence (CL) [11], and electrochemical detection using a variety of chemically mod-i�ed electrodes has also frequently been used [12].Although these methods have high sensitivity, many of them are timeconsuming and labor-intensive due to the complex pretreatment, require expensive instrumentation and high cost of personnel, and are not readily adaptable to on-site, detection.�ndoubtedly, it is of great signi�cance to develop the simple, on-site and sensitive method for hydrazine detection.
In recent years, colorimetric methods have attracted much attention due to its low cost, simplicity, and practicality.Since color changes can be read out by the naked eye, colorimetric sensor does not require expensive or sophisticated instrumentation and may be applied to �eld analysis and point-of-care diagnosis.And these methods have shown great advantages over conventional assays, particularly in sensitivity, selectivity, and practicality.
For transformation of the detection events into color changes, a number of materials have been developed, including metal nanoparticles [13,14], carbon nanotubes [15], grapheme oxide [16,17], and conjugated polymers [18].Among many materials, gold nanoparticle (AuNPs) has been regarded as the most promising candidate owing to their unique optical properties.us remarkable progress has been made on the design of AuNPs-based colorimetric biosensors owing to their intrinsically strong surface plasmon resonance (SPR) absorptions, with extremely high extinction coefficients, in the visible wavelength range.is color change effect is the result of the coupling of the SPR between particles in close proximity.Systems based on analyte induced aggregation of AuNPs have been employed for the colorimetric detection of heavy metal ions [19][20][21], melamine [22], acetamiprid [23], glucose [24], nitrate and nitrite [25], and some biological substances [26,27].
ese reported colorimetric assays generally require three steps: synthesis, modi�cation of nanoparticles, and detection of targets.e aim of the former two steps is to fabricate a sensitive colorimetric probe for targets.And the functionalization of AuNPs is vital for triggering the colorimetric response of analytes, which also can be achieved during the synthesis of monodispersed AuNPs.Herein, we proposed a novel method to realize colorimetric detection of hydrazine based on tryptophan capped gold nanoparticles (Trp-AuNPs).In our study, there is only one step in the whole formation of Trp functionalized AuNPs without seeds.In this strategy, tryptophan served not only as a reductant but also a modi�er that was capped AuNPs to avoid AuNPs aggregating autonomously to control the size of AuNPs.e colorimetric detection hydrazine was realized based on the visual color changing and ultraviolet visible (UV-Vis) absorption.In addition, the proposed method was successfully used for determination of the hydrazine in various environmental water samples.To the best of our knowledge, this is the �rst report to use this method for detecting hydrazine based on Trp caped AuNPs.(Shanghai, China).All other chemicals were of analytical reagent grade and used without further puri�cation.And all stock solutions were prepared daily with double-deionized water obtained from a Milli-� water puri�cation system (Bedford, MA, USA).

Apparatus.
UV-Vis spectra were obtained on a Lambda 950 spectrophotometer (Perkin Elmer, USA) at room temperature.e sample was thoroughly mixed with a shaker vortex (IKA Genius 3, Germany).e scanning electron microscope (SEM, JSM-6360LA, JEOL Ltd.) was also used to observe the aggregation and size distribution of Trp-AuNPs.A PHS-3CA precision pH meter (Dapu, China) was used in the experiment.e photo images of reaction solutions were recorded using a Coolpix 5400 digital color camera (Nikon, Tokyo, Japan).

Procedures.
All glassware used in the following procedure were cleaned in a bath of freshly prepared aqua regia, rinsed thoroughly in double-deionized water, and dried in air prior to use.Before detecting hydrazine, 1.2 mL premixed solution in 1.5 mL centrifuge tube containing 600 L 2.0 × 10 −2 M phosphate buffer solution (PBS, pH 5.0), 200 L 2.0 × 10 −3 M HAuCl 4 and 400 L 1 × 10 −2 M Trp were mixed for by a shaker vortex and then incubated in a 40 ∘ C water bath for 30 min.Successively, 100 L of diverse concentrations of hydrazine were added to the premixed solution, shaking 20 times at room temperature.And then the relative UV-Vis absorbance spectra and photographs of the reaction solutions were recorded.Due to electronic effect of the amino group of Trp, the formed AuNPs were capped by the Trp and could be monodisperse before the addition of hydrazine.en, the Trp-AuNPs were unstable and tended to agglomerate gradually with addition of hydrazine, which is contributed to the increasing of inter-particles aggregation [28].Meanwhile, the color of reaction solution was changed from light purple red to purple (showed in the inset of Figure 1), which is result to the absorbance of reaction solution around 540 nm increased.erefore, the concentration of hydrazine could be quanti�ed by the absorption of 540 nm (A 540 ).In addition, a hydrazine molecule contained two amino groups, and Trp also had one amino group which meant that two such units could easily form extended arrays of hydrogen bonding (NH⋯O and NH⋯N) which was particularly useful for sensing [29,30].Moreover, SEM was used to further verify the detection mechanism in Figure 2. Compared with monodisperse AuNPs (A), the interparticle distance of AuNPs decreased and resulted in obvious aggregations (B).

Optimum Conditions.
In order to obtain the optimal condition, several impact factors were optimized.As well known, color change effect was the result of the coupling effect on the strong surface plasmon resonance (SPR) between particles in close proximity [31].us we tested the pH of the reaction solution, the concentration of Trp, the temperature of the premixed solution (the premixed temperature) and the time of premixed solution (the premixed time).

3.2.�. �n�u�n�� o� p�. e pH not only in�uenced the interaction between
Trp-AuNPs and hydrazine, but also affected the stability of Trp-AuNPs.To investigate the effect of pH on the detection sensitivity, the reaction solutions at diverse pH values (3.0 to 7.0 in intervals of 1.0) were tested.Figure 3 shows that the relative UV-Vis absorption spectra in the presence of different values of pH (3.0 to 7.0) of 1.0 × 10 −2 M PBS.e best sensitivity was obtained at pH 5.0.is was probably due to the fact that the hydrogen-bonding was quite weak in strong acidic solution.However, the ionization of Trp was adverse to the stability of Trp-AuNPs when pH was higher than 5. erefore, pH 5.0 was chosen for further experiments.

�n�u�n�� o� t�� Con��nt��tion o� ��p. e selection of
Trp both as a reductant and a modi�er was due to its strong reducing capacity and amino group.e concentration of Trp was very important to the size and modi�cation of Trp-AuNPs, while the size of the Trp-AuNPs could affect the color and absorbance.erefore, the concentration of Trp played a dominant role in the detection.Various concentrations of Trp (from 0 to 4.6 × 10 −3 M) were studied on the relevant optical absorption change.Figure 4 showed the UV-Vis absorption spectra in the presence of different concentrations of Trp (0 to 4.6 × 10 −3 M) in 1 × 10 −2 M PBS (pH 5.0).e experimental results showed that compared with others, 2.3 × 10 −3 M Trp was more sensitive in the detection.us, 2.3 × 10 −3 M Trp was selected for further experiments.

�n�u�n�� o�
Op���tion�� ��mp���tu��.e operational temperature included the premixed temperature and binding temperature.Before adding the hydrazine, the Trp and HAuCl 4 should be well mixed.erefore, the premixed temperature was a key factor for the reaction between them.F 5: e effect of the premixed temperature.For conditions see Figure 3.
�n particular, the mixture temperature also in�uenced the detection sensitivity.us, we tested in the range of 20-60 ∘ C at intervals of 10 ∘ C. As shown in Figure 5, the absorption was the highest at 40 ∘ C. e possible reasons were as follows: �rst, the high temperature was bene�cial to the formation and modi�cation of Trp-AuNPs.Second, the high temperature could induce self-aggregation of Trp-AuNPs, which was adverse to the assay [32].erefore, 40 ∘ C was chosen as the premixed temperature for all experiments.When hydrazine added, the binding temperature affected hydrogen-bonding reaction.However, the self-aggregation of Trp-AuNPs attributing to high temperature should be avoided because the aggregation should be induced by hydrogen-bonding.Taking into account operational convenience, the room temperature was chosen as the binding temperature.To obtain the optimum condition, the mixture time was investigated in the range of 0-90 min at intervals of 15 min.As shown in Figure 6, the value of A 540 sharpy increased at �rst 30 min and was kept in the same level a�er 30 min.erefore, 30 min was selected as the perfect premixed time.

Colorimetric Detection of Hydrazine
3.3.1.Sensitivity.Quantitative analysis was realized by the absorption of 540 nm (A 540 ).e corresponding UV-Vis absorption spectra were recorded.A linear correlation existed between the absorption ratio A 540 and the hydrazine concentration in the range from 7.57 × 10 −6 to 2.01 × 10 −3 M (shown in Figure 7).e regression equation was   (1.359 ± 0.03982) × 10 −4 + (0.05166 ± 0.0035)  (  6) with a correlation coefficient (r) of 0.9957, where  was the absorption at 540 nm and  was hydrazine concentration (×10 −6 M). e limit of detection (LOD) calculated as 3 times the standard deviation for the blank solution was 1.0 × 10 −6 M, which was signi�cantly lower than the maximum hydrazine concentration allowed for water sources in China  As shown in Table 1, the comparison of the LOD in different methods indicated that our study provided a better LOD than most of them.Moreover, our work provides a simpler method without any sophisticated instruments and complicated experiment operations.What is more, to verify the rapidity and reliability of the proposed method, the relative UV-Vis spectra of the reaction solution were recorded at a regular interval of one minute in 10 min aer adding two different concentration of hydrazine.e reaction time-dependent response curves were shown in Figure 8. e absorption ratio kept constant aer 3 min, indicating that the detection was extremely speedy and complete in three minute.

Selectivity.
Various substances were most likely to be present in environmental water samples, such as metal ions, amino acids, and organics which could interfere with the determination of hydrazine.erefore, the interferences in the 20 times concentration of hydrazine (7.57× 10 −5 ) were used to carry out the experiment to investigate the selectivity of the proposed method.e values of A 540 were shown in Figure 9. Hydrazine obtained the largest absorption of A 540 , indicating that the determination of hydrazine had enough tolerable limit to the interferences.

Applications.
In addition, we tested its colorimetric response to tap water samples, lake water samples, sea water samples, respectively, in order to evaluate the reliability of the proposed method.Samples of tap water were from our laboratory without any additional pretreatment.Lake water samples were from the arti�cial lake in our campus, and sea water samples were obtained from the shore of Shantou.e samples of lake water and sea water were diluted with double distilled water (1 : 1, v/v).ese real water samples were supplemented with two different concentrations of hydrazine, and the collected data of analytical recoveries and RSDs were shown in Table 2.

Conclusions
Based on the experimental results above, hydrazine could be detected with this simple, rapid, direct, and sensitive method, and it was suitable for routine analysis.e concentration of hydrazine in water samples can be determined by monitoring  with the naked eye or a UV-Vis spectrometer.e method showed relatively good selectivity for hydrazine over other hydrazinium with the lowest detection concentration of 1 M.Especially, the merits made the proposed method specially useful for on-site screening hydrazine levels well below the current safety limit in drinking water.

4 F 1 :
e schematic representation of the analytical process for detecting hydrazine.Insert: colorimetric visualization of the Trp-AuNPs generated in the absence of (a) and in the presence of hydrazine (b 2.0 × 10 −4 ) in pH 5.0 PBS.
Figure 1 described the principle of the colorimetric determination of hydrazine.First, Trp was used to reduce HAuCl 4 to AuNPs.

F 2 :F 3 :
e SEM images of in the absence of (a) and in the presence of hydrazine (b).Condition: 1.2 mL premixed solution containing 1.0 × 10 −2 M PBS (pH 5.0), 3.1 × 10 −4 M HAuCl 4 , 3.1 × 10 −3 M Trp were mixed for by a shaker vortex and incubated in a 40 ∘ C water bath for 30 min.Successively, 2.0 × 10 −4 of hydrazine were added to the premixed solution, shaking 20 times.e effect of pH.For conditions see Figure 2. In�uence of the conditions in the addition of 7.57 × 10 −5 M hydrazine.

F 4 :
e effect of Trp concentration.For conditions see Figure3.

F 6 :
e effect of the premixed time.For conditions see Figure3.T 1: e comparison of the LOD between different methods..�. �n��ence of �remi�ed �ime.e premixed time also impacted the detection which was an inconvenient factor.

F 9 :
Absorption of reaction solution at 540 nm with the addition of hydrazine (7.57× 10 −5 M) or other interferences under the optimum conditions.
T 2: Application of the proposed method to determination of hydrazine in real water samples spiked with different amounts of hydrazine.