Environmentally Safe Mercury(II) Ions Aided Zero-Background and

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Environmentally Safe Mercury (II) Ions Aided Zero-Background and Ultrasensitive SERS Detection of Dipicolinic Acid Xiangru Bai, Yi Zeng, Xiaodong Zhou, Xiaohua Wang, Ai-Guo Shen, and Jiming Hu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02172 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 10, 2017

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Environmentally Safe Mercury (II) Ions Aided Zero-Background and Ultrasensitive SERS Detection of Dipicolinic Acid Xiang-Ru Bai, Yi Zeng, Xiao-Dong Zhou, Xiao-Hua Wang, Ai-Guo Shen * and Ji-Ming Hu Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P.R. China ABSTRACT: Field, reliable and ultrasensitive detection of dipicolinic acid (DPA), a general biomarker of bacterial spores and especially Bacillus anthracis, is highly desirable but still challenging in current biometric security emergency response system. Herein we report an environmentally safe mercury (II) ions-mediated and competitive coordination interaction based approach for rationally designed surface-enhanced Raman scattering (SERS)-active gold nanoparticles (AuNPs), enabling rapid, ultrasensitive and zero-background detection of DPA without the pretreatment of samples. By means of competitiveness, these papain capped gold nanoparticles (P-AuNPs) are induced to undergo controllable aggregation upon the addition of Hg2+ ions and DPA with a concentration range (1 nM~8 μM), which correspondingly cause quantitative changes of SERS intensity of cresyl violet acetate (CVa) conjugated AuNPs. The decreased Raman intensity obtained by subtracting two cases of additives that contain only Hg2+ and the mixture of Hg2+ and DPA, is proportional to the concentration of DPA over a range of 1 nM~8 µM (R2=0.9824), with by far the lowest limit of detection (LOD) of 67.25 pM (0.01 ppb, S/N=3:1). Of particular significance, mercury (II) ions actually play two roles in the process of measurements: a mediator for two designed competitive ligands (DPA and papain), and also a scavenger for the possibly blended ligands due to the different interaction time between DPA and the interferent with Hg2+ ions, which guarantees the interference-free detection of DPA even under real conditions.

Highly sensitive and selective detection of bioagents is always a vital task in order to facilitate timely and appropriate actions in the event of a biological attack. Such as bacillus anthracis, a spore-forming bacterium and the etiological agent of the acute disease anthrax, 1 is an important example. Traditional definitive identification through culture and later relatively rapid techniques such as polymerase chain reaction (PCR) 2 are still relatively slow compared with the approach aiming to look for chemical marker compounds that are characteristic of the spores. 3 Dipicolinic acid (DPA) as an excellent marker compound is most widely studied in recent years. Up to now, popular techniques useful for quantitative analysis of DPA include not only traditional instrumental methods, such as liquid and gas chromatography, 4, 5 FT-IR, 6 and mass spectrometry, 7 but also some newly constructed colorimetric 8 or fluorescent sensing assay strategies. 9-10 However, the traditional techniques require more sophisticated sample pre-treatments, necessary separation procedures and the larger clumsy laboratory appliances, which make them powerless to rapid and field detection of DPA. In contrast, the colorimetry and fluorescence-based sensing methods relying on colour-developing/fluorescence-triggering reagents and portable spectrometers are supposed to be more suitable for practical use in public security inspection. Nevertheless, the sensitivity of the former visual testing methods show obviously insufficient; while the latter

luminescent methods are susceptible to interference from other molecules leading to inhibition or luminescence quenching, 11-18 although they have received most attention due to the substantial sensitivity. Known as high sensitivity and no sample pre-treatments, also, the full width at half wavelength of a Raman peak is much narrower and low-interference due to their highly resolved spectra, surface-enhanced Raman scattering (SERS) technique has been a nice encoding element in multiplex bioassay, and portable Raman spectrometer also has been widely used in field analysis. During the past decade, SERS has been used for the direct detection of DPA by utilization of both solid phase flat substrates and liquid phase sol particles. 19-39 With regard to the former substrates, some deliberate surface nanostructure or self-assembly arrays have been rationally designed to improve the plasma coupling between particle-particle and particle-substrate, and increase the chance of DPA falling into ‘hot spots’ of SERS, which generally locate in the gap between particles. The earliest SERS substrate for DPA detection belongs to Van Duyne group, in which the obtained limits of detection (LOD) are roughly several hundred ppb. 19, 24, 26 In order to further improve LOD, this group made an important step forward in forming a self-assembled monolayer of closely arranged gold nanoparticles (AuNPs) on the gold surface, which strengthens the local electric field intensity inside the above-mentioned gaps due to the extra coupling

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between the gold surface and AuNPs, 28 and the LOD has correspondingly been reduced to the lowest 0.1 ppb till now. Thereafter, to construct non-flat SERS-enabled substrates suitable for micro-area sampling and reliable detection 39, 40 of DPA rather than to blindly pursue high sensitivity has become the predominant research trends in recent years. For the latter, the general approach is to detect DPA in solution by mixing them with the salt or other regents induced gold or silver colloidal aggregates. And the obtained sensitivity has yet to show incredible low LOD but roughly several ppb. However, nearly all those SERS-based direct methods for DPA neglect several vital points during practical use and correspondingly have two main fatal deficiencies. First, none of the above work has taken interferences in real samples into account, indicating their final detection results extremely likely unreliable. Actually, the Raman emission at 1010 cm-1 is always attributed for breathing vibration of DPA, but normally interferences in real samples, especially those structured with pyridine and benzene ring will greatly raise the uncertainty of accuracy of the measurement due to the incident spectral overlap around 1010 cm-1. In addition, all direct SERS measurements of DPA lack accuracy because only relying on random adsorption of DPA on SERS substrates may not guarantee the homogeneous distribution of DPA within detection zone even if the regularly arranged NPs are of uniform size. Moreover the competing sorption of DPA and other interfering components will imply that not all target molecules have been taken into measurement. To our knowledge, there is no study on in-direct SERS detection of DPA in the published scientific literature till now. Herein, we report for the first time a mercury (II) ion aided sensing approach for rationally designing SERSactive AuNPs, enabling reliable and highly selective indirect detection of DPA. Mercury (II) ions play two essential roles here. The stability constant of Hg2+ ions with DPA is lgK = 20.2, 41-42 therefore, Hg2+ ions are able to form stable complexes with DPA and correspondingly

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insure complete capture of DPA. Furthermore, as a scavenger, we first found that the Hg2+ ions clean out all the possible interferences owing to the immediate coordination reaction between Hg2+ ions and possibly blended ligands, such as tyrosine, phenylalanine, cysteine and DNA, which may be coexisting in spores. This significant phenomenon, which will be confirmed by the reaction kinetics curves of Hg2+ ions with the above-mentioned interferences in this paper, encourages us design an environmentally safe mercury (II) ions aided zero-background and ultrasensitive SERS detection strategy for DPA. As a consequence, both the reliability and accuracy of this assay will be greatly improved. In detailed, CVa modified AuNPs are firstly assembled with a monolayer of papain as a SERS donor, by means of the strong interaction between Hg2+ ions and sulphur groups of papain, 43 Hg2+ ions subsequently act as an “initiator” to induce SERS active P-AuNPs to undergo aggregation. As a result, the aggregation enables the formation of hot spots, which are located between two adjacent P-AuNPs, and correspondingly causes dramatically enhanced Raman signals of CVa in these hot spots. Once DPA and Hg2+ are synchronously introduced into SERS donor solution, P-AuNPs are not inclined to aggregation because DPA prior to coordinate with Hg2+ rather than papain.44-45 Raman signals of CVa would decline along with the addition of DPA. Particularly, when analysing in complicated mixed real samples, the excess Hg2+ ions were firstly added into the mixture to coordinate with the possible interferences. Due to the different interaction intensity and time between Hg2+ with the possible interferences and Hg2+ ions with DPA, a facile and quantitative SERS assay with high selectivity for detection of DPA in homogeneous solution is fabricated. The limit of detection (LOD) is 67.25 pM (0.01 ppb) over a range of 1 nM~8 μM (R2=0.9824), in which the indirect detection of DPA based principle has not been reported before.

Scheme 1. Schematic demonstration of Hg2+ ions aided SERS detection of DPA and SERS measurements.

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EXPERIMENTAL SECTION Chemical reagents. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O, Au 23.5-23.8%), tri-sodium citrate (C6H5Na3O7, 99%), cresyl violet acetate (CVa, C18H15N3O3, analytical grade) were purchased from Aladdin. Papain (BR, 800U/mg), hydroquinone (HQ, C6H6O2, 99%), dipicolinic acid (DPA), mercuric nitrate (Hg(NO3)2) and glucose were of analytical grade and offered by Sinopharm Chemical Reagent CO., Ltd. Cysteine, tyrosine, phenylalanine, ATP, BSA, cholesterin, lecithin, trypsin and lysozyme were of biological reagent and purchased from Sinopharm Chemical Reagent CO., Ltd. Phosphate buffer (PB) was used as the buffer. Ultrapure water with an electrical resistance larger than 18.2 MΩ cm was used throughout the experiment. Synthesis of AuNPs with 30±2 (40±2) nm diameter. The 30±2 (40±2) nm AuNPs were prepared by the chemical reduction of chloroauric acid with sodium citrate. In a typical synthesis, 1.4 (1.2) mL of sodium citrate solution (38.8 mM) was added into 99 mL of aqueous HAuCl4 solution (0.29 mM) with stirring in boiling condition, and the mixture was boiled for 20 min to turn into a wine red solution. 46 After that, the sol was standing to cool down to room temperature with stopping heating and stirring. Then, 200 μL of CVa (1 mM) solution was added into the solution, followed by centrifugation three times to remove the excess, and then re-dispersed in deionized water. Synthesis of AuNPs with large diameter (>40 nm). In a typical synthesis, a gold seed solution was prepared by sodium citrate reduction. 30 mL of ultra-pure H2O and 300 μL of 1% (W/V) HAuCl4 (Sigma-Aldrich) were added to a 250 mL Erlenmeyer flask with a clean stir rod. This was rapidly brought to a boil while stirring at the maximum speed possible that did not cause splashing of the solution. As soon as the solution was boiling, 900 μL of 1% (W/V) sodium citrate trihydrate (Sigma-Aldrich) solution was added. The flask was removed from heat after 10 min, once nanoparticle maturation was complete as indicated by colour transition. Larger diameter particles were synthesized in 10 mL batches. Hydroquinone and sodium citrate solutions were prepared fresh each day. Gold chloride (Sigma-Aldrich) solution was centrifuged at 18000 r/m for 60 min in a centrifuge tube before use to remove any aggregates, after which volumes were obtained off the top. For a given synthesis, 100 μL of a 1% (W/V) HAuCl4 solution was added to 9.4-9.8 mL of ultra-pure H2O (depending on balance of water added with seeds) in a 20 mL scintillation vial. The appropriate volume of particle seeds were then added (minimum of 7.7 μL and maximum of 406 μL). The solution was then stirred rapidly at room temperature. 22 μL of a 1% sodium citrate solution was then added, immediately followed by 100 μL of 0.03 M hydroquinone (Sigma-Aldrich). Reduction was immediately apparent in reactions containing a high ratio of seeds, and completed within approximately 10 min, whereas reactions with low ratios of seeds took up to 60 min to complete. 47

Synthesis of CVa modified AuNPs. 2 μL of CVa (1 mM) per 1 mL gold colloid solution as the internal standard was added into the gold solution with 30±2 (40±2) nm diameter, while 1 μL of CVa (1 mM) per 1 mL gold colloid solution was added into the gold solution with larger diameter (>40 nm). The mixture solution was stood overnight and followed by centrifugation three times to remove the excess, and then re-dispersed in deionized water. Preparation of CVa labelled P-AuNPs. The preparation of P-AuNPs was according to the reported method. 48 Firstly, the pH value of CVa labelled AuNPs solution was adjusted to 11 by 1 M NaOH solutions. Then, an excess amount of papain solution was added into the AuNPs solution to ensure that papain completely functionalized the AuNPs. The mixture was shaken for about 30 min and stood for about 24 h. The P-AuNPs were centrifuged at certain rpm for 30 min. After the removal of the supernatants, the sediment was washed several times and suspended in 10 mM PB buffer (pH 6.0). Prior to use, the stock solution of P-AuNPs was kept at 4℃. Measurement procedure. The SERS sensing of DPA were carried out under the following procedures. First, 20 μL of various concentrations of DPA were mixed with 8 μL of 4 μM Hg2+ ions at room temperature for 15 min allowing for the complete coordination between DPA and Hg2+ ions. Then, the reaction mixture was added to the 60 μL of CVa labelled P-AuNPs and 112 μL of PB buffer were added to the above mixture. And then the whole solution was allowed to react for 5 min at room temperature for SERS measurements. Characterization and SERS measurement. SEM and TEM images were taken by field-emission scanning microscopy (FE-SEM, SIGMA) and transmission electron microscopy (TEM, JEOL JEM-2100 microscope), respectively. Raman measurements were conducted with Renishaw in Via-Plus Raman microscope quipped with a 632.8 nm excitation laser and a 50L× objective. The signals were integrated for 5 s with 2 accumulations. UV-vis spectrophotometer was used by (Shimadzu, UV2550). Zeta potentials were measured with a Nano-ZPS zetasizer (Malvern Instruments). RESULTS AND DISCUSSION Feasibility of SERS detection. The design rationale for DPA detection is illustrated in Scheme 1. In the absence of DPA, the addition of Hg2+ ions to P-AuNPs would induce the aggregation of AuNPs because of coordination interaction between Hg2+ and P-AuNPs. In this way, the working curve of Hg2+ ions could be constructed. The aggregation level of AuNPs will be weakened if DPA is introduced, in which DPA could bind Hg2+ ions selectively with a higher force and form a coordination complex. 44-45 Because of the different interaction intensity and time between DPA and the interference with Hg2+ ions, when DPA and other interferences possibly existing in bacterial spores were introduced, the interference can be effectively removed and quantified. Other interferences possibly existing in bacterial spores, such as tyrosine, phenylalanine, cysteine

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and DNA, which could coordinate, with Hg2+ will immediately finish the coordination within 1 min while the interaction time between Hg2+ and DPA is 15 min. In this way, the interferences in bacterial spores and DPA could be effectively detected, in fact, the Hg2+ also plays the role of masking agent to remove the interference.

P-AuNPs lead to the reduced aggregation, as shown in Figure S1 (A1, B1). Two different sizes of AuNPs all confirm this phenomenon. Hence, based on the SERS intensity change of CVa due to the Hg2+ ions-induced aggregation of P-AuNPs, detection of DPA was believed to have feasibility through this strategy.

To estimate the mutual interaction of Hg2+ ions with DPA and papain, a series of contrastive studies have been suggested. As displayed in Figure 1 (A), the addition of 4 μM Hg2+, Cu2+ and Pb2+ ions respectively triggered the aggregation of P-AuNPs through the coordination between the three metal ions and papain, resulting in an absorption peak near 650 nm, while the P-AuNPs were more easily aggregated with Hg2+ ions than the other two metal ions, therefore, Hg2+ is chosen as the competitive inducer for P-AuNPs and DPA. When DPA was introduced, as shown in Figure 1 (B), with the increase of

Optimization of modification and experimental parameters. The AuNPs with the diameter of ~70 nm as shown in Figure S2 (A), were further functionalized with Raman reporter CVa, the modified parameters of papain onto CVa labelled AuNPs depends on the pH and the concentration of papain. The successful preparation of P-AuNPs conjugate was illustrated by transmission electron microscope (TEM) images in Figure S2 (B), from the TEM image, it can be clearly seen that there was a thin layer outside the CVa labelled AuNPs. All of these suggest that papain was coated successfully on the surface of AuNPs, meanwhile the probe owns the preferable stability as the SERS intensity of CVa changed almost nothing when they were placed for one month as shown in Figure S2 (C). As shown in Figure S3 (A, B), the optimal pH and the concentration of papain were approximately 11 and 5 mg/mL, respectively. P-AuNPs can be aggregated with 4 μM Hg2+ ions. We also carried out control experiments using the citrate-AuNPs, which did not show any response to 4 μM Hg2+ ions.

Figure 1. (A) UV-vis spectra of P-AuNPs with different metal ions with certain concentration, the concentration of metal ions: 4 μM. (B) UV-vis spectra of P-AuNPs with 4 μM Hg2+ ions towards different concentrations of DPA. The concentration of DPA: (1) 0.1 μM, (2) 0.3 μM, (3) 0.5 μM, (4) 0.7 μM, (5) 0.9 μM, (6) 1 μM, (7) 2 μM, (8) 3 μM, (9) 5 μM. DPA introduced, the P-AuNPs solution changed from aggregation to dispersion, along with colour changes from blue to violet and a new absorption peak was observed at around 530 nm, which implied the interaction between Hg2+ and DPA. It can also be confirmed by TEM images, as shown in Figure S1, upon the addition of Hg2+ ions, aggregation of P-AuNPs occurred, according to Figure S1 (A2, B2). However, once the DPA was first incubated with Hg2+ ions for 15 min, the addition of the mixture to

For this SERS assay of DPA, the experimental parameters include the concentration of Hg2+ ions and PB buffer, the pH value and the reaction time between Hg2+ ions and DPA. We investigated the pH and the concentration of PB, as shown in Figure S4 (A), below pH 6, we found that the addition of DPA cannot disperse the P-AuNPs; at pH 6, the P-AuNPs was in dispersion; we also found that the higher pH (>6), the better dispersion. However, the hydrolysis reaction is dominant for Hg2+ ions with higher pH than 6 to form precipitate and break the coordinate interaction of DPA and P-AuNPs toward Hg2+ ions. Therefore, pH 6 of PB is the suitable condition for this SERS sensing assay. The higher concentration of PB buffer always owns the stronger buffer capacity, however, as shown in Figure S4 (B), when the concentration of PB buffer was more than 10 mM, the P-AuNPs were easily aggregated. The suitable concentration of PB buffer is 10 mM. The concentration of Hg2+ was optimized to obtain an obvious colour change. It is worthy to notice that there was an obvious aggregation of P-AuNPs for Hg2+ ions concentration up to 4 μM, as shown in Figure S4 (C). As well known, the maximum concentration of Hg2+ ions in the waste water, which is allowed to be emitted nationally, is no more than 0.05 mg/L. Herein, 8 μL of 4 μM Hg2+ ions was added into the detection system with the final volume of 200 μL. The final concentration of Hg2+ in the whole system is 0.032 mg/L that is obviously lower than the national standard. Therefore, 4 μM Hg2+ ions was chosen for the SERS sensing assay in the following experiments, which is totally environmentally safe but just unfit to drink. Finally, detection of DPA was further studied by treating certain amount of DPA with 4 μM Hg2+ ions for different time. As shown in Figure S4 (D), it is apparent that SERS intensity of CVa decreased by prolonging the incubation time

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between DPA and Hg2+ ions, indicating that more DPA was coordinate with Hg2+ ions and then the interaction between P-AuNPs and Hg2+ ions was weakened. It has also been found that after 15 min, one can observe a slower decline in Raman intensity, indicating that the interaction between DPA and Hg2+ ions was mostly finished. Therefore, to realize detection of DPA at a short time, the incubation time of DPA and Hg2+ ions was optimized at 15 min, it is believed that a short time coordinate reaction could be applied for fast analysis. Optimization of the size of AuNPs. The particle size of AuNPs used in this experiment was also investigated. We synthesized AuNPs with different sizes, from the SEM image of the AuNPs, as shown in Figure S5, with the increase of the particle size, the better homogeneity of the AuNPs, it can also be confirmed from the particle size distribution as displayed in Figure S6. The UV-vis absorbs spectrum of the AuNPs with different sizes was shown in Figure S7, the UV-vis absorption spectrum turned red-shift with the increase of the size. The SERS intensity of CVa labelled on AuNPs with different sizes reached the maximum when the size of AuNPs were 70 nm as shown in Figure S8. Therefore, the 70 nm AuNPs were chosen as the optimal substrate during the experiment. SERS and Colorimetric detection of DPA. Consequently, under the optimized conditions above, quantitative and sensitive detection of DPA was achieved by monitoring the SERS peaks of CVa labelled on AuNPs on different spots of three parallel samples at varied concentrations. Figure 2(A1) presented the SERS spectra of CVa upon the addition of Hg2+ incubated with different amount of DPA after reaction for 15 min. One clearly observes that the Raman intensity of peak at 595 cm-1 drastically decreased with increased concentration of DPA from 1 nM to 8 μM, indicating that with more DPA

introduced, the coordination between DPA and Hg2+ was enhanced and more Hg2+ were consumed in the coordinate interaction. Figure 2(A2) showed the SERS intensity at 595 cm-1 varying with the concentration of DPA from 1 nM to 8 μM in the presence of 4 μM Hg2+, in which there is a nearly exponential decreasing curve in the tested concentration range, and an appropriate calibration curve was developed. The regression equation is y = 17.56x-95.11 (where x is the negative logarithm of the target DPA concentration (M) and y is the SERS intensity (R =ΔI/1000) of peak at 595 cm-1 with a squared correlation of 0.9824). Calculation from the standard deviation of the blank was used in determination of LOD, the LOD of the SERS sensor for DPA is calculated at 0.01 ppb. Under the same optimized conditions, to evaluate the detectable minimum concentration of DPA in an aqueous solution by means of colour change, different concentrations of DPA were added into the P-AuNPs solution, as shown in Figure 2(B1, B2), with the addition of DPA and the increase of its concentration, obvious blue-shift of peak were observed, along with a series of color change from blue to violet. I650/I530 ratio increased gradually with the increase of DPA concentration, indicating the increase of P-AuNPs dispersion degree due to the interaction of Hg2+ to DPA. Hence, it was possible to detect DPA in the range from 0.01 to 40 μM with a limit of detection of 25.7 ppb. The proposed SERS assay for DPA has satisfactory sensitivity in comparison with colorimetric assay due to the stronger Raman signals of the aggregated functionalized AuNPs. Compared with reported methods, the proposed method has the best sensitivity. Because of the broad application of AuNPs, We also investigated the LOD of DPA with different particle sizes, as shown in Figure S9, the LOD of DPA is the minimum when the AuNPs used in the experiment is 70 nm, as shown in Figure 3.

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Figure 2. (A1) Raman spectra of CVa varied with different concentrations of DPA: (1) 1 nM, (2) 3 nM, (3) 5 nM, (4) 7 nM, (5) 9 nM, (6) 20 nM, (7) 40 nM, (8) 0.2 μM, (9) 0.6 μM, (10) 0.8 μM, (11) 2 μM, (12) 4 μM, (13) 8 μM and (0) blank sample of CVa. (A2) the quantitative calibration curve of normalized Raman band intensity at 595 cm-1 against negative logarithm over different concentrations of DPA. (B1) The UV-vis spectra of the P-AuNPs towards different concentrations of DPA, the concentration of Hg2+ ions: 4 μM. The concentration of DPA: (1) 0.01 μM, (2) 0.03 μM, (3) 0.05 μM, (4) 0.07 μM, (5) 0.09 μM, (6) 0.2 μM, (7) 0.4 μM, (8) 2 μM, (9) 6 μM, (10) 8 μM, (11) 20 μM, (12) 40 μM; (B2) The linear relationship between the A528/A675 ratio and the concentration of DPA. cholesterin, lecithin, glucose, trypsin and lysozyme generate non-obvious changes of Raman signals of the functionalized P-AuNPs at 595 cm-1 while all of this interference may be the component of bacterial spore. It is well known that the Raman intensity is linked closely with the aggregation level of the functionalized P-AuNPs, therefore, it could be deduced that the increase in Raman intensity is attributed to the interaction between Hg2+ ions and tyrosine, phenylalanine, cysteine and DNA, which is similar to interaction of Hg2+ ions with DPA. In this case, this method is demonstrated not suitable for the real mixture.

Figure 3. Comparison for LOD of DPA with different sizes of AuNPs. Selectivity of the SERS sensor for DPA. The selectivity of this method has been investigated by testing the response of the functionalized P-AuNPs/Hg2+ in the presence of interferences including tyrosine, phenylalanine, cysteine, DNA, ATP, BSA, cholesterin, lecithin, glucose, trypsin, lysozyme under the above mentioned optimal conditions. As shown in Figure 4, it is

Figure 4. Evaluation of the specificity of the SERS biosensor. Variation of SERS intensity of CVa at 595 cm-1 after separate incubation of Hg2+ with: (1) DPA, (2) Tyrosine, (3) Phenylalanine, (4) Cysteine, (5) DNA, (6) ATP, (7) BSA, (8) Cholesterin, (9) Lecithin, (10) Glucose, (11) Trypsin, (12) Lysozyme and (13) blank sample of CVa. The concentration of DPA and several kinds of amino acid: 2 μM, others: 2 mM. obviously illustrated that the Raman intensity of functionalized P-AuNPs at 595 cm-1 is the strongest when the SERS donor is only incubated with 4 μM Hg2+ ions. Meanwhile, it is weakened immensely when SERS donor is incubated with the mixture of 4 μM Hg2+ ions and 8 μM DPA. However, except tyrosine, phenylalanine, cysteine and DNA, other interference, such as ATP, BSA,

Figure 5. (A) The reaction kinetics curves of Hg2+ ions with different molecules; (B) the quantitative calibration curve of Raman band intensity at 595 cm-1 against logarithm over different concentrations of Hg2+ ions. To solve this problem, we studied the reaction kinetics curves between Hg2+ ions and DPA, tyrosine, phenylalanine, cysteine, DNA. As shown in Figure 5(A), When tyrosine, phenylalanine, cysteine and DNA have mixed with Hg2+ ions, the interaction will start upon mixing and all the reaction kinetics curves present a sharp drop within 1 min. In contrast, the reaction kinetics curve of Hg2+ ions with DPA presents a gentle slope and the SERS intensity decreases slowly until 15 min. This figure

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Analytical Chemistry

demonstrates the different coordination velocities of Hg2+ ions with DPA and other biomolecules. Unlike DPA, some

Analysis of DPA in simulation of mixture. We studied the quantitative calibration curve of Raman band intensity at 595 cm-1 against logarithm over different concentrations of Hg2+. As shown in Figure 5(B), the regression equation is y = 17.46x-9.92 (where x is the logarithm of Hg2+ ions concentrations (μM) and y is the SERS intensity (R =ΔI/1000) of peak at 595 cm-1 with a squared correlation of 0.9389). Firstly, excess Hg2+ was added into the mixture to remove interferences, then the rest of Hg2+ ions would cause the aggregation of the SERS donor added later, finally we can actually calculate the consumed amount of Hg2+ through regression equation of Hg2+ ions. That also means the amount of interferences can be calculated. We tested the influence of complex sample in this method using the simulation of mixed samples. The simulation of mixed samples included ATP, BSA, cholesterin, lecithin, glucose, trypsin and lysozyme, the concentration of every component is 1.0×10-3 M while the concentration of tyrosine, phenylalanine and cysteine are all 1.0×10-6 M, the three spiked concentration of DPA is 0.1, 1.0 and 5.0 μM respectively. The sample was diluted in PB buffer. The mixture was divided into two parts. At first, certain amount excess Hg2+ ions were added, after 1 min, the P-AuNPs was added to calculate the consumed Hg2+, while the other mixture was detected following the same detection procedure for DPA in PB buffer. A quantitative analytical method was established for comparison by using external reference method of chromatogram, the linear relation of peak area and concentration of DPA was shown in Figure 6. As shown in Table 1, towards DPA detection in simulation of mixture, there is obvious difference between SERS1 and SERS2. We see that SERS1 are much larger than actually added value. The reason for this apparent discrepancy is that the interference acts as “spy” of DPA to coordinate with Hg2+ ions. In this way, the totally consumed Hg2+ ions are more than what the actually consumed by DPA because the extra Hg2+ ions rapidly coordinate with the interference first. SERS2 of three different concentrations are comparable to the added concentration of DPA when Hg2+ ions first clear out all the interferences. Moreover, the value obtained by SERS1 subtracting SERS2 represents the concentration of interferences, it can be easily seen that the value for all the three different concentrations are almost the same. Therefore, our method demonstrated the potential of the method for reliable application in complex mixture.

Figure 6. (A) The chromatogram of DPA with different concentrations; (B) The linear relationship between the peak area at 5.134 s and the concentration of DPA. coexisting biomolecules don’t need much time (less than 1 min) totally coordinate with the dissociative Hg2+ ions. Upon mixing them with Hg2+ ions, the amount of the remaining dissociative Hg2+ ions will rapidly achieve a constant value (the minimum value). However, Hg2+ ions still don’t completely react with DPA within 15 min. The remaining dissociative Hg2+ ions at this moment can still induce high degree of aggregation of P-AuNPs. During this 15 min, SERS peak intensity at 595 cm-1 for DPA will decrease along with the gradually reduced dissociative Hg2+ ions. Under this circumstance, Hg2+ ions could be first served as a scavenger to remove the possible blended ligands, such as tyrosine, phenylalanine, cysteine and DNA, which could effectively eliminate the interference in the complicated mixtures.

CONCLUSION

Table 1.Results of the recovery of DPA from spiked samples in mixture.

Added (µM)

Detected (µM)

Recovery (%)

RSD (%)

HPLC

SERS1

SERS2

HPLC

SERS1

SERS2

HPLC

SERS1

SERS2

1

0.1

0.09993

3.13

0.105

99.93

3130

105.0

0.026

38.94

4.25

2

1.0

0.9996

4.32

1.08

99.96

432

108

0.026

22.07

5.96

3

5.0

4.9965

8.03

4.83

99.93

160.6

96.6

0.025

9.90

4.34

SERS1 is the quantitative results without the remove of interferences; SERS2 is the corresponding quantitative results after the remove of interferences.

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In conclusion, a novel SERS assay platform for DPA detection with high sensitivity and selectivity was fabricated based on DPA-Hg2+ complex and Raman labelled P-AuNPs, which was used for the first time for the indirect SERS detection of DPA with the lowest LOD of 0.01 ppb. Compared with other assay for DPA, the SERS sensor meets the requirement of high sensitivity and no complicated sample pre-treatments. Our proposed method can achieve the completely sufficient capture of DPA and the interference-free detection under complex ambient conditions, which have greatly raised the accuracy and reliability when it is compared with the current assay of DPA. Because of the dual-function of Hg2+ ions, the SERS sensor have been successfully used for quantitative detection of spiked DPA in mixture, the results can be comparable with the methods of liquid chromatography, which may broad the rapid and field analysis of detection in current biometric security emergency response system. Due to exclusive coordination between heavy-metal ions and certain biomolecules, several promising strategies have been developed in our group, 49, 50 for example, some new sensors for Ag+ ions, Hg2+ ions, and cysteine have been freshly fabricated based on the interaction between peptide, DNA and heavy-metal ions. In the near future, this field is expected to be fruitful on the heavy metal ions-mediated sensors for various biomolecules (e.g., DNA, peptides and etc.) besides the simplex detection of heavy metal ions.

AUTHOR INFORMATION Corresponding Author *

Prof. Dr. Ai-Guo Shen: [email protected]. Notes The authors declare no completing financial interest.

ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (Nos. 21475100, 81471696 and 21175101).

Supporting Information TEM images, SEM images, Raman spectra, and size distribution. This material is available free of charge via the Internet at http://pubs.acs.org.

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