Inconspicuous Reactions Identified by Improved Precision of

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Inconspicuous reactions identified by improved precision of plasmonic scattering dark field microscopy imaging using silver shell-isolated nanoparticles as internal references Wei Feng, Wei He, Jun Zhou, Xiao Ying Gu, Yuan Fang Li, and Cheng Zhi Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05285 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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Analytical Chemistry

Inconspicuous reactions identified by improved precision of plasmonic scattering dark field microscopy imaging using silver shell-isolated nanoparticles as internal references Wei Feng,a Wei He,b Jun Zhou,c Xiao Ying Gu,a Yuan Fang Li,a and Cheng Zhi Huangad aKey

Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest

University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China. of Chemistry and Chemical Engineering, Yangtze Normal University ，

bCollege

Chongqing 408100, P. R. China. c College

of Computer and Information Science, Southwest University, Chongqing

400715, China dCollege

of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P. R.

China. Abstract Investigating a reaction that is inconspicuously weak, slow or coexists with other fast reactions is interesting since that can supply a capability to find new reactions or explore indecisive mechanisms, but the strategies for the investigations are still greatly limited. Herein, we apply the strategy of “finding from the data” to discuss inconspicuous reactions through improving the confidence level of detected signals.

* Corresponding author. Tel.: (+86) 23-68254659. Fax: (+86) 23-68367257. E-mail address: [email protected] (Cheng Zhi Huang)

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In dark-field microscopy (DFM) imaging analysis, plasmonic nanoprobes such as silver nanoparticles (AgNPs) have been applied at single nanoparticle level with high sensitivity, but correspondingly the deviations caused by instrumental and operational errors are inevitable. Thus, silver shell-isolated nanoparticles (AgSHINs) as an internal reference (IR) are introduced to calibrate the scattering signals of AgNPs probe during the reactions. And two calibration factors,  and , are used to calibrate the plasmonic scattering intensity and RGB values of AgNPs probe of the DFM images, respectively, making the confidence level of the DFM imaging analysis greatly improved and thus supplying a possibility to monitor inconspicuously weak or slow reactions. As a proof of concept, the inconspicuous amalgamation of AgNPs probes bathed in dilute solution of mercury higher than 1.01010 mol/L was successfully monitored. In the same way, we identified the very weak oxidation process of AgNPs probes by dissolved oxygen in water. These successful monitoring of inconspicuous weak or slow reactions, which might be possible to be regarded as detection deviations mistakenly, shows that the use of AgSHINs as an IR can provide a precise method to discern or discover inconspicuous slow reactions in nature through the DFM imaging analysis. Keywords: shell-isolated nanoparticles (SHINs), internal reference (IR), silver nanoparticles (AgNPs), dark-field microscopy (DFM) imaging. Introduction Reactions that are inconspicuously weak, slow or coexist with other fast reactions are easily overlooked owing to their poor display of detectable characteristics.1-3 A 2


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good strategy is to discern, find or figure out these reactions so as to make precise monitoring or possible measurements. For example, finding from the data, a smart strategy long ago, has been unconsciously adopted in the discovery of argon element by Rayleigh and Ramsay.4 In fact, with the fast development of science and technology, obtaining information from the reactions that are inconspicuously weak, slow or coexist with other fast reactions and deeply understanding the essential mechanism or find new reactions becomes much indisputably easier. Although silver nanoparticles (AgNPs) have been applied in real-time monitoring of target molecule delivery5 and biological imaging6 with dark-field microscopy (DFM) imaging owing to their sensitive localized surface plasmon resonance (LSPR) scattering properties and long-term photostablity without photobleaching,7 the inconspicuously slow oxidation process of AgNPs in aqueous solution has been scarcely concerned, which unquestionably leads detection deviations in these studies, particularly when AgNPs are used as the DFM imaging probe at single plasmonic nanoparticle level. 8 DFM imaging analysis has attracted more and more attention for the smart LSPR scattering signals of plasmonic nanoparticles (PNPs) probes.7,9,10 Long et al developed a chrominance-to-dimension method for on-site estimation of the diameters of single GNPs, which represented the advantage of the RGB value algorithm in DFM images.11 The LSPR scattering properties of PNPs probes that real-time monitoring of single plasmonic nanoprobe such as AgNP under DFM imaging system has become an important tool for sensing,12 hot-electrons transfer investigations13 and ultra-micro target detection14. Recently, Xu and Chen et al designed new plasmon rulers for 3



continuous observation of toehold mediated strand displacement progress, and then for in vitro monitoring of a DNA/RNA strand displacement process sensitively at the single-molecule level.15 Fan et al developed a smart plasmonic nanobiosensor for microRNA21 detection at the single-molecule level.16 Although researchers have developed many sensitive methods by analyzing the scattering intensity and RGB features of single plasmonic nanoprobe, these problems of objective and subjective factors that always lead to obvious deviations in the DFM imaging analysis, such as the skills of operators, instrumental conditions, working environments, can be tackled to some degree, which we supposed to be more and more worthy of attention as the increased sensitivity of analytical methods. Attempting to get much more confidence level of the analytical methods in our previous researches, gold nanoparticles with good chemical stability have been applied as the IR to calibrate the scattering light of silver nanoprobes in the dark field microscopy (DFM) images.17 This strategy, however, can only calibrate the scattering intensity, and does not involves the color calibration which is greatly dependent on the scattering wavelengths of gold nanoparticles and silver nanoparticles. In such case, herein we introduce shell-isolated nanoparticles18 (SHINs) as the internal reference (IR) which can effectively resolve these problems since SHINs have high stability and their scattering wavelengths are close to the corresponding nanoprobes. SHINs as the IR can calibrate the scattering intensity and the scattering light color of nanoprobes in the DFM images on one side, and can be further applied for finding or figuring out new inconspicuously weak or slow chemical reactions on the other, just like the famous finding of argon (Ar) by 4


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Rayleigh and Ramsay in history.4 SHIN, first proposed by Li and Tian in 2010,19 is composed by a plasmonic nanocore coated with a controllable ultrathin shell of SiO2 so as to keep the optical features of the plasmonic nanocore from external interference, and to have stable chemical property with more and more wide applications in complicate matrix. The creation of SHINs has greatly promoted the development of Raman scattering spectroscopy, resulting in a new field of shell isolated nanoparticle-enhanced Raman spectroscopy (SHINERS).18,20 Thanks to the very thin SiO2 shell of SHINs, the LSPR scattering properties of the plasmonic nanocore are almost kept. Actually, it is so interesting to explore different PNPs with same scattering properties and to explore identical PNPs but having different scattering properties, since that two explorations can endow us much more information about their plasmonic natures and optical activities, so as to make the real-time monitoring the reaction progress under DFM imaging, such as, target molecule delivery and biological binding8 , much more effectively. So, we prepared and applied AgSHINs with stable optical properties that are similar to the initial optical properties of plasmonic nanoprobe (herein, AgNPs applied), as an internal reference (AgSHINs IR) to calibrate the real time scattering imaging signals of plasmonic nanoprobes since the plasmonic nanoprobes might be changed or sacrificed during the reactions. This strategy is universal and can find wide applications because the various plasmonic nanoprobes can be easily developed as their corresponding SHINs IR by coating a controllable ultrathin shell of SiO2.19

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Scheme 1. Working protocol of calibration of imaging data by using the strategy of “finding from the data” to improve the confidence level so as to be further applied for investigating inconspicuously weak and slow reactions. Both AgSHINs IR and AgNPs probes have the same initial scattering features under DFM system. Inevitable deviations of signals such as operations or the accidental changes of instruments during the reactions could be calibrated by two calibration factors,  and , which calibrate the scattering intensity and RGB color of the images, respectively.

Scheme 1 shows our working protocol. Both AgSHINs IR, which are prepared according to the experimental descriptions and act as an internal reference, and AgNPs, which act as plasmonic nanoprobe, are successively deposited on a same slide and should have about the same initial plasmonic features in terms of the scattering intensity and color. With the reaction proceeding, the plasmonic scattering properties of AgSHINs IR are retained theoretically owing to their good chemical stability, while that of AgNPs probe get varied. That is, the introduction of SHINs IR can simultaneously calibrate inconspicuously weak, slow reactions or that coexisting with some fast reactions, greatly improving the confidence level of DFM imaging analysis, 6


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and thus, as proofs of the concept, the monitoring of the inconspicuous amalgamation of AgNPs probes in the presence of low concentration of mercuric ions and the very slow oxidation of AgNPs in water is successfully made. Experimental Section Preparation of AgNPs. AgNPs were prepared according to ref21 with some modifications. First, 50 mL glycerol/water mixture (20 vol % glycerol) was heated to 97 °C. With stirring for 1h, 9 mg of silver nitrate and 1 mL of sodium citrate (3%) was injected successively. The as-prepared AgNPs colloid was stored at 4 °C for next step. Preparation of shell-isolated Ag nanostructures. AgSHINs were principally prepared according to the method of Tian group.22 43 nm as-prepared Ag sol was diluted

two

times

with

ultra-pure

water,

then

NaBH4,

(3-aminopropyl)

trimethoxysilane (APTMS), and sodium silicate solution were added. Here, 0.1 M H2SO4 solution was employed to adjust the pH value. After reaction, the colloid was stored at 4 °C. Preparation of the flow cell. The flow cell consists of slide glass and cover glass. The potential of AgSHINs and AgNPs are negative, thus the positive slide glass is used for the electrostatic interaction in this experiment. In order to monitor the same area AgSHINs and AgNPs in DFM imaging, a crossed scratch was marked on the slide glass. firstly. Then, AgSHINs solution was dropped onto the slide glass nearby the scratch, 10 min later, the slide glass was washed by 18.2 M water and was blown dry by nitrogen. The left AgSHINs deposited on the slide were observed in the 7



DFM imaging by covering a cover glass under the slide. AgNPs were then deposited in the same area using similar operations. Results and discussion Development of AgSHINs IR for Calibrating DFM Imaging. The basic structure of AgSHINs IR is composed of an AgNPs core and a controllable ultrathin shell of SiO2, therefore, two basic criteria for AgSHINs acting as the IR in DFM imaging are required. One is the chemical stability during the reaction, and the other is the similarity to the initial plasmonic scattering properties of the AgNPs probe. Hence, the AgSHINs IR should hold two features. Firstly, the coating is as thin as possible so that the surface coating of SiO2 can scarcely exert effect on the plasmonic scattering properties of the AgNPs core. Secondly, the coating should be chemically stable and can keep from the physical, chemical and biological interferences during the reactions. In such case, we at first prepared stable and thin-coating AgSHINs IR. By coating the average size of 43 nm AgNPs (Figure S1AB) with 2 nm thickness SiO2 shell (Figure S2AB), and the characteristic LSPR absorption peak kept at 410 nm (Figure S1C, Figure S2C). When bathed in the solution of a 6 wt% H2O2, the characteristic LSPR absorption of the SiO2-shell coated AgNPs got decreased to 64% within 1 hour (Figure S2C), indicating that there exits dissolution of silver atoms for H2O2 can easily oxidize the Ag atoms.23-24 That is to say, it is most likely that the coating of AgNPs by the SiO2 is not complete, or there are some pinholes on the SiO2-coating layer outside the core of AgNPs.20 8


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Since AgSHINs act as an IR for the researches at single particle level in the DFM imaging, thus it has to guarantee the quality of AgSHINs. A noticeable phenomenon is that no further decrease of the characteristic LSPR absorption of the SiO2-shell coated AgNPs occurs, and the 64% of LSPR characteristic absorbance of the SiO2-shell coated AgNPs are always kept either with the time proceeding (Figure S2C). That is, the treatment of the SiO2-shell coated AgNPs with 6 wt% H2O2 can produce very stable core-shell structured plasmonic nanoparticles. Greatly different, the bare AgNPs are almost etched and the LSPR characteristic absorption band gets totally disappeared within 2 min (Figure S1C). In such case, we can conclude that the SiO2-shell coated AgNPs after the treatment of 6 wt% H2O2 can act as the AgSHINs IR, wherein the treatment of 6 wt% H2O2 is critical to the completion of the outside layer coating of the AgNPs core. The evidence for the importance of the treatment with 6 wt% H2O2 can be identified by amalgamation of silver atoms. DFM imaging results showed that the scattering light of all AgSHINs IR are very stable since no amalgamation of AgSHIN IR (the red circle marked in Figure 1A) occurs within 30 min when bathed in the solution of 1.0×103 M mercury (Figure 1ABC). As compared, the scattering intensity of some SiO2-shell coated AgNP (the red circle marked in Figure 1D) without the treatment of H2O2 get gradually increased (Figure 1E) and color red-shift (Figure 1F) within 30 min, making the DFM imaging spots brighter and brighter with time going on. Some SiO2-shell coated AgNPs (the blue circle marked in Figure 1D) without the treatment of H2O2 is stable (Figure S3) within 30 min. 9



Figure 1. DFM imaging analysis to identify the successful preparation of AgSHINs IR. (A) DFM images of AgSHINs IR treated by 1.0103 M mercury solution, and the real time analysis of the plasmonic scattering intensity (B) and RGB values (C) of a typical AgSHIN IR (the circled one in A) against time scale. (D) DFM images of SiO2-shell coated AgNPs without the treatment of 6 wt% H2O2 when treated by 1.0103 M mercury solution, and the real-time analysis of the plasmonic scattering intensity (E) and RGB values (F) of a typical SiO2-shell coated AgNPs (the red circled one in D) against time scale. The scale bar is 4 μm for all DFM images.

In the DFM images shown in Figure 1A, there have been no obvious changes in both scattering intensity (Figure 1B) and RGB values (Figure 1C) of AgSHIN IR in the presence of 1.0103 M mercury solution within 30 min, indicating that the coating of AgNPs with the SiO2 shell is well owing to the treatment of H2O2. It has known that the occurrence of amalgamation of AgNPs in mercury solution is very easy,25 thus, the stable plasmonic scattering properties of AgSHINs IR bathed in mercury solution are very easily understood because the SiO2 shell could completely isolate silver atoms from the mercury after the treatment of H2O2. 26-28 Differently, the DFM images of some SiO2-shell coated AgNPs without treatment of H2O2 show much brighter plasmonic scattering spots with time going on in the 10


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presence of 1.0103 M mercury solution (the red circle marked in Figure 1D), which can be expressed by the increased scattering intensity (Figure 1E) and red-shift (Figure 1F). Particularly, the percentages of blue in the RGB analysis are gradually decreased from 95% to 68%, but the percentages of green are increased from 2% to 31%. During which, the percentages of red have no significant change. That is, the color of blue is dominant as compared to both green and red. The occurrence of the much brighter plasmonic scattering spots (the red circle marked in Figure 1D) in the images is attributed to the direct amalgamation of sliver atoms through the incompletion of the SiO2 shell. And stable scattering properties spots (the blue circle marked in Figure 1D) is due to the completion SiO2 shell. Therefore, the treatment of 6 wt% H2O2 can verify whether each individual SiO2-shell coated AgNPs can act as AgSHINs IR as stated above or not, and in the following study, we apply blue values as the main color signal to research the reaction in DFM imaging by using AgSHINs stored in 6 wt% H2O2 as an IR.

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Figure 2. Plasmonic scattering colorimetry of mercury ions. The DFM images of AgNPs probes before (A) and after (B) the amalgamation reaction for 5 seconds as compared to the AgSHINs IR which are displayed at the upper right corners. It is obviously that the plasmonic scattering intensity of AgNPs probes gets increased and color gets red-shifted with the occurrence of amalgamation reaction. With the plasmonic scattering colorimetry, mercury ions in the range of from 1.0×1010 M to 1.0×104 M could be detected. The scale bar is 4 μm.

AgSHINs IR for Sensitive Detections of Mercuric Ions. At first, in order to identify that both AgSHINs IR and AgNPs probe are successfully deposited on a same glass slide since both of them display nearly same initial blue scattering features, AgSHINs IR and AgNPs probe has to be deposited successively (Figure S4A). Then, highly sensitive plasmonic scattering colorimetry of mercuric ions could be developed. For both AgSHINs IR (the blue circle marked in Figure S5A) and AgNPs probes (the red circle marked Figure S5B) deposited in the same region, comparable difference of the plasmonic scattering features came out immediately after the slide was bathed in 1.0×106 M mercury solution for 5 seconds (Figure S5C).25 Since AgSHINs IR are very stable as stated above (Figure 1ABC), it is obviously that significant difference is owing to the amalgamation of AgNPs probes. It has known that imaging effect depends on the exposure time. That is, the scattering intensity and color of plasmonic scattering images are very relevant to the 12


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exposure time of DFM imaging. In this case, if the exposure time of DFM imaging is set at 455 ms (Figure S5C), the image of AgSHINs IR and AgNPs probes could be available satisfactory. If set at 191 ms (Figure S5D), the scattering images of AgSHINs IR and AgNPs probes get very faint and it is hard to discern the scattering images of AgSHINs IR and AgNPs probes from the dark background. The faint scattering images of AgNPs probes available at 191 ms will give weak scattering intensity and the changeable scattering color, leaving a misconception that no amalgamation of AgNPs occurred. That is to say, improper set of exposure time will give results with deviations and even if with big errors, which might make researchers ignore the reaction or misunderstand reaction mechanism. On the basis of above discussions, we developed a novel plasmonic scattering colorimetry of, such as mercury ions, with DFM imaging (Figure 2). Owing to the use of AgSHINs IR, even if fluctuations resulting from the slightly deviation of exposure time, the significant comparison between the scattering signals of AgNPs probes and that of AgSHINs IR can easily stand out with the occurrence of the amalgamation of AgNPs probes. With that, we can successfully identify that the amalgamation of AgNPs probes can quickly occur at the mercury ions of 1.0×1010 M. In fact, it is unthinkable that so low concentration of detection of mercury ions can make the amalgamation of AgNPs probes occur. Therefore, using AgSHINs as an IR can disclose sensitive information which might be mistakenly regarded as detection deviations.

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Figure 3. Deviations caused by exposure time and focusing in DFM images and calibrations in terms of scattering intensity and color. (A) DFM Images of AgSHINs IR (a) and both AgSHINs IR and AgNPs probes (b-f) under different exposure time (b, c, d) and focusing (b, e, f). (B) Calibrated scattering intensity as compared to the uncalibrated ones. (C) Calibrated blue values in RGB system as compared to the uncalibrated ones. Two scattering spots (circled by red and green) in the images are selected to assess the calibration. Red and green pillars correspond to AgNPs circled with red and green in A respectively. The scale bar is 2 μm for all DFM images. Taking one of AgNPs (the spot circled with red in c) as the example,  was calculated to be 1.511 by Equation 1 according to the calculated  and Equation 3, we can obtain I cal . Similarly, with Equation 2 and 3,  could be calculated to be 1.367 and Vcal was expressed (the detail calculation progresses

see the Table S1). In order to display the results after calibration intuitively, different

scattering intensity of AgNP are used for comparison. The I / I 1.509 and the V / V

0

0

(red pillar in Figure 3B) is

(red pillar in Figure 3C) is 1.356, which means there is deviation when the

14


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focal plane is changed. The I cal / I 0 is 0.998 (red shadow pillar in Figure 3B) and the Vcal / V 0 is 0.992 (red shadow pillar in Figure 3B), both of which are about 1, implying the deviation from exposure time the focal planes can be eliminated. We treated other spots of AgNPs in DFM images (c, d, e, f) and obtained the same conclusions which means the calibrated Equations are feasibility (Figure 3BC).

AgSHINs IR for Confidence Level Improvements of DFM images. As our previous literature reported, there still are some inevitable deviations in the single nanoparticle level of DFM imaging analysis,17 which have become a technique obstacle for high sensitive detections because they inevitably exert effects on the confidence level of the detection results. For example, slight changes of the focal plane and exposure time would lead to significant different results in DFM imaging (Figure 3A). Even if the operators skillfully try their best to make the focus plane and exposure time suitable in DFM imaging, it still cannot guarantee that the DFM images have high qualities such as high contrast, resolution, and visibility because there still are some deviations from instruments in a long time monitoring (Figure S6). In order to improve the confidence level of the DFM images for quantitative analysis, two factors to calibrate the scattering intensity, , and to calibrate the color,

, are introduced to calibrate imaging deviation by analyzing the data in the DFM images, and the two factors can be defined as follow at the time of t.

 (t ) 

I IR (t ) I IR0

(1)

 (t ) 

VIR (t ) VIR0

(2)

We define the plasmonic scattering of AgSHINs IR at the initial time (t=0) as the 15



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0 standard ( I IR and VIR0 ), while that at other time (t) need to be calibrated for the

deviations ( I IR (t ) and VIR (t ) ), so α can be expressed as the ratio of the uncalibrated scattering intensity of AgSHIN IR ( I IR (t ) ) to the standard sacttering intensity of 0 AgSHIN IR ( I IR ) (Equation 1). Similarly,  is expressed as the ratio of the

uncalibrated RGB values of AgSHIN IR ( VIR (t ) ) to standard RGB values of AgSHIN IR ( VIR0 ) (Equation 2). It should be noted that there are R, G, B for any images in the RGB system. With the calibration factors, the calibrated scattering intensity of AgNPs probe ( I cal (t ) ) can be expressed as the ratio of the uncalibrated scattering intensity of AgNPs probe ( I (t ) ) to α (Equation 3) since at the time of t the conditions to affect the DFM imaging of both AgSHINs IR and AgNPs probe are the same.

I cal (t ) 

I (t )  (t )

(3)

Vcal (t ) 

V (t )  (t )

(4)

Correspondingly, the calibrated RGB values of AgNPs probe ( Vcal (t ) ) also can be expressed by the relationship between the uncalibrated RGB values of AgNPs probe ( V (t ) ) and  (Equation 4). That is, we can calibrate the plasmonic scattering signals of the AgNPs probe by using AgSHINs IR. Since the blue values of both AgSHINs IR and AgNPs probe spots dominate 92.91% and 93.96% in the DFM image (Figure S4), blue value of both AgSHINs IR and AgNPs probe were adopted as the primary color signal to calibrate in the following experiment for convenience. In order to verify the feasibility of the above Equations, the plasmonic scattering 16


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light of AgSHINs IR and AgNPs probe deposited in the very close region of a same glass slide were analyzed (Figure 3A), AgSHINs IR is marked with blue circle, the red circle and green circle represent two AgNPs which would be analyzed and calibrated. We define the DFM image b as standard, then we got the DFM images with increased and decreased exposure time (c, d in Figure 3A), and changed focal planes (e, f in Figure 3A) to simulate the deviations (Figure 3BC). The standard 0 plasmonic scattering light of AgSHINs IR ( I IR and VIR0 ) and AgNPs ( I 0 and V 0 )

were acquired from b, the uncalibrated plasmonic scattering light of AgSHINs IR ( I IR and VIR ) and AgNPs ( I and V ) were acquired from c, d, e, f respectively. In such case, the plasmonic scattering images of both scattering intensity and blue values of AgNPs probe could be calibtated by using AgSHINs IR, and thus a method could be established to eliminate deviations caused by the unsuitable exposure time and focusing, making us accurately calibrate the plasmonic scattering light of plasmonic nanoprobes. AgSHINs IR for Weak Oxidation Reaction Monitoring of AgNPs. It is well known that the oxidation of AgNPs in the presence of oxygen, even if in the air, results in the decrease of scattering light.17 However, the slow oxidization of AgNPs in solution resulting from low oxygen content at room temperature, the dissolved oxygen, for example, has been generally ignored for the slow oxidization fails to give off obvious signals. By using the calibration factors of  and , herein we present the real time information about the slow inconspicuous oxidation process of AgNPs in solution. 17



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Figure 4. Real-time monitoring weak oxidation of AgNPs at DFM imaging. (A) a represents the DFM image of AgSHINs IR, and b represents the DFM image of AgSHINs IR and AgNP deposited later. The DFM images of b, c, d, e and f are obtained at different reaction time. (B) Uncalibrated scattering intensity as compared to the calibrated ones. For the uncalibrated 0

scattering data, I / I values rise from 1.015 to 1.037 (red dots) and the data in the confidence interval (red shadow) of I / I

0

values increase within 120 min. For the calibrated ones, I cal / I 0

values decrease from 0.982 to 0.913 (blue dots) and the data in the confidence interval (blue shadow) of I cal / I 0 values decrease within 120 min. system as compared to the uncalibrated ones. V / V

0

(C) Calibrated blue values in RGB

values rise from 1.013 to 1.034 (red dots)

and the confidence interval (red shadow) reflects increased trend within 120 min. Vcal / V 0 values decrease from 0.982 to 0.913 (blue dots) and the confidence interval (blue shadow) also reflects decreased trend within 120 min. The scale bar is 2 μm for all DFM images.

As shown in the Figure 4A, the oxidization reaction was real-time monitored in solution at room temperature with DFM imaging. From these DFM images acquired by every five minutes until 120 min, and it is hard for us to visually find they have 18


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differences in terms of the scattering intensity and the scattering color (b, c, d, e, f in Figure 4A), misleading us that the slow oxidation reaction of AgNPs probe does not occur. With the introduction of calibration factors,  and , however, the scattering intensity and blue values of the AgNPs (the spot circled with red in Figure 4A) and the AgSHIN (the spot circled with blue in Figure 4A) were analyzed from all DFM images (Figure 4BC). For the uncalibrated data, I / I 0 and V / V 0 gets increase with time going on (red dots in Figure 4BC), the corresponding confidence intervals accordingly turn out increasing trend (red shadows in Figure 4BC). These results do not correspond to the fact that scattering light of AgNPs decreases gradually owing to oxidation progress. Delightfully, the calibrated data of I cal / I 0 and Vcal / V 0 reflect the slight decreased trend (blue dots and shadows in Figure 4BC). Thus we improved the confidence level of DFM imaging analysis and characterized the weak oxidization progress of AgNPs by calibrated the scattering intensity and blue values of AgNPs using AgSHINs IR. Conclusion In conclusion, AgSHINs as an internal reference are introduced to calibrate both the scattering intensity and scattering color of AgNPs probe during the reactions. Two calibration factors,  and , are successfully used to calibrate the scattering intensity and RGB values of AgNPs probe in the DFM images, respectively, making the confidence level of the DFM imaging analysis greatly improved. By using AgSHINs as the IR, the inconspicuous amalgamation of AgNPs probes bathed in dilute solution of mercury higher than 1.01010 mol/L and the very weak oxidation process of 19



AgNPs probes by dissolved oxygen in water have been identified. It is worth noting that almost all nanoparticles have the corresponding SHINs, and the use of SHINs as an IR thus can provide a precise data to discern or discover many inconspicuous slow reactions in nature through the DFM imaging analysis. Acknowledgements The authors are grateful to the National Natural Science Foundation of China (NSFC, No. 21535006). Supporting Information Apparatus, Reagents and Materials, various characterizations and comparison of scattering light of AgSHINs IR and AgNPs. References (1) Zhou, J.; Lei, G.; Zheng, L. L.; Gao, P. F.; Huang, C. Z. HSI colour-coded analysis of scattered light of single plasmonic nanoparticles. Nanoscale. 2016, 8 (22), 11467-11471. (2) Han, Y.; Lupitskyy, R.; Chou, T. M.; Stafford, C. M.; Du, H.; Sukhishvili, S. Effect of oxidation on surface-enhanced Raman scattering activity of silver nanoparticles: a quantitative correlation. Anal. Chem. 2011, 83 (15), 5873-80. (3) Smith, J. G.; Yang, Q.; Jain, P. K. Identification of a Critical Intermediate in Galvanic Exchange Reactions by Single-Nanoparticle-Resolved Kinetics. Angew. Chem. Int. Ed. 2014, 53 (11), 2867-2872. (4) Aris Spanos. The Discovery of Argon: A Case for Learning from Data? Philos. Sci. 2010, 77 (3), 359-380. (5) Lei, G.; Gao, P. F.; Yang, T.; Zhou, J.; Zhang, H. Z.; Sun, S. S.; Gao, M. X.; Huang, C. Z. 20


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For TOC only

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Inconspicuous Reactions Identified by Improved Precision of

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