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Rapid and Reliable Detection of Alkaline Phosphatase by a Hot Spots Amplification Strategy Based on Well-Controlled Assembly on Single Nanoparticle Yi Zeng, Jia-Qiang Ren, Shaokai Wang, Jiaming Mai, Bing Qu, Yan Zhang, Ai-Guo Shen, and Jiming Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09336 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

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ACS Applied Materials & Interfaces

Rapid and Reliable Detection of Alkaline Phosphatase by a Hot Spots Amplification Strategy Based on Well-Controlled Assembly on Single Nanoparticle Yi Zeng, a Jia-Qiang Ren, a Shao-Kai Wang, a Jia-Ming Mai, a Bing Qu, b Yan Zhang, c Ai-Guo Shen a, * , and Ji-Ming Hu a a

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. E-mail: [email protected]

b

Department of General Surgery, China Resources & WISCO General Hospital, Wuhan, 430080, P. R. China

c

Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan 430060, P. R. China

ABSTRACT: The first appeal of clinical assay is always accurate and rapid. For alkaline phosphatase (ALP) monitoring in medical treatment, a rapid, reliable surface-enhanced Raman scattering (SERS) test kit is designed based on a “hot spots” amplification strategy. Consisting of alkyne-tagged Au nanoparticles (NPs), Ag+, and enzyme substrate, the packaged test kit can achieve one-step clinical assay of ALP in human serum within several minutes, while the operation is simple as it directly inputs the sample into the test kit. Here, Ag+ ions are adsorbed onto the surface of Au core due to electrostatic interaction between Ag+ and the negatively charged donor surface, then enzymatic biocatalysis of ALP triggers the reduction of Ag+ and subsequently silver growth occurs on every Au core surface in a controllable manner, forming “hot spots” between the Au core and Ag shell, in which the SERS signal of alkyne Raman reporters would be highly amplified. Meanwhile, ALP mediates a redox reaction of Ag+ as well as the dynamic silver coating process so the increase of SERS intensity is well-controlled and can be recognised with increasing amounts of the targets. Instead of conventional NP aggregation, this leads to a more reproducible result. In particular, the distinct Raman emission from our self-synthesized alkyne reporter is narrow and stable with zero background in the Raman silent region, suffering no optical fluctuation from bio-system inputs and the detection results are therefore reliable with a limit of detection of 0.01 U/L (2.3 pg/mL). Along with ultra-high stability, this SERS test kit therefore is an important point-of-care candidate for a reliable, efficacious, and highly sensitive detection method for ALP, which potentially decreases the need for time-consuming clinical trials. KEYWORDS: alkaline phosphatase, SERS, one-step clinical assay, rapid detection, test kit, self-assembly on single NP

INTRODUCTION Alkaline phosphatase (ALP), widespread in mammalian tissues, catalyses dephosphorylation in various biomolecules and plays a vital role in clinical diagnosis. 1 ALP serves as a biomarker and indicates diseases in bone, kidney, liver, and even certain cancers. 2 Generally, ALP levels in adults are approximately 20-140 U/L,3 while abnormal mainly elevated levels of ALP are correlated to osteoporosis, leukaemia, hepatitis, lymphoma, etc. 4 Besides, ALP is widely used as an antibody binding label for enzyme immunoassays, 5 and is commonly used in the dairy industry as an indicator of successful pasteurisation, which has led to significant interest in developing a method for simple, accurate detection of this group of isoenzymes in clinical care or dairy production. Actually, nanomaterials for diagnostics and therapeutics based on various methods have been widely investigated. 6-10 Specifically, substantial efforts have been made in the development of analytical methods for monitoring ALP, such as fluorescence, 11-13 colorimetry, 14-17 surface-enhanced Raman scattering (SERS), 18-20 and electrochemical means, 21, 22 mainly based on the unique signal output from products generated by enzymatic hydrolysis of substrates. Taking a panoramic view of the current analytical approaches, several research projects have exploited the intrinsic virtue of those substrates and the corresponding optically active or electroactive products, while others focus on transformation capacity (reducibility) from the products of ALP-mediated dephosphorylation. Typically, these sensing platforms always demand sophisticated preparation techniques or long incubation times. Thus, it is deemed necessary to explore simple, rapid, reliable sensing systems without complicated fabrication demands for further application in actual operations.

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Aiming at a fast, convenient, accurate ALP strategy for medical diagnosis, we designed a novel surface-enhanced Raman scattering (SERS) test kit based on a “hot spots” amplification strategy. Consisting of Au nanoparticles (NPs), Ag+, and an enzyme substrate, the packaged test kit can achieve one-step clinical diagnosis of ALP in human serum within several minutes. Meanwhile, the operation is simple, involving directly sample input to the test kit. In the SERS-based methods for ALP reported before, 18, 20 complex preparation and long detection time were always in demand. The construction of sensing platform was complicated and time-consuming, and the poor reproducibility made the platform less reliable, along with overlong detection time (over 1h), it seems difficult for those platforms to be practical in clinical application. Here, our developed system showed better performance with extremely simple operation and short detection time, which is important for clinical application of biomolecules. Together with linear range and sensitivity suited for real sample detection, this SERS test kit is an important point-of-care candidate for a reliable, efficacious, and sensitive detection method for ALP. Apart from the appeal for convenience, we combine enzyme-mediated silver growth at nanoscale with a well-controlled “hot spots” amplification strategy to propose a more reliable SERS sensing platform. Reproducible and controllable growth of noble metal nanostructures can be mediated by DNA and enzyme, resulting in different nanostructure with well-defined physical and chemical properties such as high SERS enhancement.23-26 The enzyme-mediated redox active metallisation, 27 including enzyme induced growth of gold or copper NPs and silver deposition, for ALP particularly, often originates from the reducing product of enzymatic dephosphorylation. In general, SERS platforms are of high sensitivity but suffer from poor reproducibility due to typical NP aggregation. Thus, in this novel SERS test kit, instead of normal aggregation/segregation theory, a more reliable detection rationale is created

Figure 1. (A) One-step ALP detection by the as-prepared SERS test kit, which consists of alkyne-tagged Au NPs, Ag+, and AAP. The detection is based on enzyme-mediated redox active silver growing on the surface of single Au NPs. The figure shows the specific process of increasing “hot spot” counts generated between the Au core and Ag shell. The insets show corresponding TEM and HRTEM images. (B) Dynamic diameter distribution of the SERS sensing platform before, and after, addition of ALP. Average hydrate particle sizes: 25.85 nm and 30.26 nm respectively. (C) Stability and reproducibility test over a one month period (CV % = 5.09). by focusing on the self-assembly on single NPs. The enzymatic hydrolysis product could induce well-organised silver growth on the surface of every single alkyne-tagged Au NPs, which would generate “hot spots” between the Au core and Ag shell, as a consequence an obvious SERS signal can be recognised in response to the addition of ALP. 28 In this way, biomolecule quantification can be successfully transferred to a controllable enzyme-mediated silver coating process, and finally, an optical signal output. Our developed assay is rapid and simple as the whole process can be finished within several minutes. This SERS test kit is therefore an important point-of-care candidate for a reliable, efficacious, and highly sensitive ALP-detection method, which potentially decreases the need for time-consuming clinical trials.


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EXPERIMENTAL SECTION Chemicals and Reagents. Chloroauric acid (HAuCl4), ascorbic acid (C6H8O6), silver nitrate (AgNO3), diethanolamine (C4H11NO2, DEA), were purchased from Sinopharm Chemical Reagent Company (Beijing, China). Trisodium citrate (C6H5Na3O7, TSC), Bis (p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt (C18H13K2O6PS2·2H2O, BSPP), 2-Phospho-L-ascorbic acid trisodium salt (AAP) were obtained from Sigma-Aldrich (St Louis, MO). Alkaline phosphatase (ammonium sulfate suspension, ALP) was supplied by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Human serum samples were purchased from Y-J Biological, Shanghai. Clinical specimens were obtained directly from the Department of General Surgery, China Resources & WISCO General Hospital in Wuhan. The alkyne reporter: 4, 4'-(1, 4-phenylenebis (ethyne-2, 1-diyl)) dibenzenethiol was synthesised in our laboratory (see Supporting Information). All reagents were of analytical reagent grade and were used without further purification. Ultrapure water was obtained by using a Milli-Q ultrapure (resistance > 18.2 MΩ cm) system. Instruments. The Raman spectra were collected by a Renishaw inVia-Plus Raman microscope with excitation at 532 nm with the solution installed in a capillary glass tube. The laser output was focused on sample by a 20 × objective lens. The slit width of the spectrograph was 75 μm. The morphology and microstructure of NPs were characterised by high-resolution transmission electron microscope (TEM, JEM-2100, JEOL, Japan) operating at a 200 kV accelerating voltage. UV–vis studies were performed on a UV–vis spectrophotometer (UV-2550, Shimadzu, Japan) at room temperature using a 600-μL black body quartz cuvette with a 1 cm path length. Preparation of alkyne-coded BSPP-protected Au NPs. The specific synthesis procedure for alkyne reporter: 4, 4'-(1, 4-phenylenebis (ethyne-2, 1-diyl)) dibenzenethiol is illustrated in the Supporting Information and previous reports. 29, 30 Au NPs with a diameter of 20 nm were prepared by reduction of HAuCl4 using sodium citrate. BSPP (3 mg) and alkyne reporter (30 μL, 2 mM) were added to a 10 mL solution of Au NPs, followed by shaking overnight at room temperature. The resulting mixture was centrifuged at 6000 rpm for 20 min, and the supernatant was removed. BSPP-protected Au NPs were then re-suspended in 100 μL water, and stored in a refrigerator at 4°C until used. SERS detection of ALP. The detection system consists of AgNO3, Au NPs, and AAP in a DEA buffer (50 mM, pH 9.8, containing 0.1 mM MgSO4). The SERS sensing platform was constructed by mixing these reagents in certain concentrations. Different amounts of ALP were added to the mixture to make a final volume of 50 μL and then the SERS spectra of these suspensions were recorded directly after incubation at 37°C for a designated time. Experimental parameters of the enzymatic reactions, such as the pH of the buffer, reagent concentration, and incubation time, were all considered.

RESULTS AND DISCUSSION Sensing Mechanism of the One-Step Clinical Assay for ALP by SERS Test Kit. The one-step clinical assay process for ALP detection is shown in Figure 1A. Firstly, according to our previous work, 29, 30 taking full advantage of SERS, alkyne reporter molecules were employed here instead of conventional Raman reporters by attaching them onto the Au NP surface, thus the SERS spectrum for quantification originates with the alkyne tags (the entire SERS spectrum of the alkyne reporter is shown in Figure S1). The distinct Raman emission from our self-synthesised alkyne reporter (4, 4'-(1, 4-phenylenebis (ethyne-2,1-diyl)) dibenzenethiol) is narrow and stable in the Raman silent region with almost zero background, and it suffers no fluctuation from bio-system input, and thus shows high reliability compared to other optical methods. We designed an efficacious SERS test kit that simply consists of alkyne-tagged Au NPs, Ag+, and the ALP substrate AAP. The detection platform is rapid, and convenient, because it works as a one-step assay (adding the ALP-containing sample and mixing thoroughly). It only takes a few minutes for the test kit to respond, as demonstrated by a change in solution colour from red to orange and the amplification of SERS intensity. In detail, first and foremost, as high enzyme activity in ALP requires optimal alkaline conditions, to avoid aggregation of NPs in our experimental settings (pH>9), we protect Au NPs with BSPP to ensure that they are stable in an alkaline buffer environment, meanwhile the Au NPs are functionalised by the alkyne reporter. Then the test kit is packed as mentioned, and during a pre-incubation phase, sufficient Ag+ ions are adsorbed onto the surface of the Au core due to electrostatic interaction between the positively charged Ag+ and the negatively charged donor surface. With this test kit, once ALP is added, enzymatic biocatalysis will turn the enzyme substrate AAP into L-ascorbic acid (AA), which triggers the reduction of Ag+ into Ag0 and generates an Ag shell around the Au core. Here, “hot spots” are formed between the Au core and Ag shell, in which the SERS signal of alkyne Raman reporters would be highly amplified. Specifically, with incremental addition of ALP, the silver coating on each Au NP is well-organised and controllable. The specific process behind this outer silver shell formation and the corresponding TEM and HRTEM images are shown in Figure 1A. Since a certain optimal amount of Ag+ has already adsorbed on the surface of the Au core during



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pre-incubation, the reduction of Ag+ occurs on the surface of the Au rather than forming nucleation sites in solution, 31 so that the core-satellite structure of the Au–Ag composite NPs is formed ab initio. With increasing target concentration, the structure gradually grows from an Au-Ag core-satellite nano-assembly into Au@Ag NP with partly-coated shells, and finally fully-coated Au@Ag core-shell NPs. TEM and HRTEM are used to characterise the process of silver growth at the nanoscale, which demonstrates strong correlation with the inferred rationale. The integrated HRTEM images are shown in Figure S2. According to the sensing mechanism and HRTEM image, we considered that the Au@Ag structure was gapless. Gapless nanostructure can also induce strong field enhancement, although it is weaker than nanostructure with gap inside.28, 32 The synthesis of hollow nanostructure with nanogap requires special reaction condition such as DNA-mediated growth and via the kirkendall effect. 28, 32 But the sensing mechanism in this platform was the reduction of Ag+ ions on the surface of Au core, thus no visible gap was formed. Here, the alkyne Raman reporters adsorbed on the surface of Au NPs so there was a self-assembly reporter molecular layer between Au core and Ag coating. And the chemical environment of alkyne reporter molecules is gradually converted from the surface of the Au NPs to the “hot spots” between the Au core and Ag shell, which leads to large amplification of the SERS signal. Hence, a well-controlled self-assembly on single NPs has been accomplished to achieve reliable ALP sensing. To confirm this point further, a comparison of dynamic diameter distributions of the sensing platform before, and after, ALP addition is carried out (Figure 1B). The particle diameter distribution of hydrated NPs remains steady within a certain range, with only slight diameter changes observed from 25.85 nm to 30.26 nm, which confirms the silver-coating process as having occurred on each Au NP. Meanwhile, no aggregation occurred in this process since there were no larger nanoclusters found in the particle diameter distribution and TEM images. Integral TEM images are also shown in Figure S3 to reflect the condition of NPs in solution and to trace the reaction trends. Aiming at practical use, any test kit should be stable and easy to store, thus the stability of the developed test kit is evaluated by 30 Raman measurements over a one month period (Figure 1C). During one month, 30 SERS spectra are collected every day from one as-prepared test kit solution. In particular, the mapping image denotes the 30 spectra merged together from bottom to top, while the bright-white zone represents the location of the quantifiable peak, specifically at 2209 cm−1. The results indicate that the test kit was stable, with the percentage coefficient of variation (CV %) calculated as being 5.09. From the morphological perspective (Figure S3), the as-prepared stabilised Au NPs remain well-dispersed, both in the absence, and presence, of ALP, basically owing to the simple, compact composition of the test kit. To understand the sensing rationale behind this strategy, here two conceivable reaction mechanisms are taken into consideration. Technically, as shown in Figures 1A and 2, there are two hypothetical growth patterns of Ag+: “gradual coating” as mentioned above and “one-step full coating”, which depends on the surface conditions of the Au core. The surface charge density of the Au core plays an important role here by influencing the amount and density of Ag+ adsorbed on the surface. With more Ag+ being adsorbed, a reducing reaction on every preformed Au core surface triggers silver growth at nanoscale. Generally, the as-prepared Au NPs remain stable owing to the protective effect of the negatively charged citrate and BSPP, which is relatively unconsolidated. To reveal the effect of surface charge density, heparin, with the highest negative charge density of any known biological molecule, is used here to functionalise the Au core, which is an attempt to change the surface charge density of Au NPs to the greatest possible extent. Parallel experiments are performed and it works slightly differently in each trial. For the heparin-functionalised Au NPs, the reaction scheme, TEM images, and SERS response are shown in Figures 2A-C, which demonstrate the morphological and structural changes involved during enzyme-mediated metallisation. Owing to the high negative charge density of heparin, Ag+ ions are densely and uniformly adsorbed on the surface of the Au NPs, resulting in an entire surrounding silver coating layer being formed ab initio, and the increasing target concentration only generates a thicker Ag layer, which shows no amplification of the SERS intensity and therefore contributes virtually nothing to ALP sensing. The parallel SERS experiments prove that the signal intensity remained constant after a short initial increase (Figures 2D and 2E). The surface charge property of Au NPs plays an important role in stability and deposition of Ag+ in the test kit, and further is one of the key factors that influence the quality control of the proposed SERS test kit.


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Figure 2. (A-C) Reaction scheme showing morphological, and structural, changes involved in the heparin-mediated ALP sensing platform. (D-E) The corresponding SERS result of ALP sensing: this was found to have been invalid. To test both the feasibility and reliability of this system, UV–vis and Raman spectroscopy are used to explore the changes that Raman reporter functionalised Au NPs experience during ALP-mediated dephosphorylation. UV–vis absorbance and SERS spectra of Au NPs before, and after, the addition of the targets are shown in Figure S4. Generally, after ALP triggers Ag+ reduction, a single, narrow SERS band at 2209 cm-1 originating from the alkyne reporter is highly enhanced, and the SPR absorption bands at c. 520 and 390 nm suggest the existence of Au NPs and an Ag shell. The necessity of Au NPs has been proved by control experiment. The Au core is essential for the Ag+ to attach, or the Ag+ would be reduced in the buffer solution and self-nucleate into nanoclusters with no “hot spot” effect, as SERS enhancement demands a sufficiently large substrate size. In fact, the reduction of Ag+ to Ag0 in the presence of Au NPs can also lead to obvious colour changes in the solution, which establishes the foundation of this colorimetric assay, so a series of preliminary experiments was performed with the aid of UV–vis spectroscopy (Figure S5). As a consequence, with increasing ALP concentration, a peak denoting the presence of silver at c. 390 nm is observed while the peak for the Au NPs blue-shifts to within 500-520 nm owing to the growing silver coat. However, the peak denoting the silver that ought to be used for quantification is unsuitable. As we collect three groups of detection systems during different reaction times (1 min, 10 min, and 30 min), SPR absorption peaks are all broad and continued to red-shifting within a certain range (c. 370-400 nm), thus it would be inaccurate when a user were to select a peak among the range for subsequent quantitative analysis. That is why we doubted the reliability of quantification in this way and thus chose SERS as a more convincing strategy. Generally, SERS allow quantification from a narrow peak which ensures excellent reliability. Here, our as-prepared zero-background strategy is accomplished utilising an alkyne reporter, whose SERS peak is narrow and singular in the Raman silent region (c. 1800-2800 cm-1), with no need to suffer from uncertainties in quantifiable information or interferences from bio-systems. Optimisation of Experimental Parameters. According to the detection rationale, experimental parameters that influence the reaction efficiency are clear. The amplified SERS intensity at 2209 cm-1 comes from the silver deposition caused by reducing product AA generated from enzymatic hydrolysis of the AAP substrate. Thus, the concentration of reacting agents AAP and Ag+, the concentration, and pH, of the buffer solution, reaction temperature, and other variables, are all taken into account and optimised.



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Figure 3. The influence of changes in the experimental parameters on SERS intensity at 2209 cm-1: (A) AAP concentration, (B) Ag+ concentration, (C) pH of the buffer solution, (D) DEA concentration, and (E) reaction temperature. The error bars represent standard deviations based on three independent measurements. As shown in Figure 3, the amplitude of the corresponding SERS signal is calculated from the difference R - R0 (R0 and R are the SERS intensities before, and after, ALP addition, respectively). Within a certain range, higher concentrations of AAP and Ag+ could accelerate the hydrolysis and reduction processes, and in consequence, a higher reaction efficiency is achieved. However, excess Ag+ may lead to a high ion-strength system and render the Au NPs unstable, resulting in a higher R0 (for a system without ALP), so the amplified SERS value R - R0 decreases. In particular, the silver concentration with the best performance is the point at which enough Ag+ ions have been absorbed onto the surface of the Au NPs without a surplus thereof remaining in solution to form silver nanoparticles (see dynamic range in Figure 1B). Besides, as ALP is most effective in an alkaline environment, considering the stability of the test kit, and to prevent hydrolysation of metal ions, DEA is selected as the buffer solution because it can form complexes with Ag+ to ensure the participation of Ag+ in the reduction reaction instead of forming AgOH precipitate in alkaline conditions. Moreover, the reaction temperature is also evaluated as enzymes are temperature-sensitive. ALP, existing in various mammal organisms, shows best activity at an inflection point of 37°C, after which enzyme inactivation takes the place of an acceleration effect from higher temperatures. As a result, the experimental parameters are set as follows: 1 mM AgNO3, 5 nM Au NPs, and 3 mM AAP in 50 mM pH 9.8 DEA buffer, at a reaction temperature of 37°C. Quantification of ALP by SERS Assay. To evaluate the influence of time on the reaction, a series of control experiments were conducted (Figure S6). The SERS intensity at 2209 cm-1 was continuously recorded for 40 min after adding different concentrations of ALP under optimal conditions. With increasing ALP concentration, the SERS signals were gradually amplified. When the concentration of ALP was less than 1 ng/mL, the signal increased continuously at different rates for over 40 min, but at concentrations of greater than 1.8 ng/mL, the reaction was too intense to be measured, resulting in uncontrolled aggregation of NPs, as evinced by the grey-black colour of the solution and suddenly declining SERS signal after 15 min. Based on that, a Raman spectroscopic homogeneous assay was performed for quantitative SERS detection of ALP in an aqueous buffer solution. Typical SERS spectra of ALP at different concentrations are shown in Figure 4A. The enhanced SERS response of ALP-induced “hot spot” generation is observed upon the addition of ALP to the SERS-based sensing platform, which also clarifies the specific Ag growth process. Figure 4B shows the SERS intensity at 2209 cm-1 versus the concentration of ALP after 10 minutes, and the inset shows the corresponding linear plot. Here, the enhanced SERS intensity at 2209 cm-1 varied with ALP concentration in an exponential manner, thus a semi-log plot was used to show that the constant logarithm of SERS intensity at 2209 cm-1 displays an excellent linear correlation with ALP concentrations from 0.18 to 0.75 ng/mL (0.72 to 3 U/L) with the calibration equation being: y = 0.87x + 2.83 (R2 = 0.996). The limit of detection (LOD) is 2.3 pg/mL (0.01 U/L) at a signal-to-noise ratio of 3, which is four to five orders of magnitude lower than the clinical ALP levels in normal adults (about 20–140 U/L). The detection range seems to be limited due to the rationale. The detection is valid during the silver coating forming stage. After the silver coating has occupied all the


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surface of the gold core as Au@Ag NPs, the SERS signal would not increase anymore even the added ALP leads to thicker silver shell. And actually, the LOD can be further optimised by extending the incubation time, because it can be inferred that this longer time will allow a lower concentration of ALP to react more thoroughly and present recognisable signal amplification according to the time-based kinetics study. However, taking practical application into account, we chose a relatively short reaction time (10 min) over which to analyse the response. Clinical diagnosis often regards rapid operation and accurate results as most appealing. To develop a sensing strategy aimed at practical use, we sacrificed LOD in the name of rapidity, and simplicity, of detection given that the LOD is already low enough for the standard of clinical application as most diseases are correlated to elevated ALP levels.

Figure 4. (A) Typical SERS spectra from the as-prepared test kit in the presence of ALP with different concentrations from a to f: 0.09, 0.45, 0.54, 0.75, 1.80, and 3.60 ng/mL; (B) SERS intensity change at 2209 cm-1 against concentration of ALP, where the inset shows the linear plot of the constant logarithm of Raman intensity against ALP concentration. The error bars represent standard deviations based on three independent measurements. Selectivity Test. To evaluate the selectivity of this SERS platform, a series of control experiments between ALP and other sources of interference were carried out (Figure S7). Potential co-existing substances are separately detected under optimised conditions, including bovine serum albumin (BSA), adenosine triphosphate (ATP), glucose (Glu), glucose oxidase (GOx), horseradish peroxidase (HRP), trypsin (TRY), and uric acid (UA), and the concentrations were set so as to be five times greater than that of the ALP. As a consequence, except for ALP, no silver deposition and signal amplification was observed in the presence of such interferences. This indicated that the as prepared SERS assay was highly specific for ALP due to the enzyme catalytic selectivity towards substances.



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Figure 5. (A) The determination of ALP in commercialised human serum samples using the method. (B) The determination of ALP in clinical human serum samples using the method. Real Sample. To test the utility of this approach in real clinical diagnoses, we trialled it on not only commercialised human serum samples but also clinical blood specimens (Figure 5). For the former one, considering the form of the matrix in serum, the samples were first filtered through 0.22 μm Millipore filters to remove any particulate suspension. The samples were then diluted (100-fold) in water. As no response was observed directly from this diluted sample, it was clear that, in human serum samples, the amount of ascorbic acidthe potential source of interferencewas too small to have an effect. As a consequence, our real sample was spiked with a series of ALP solutions at different concentration for further detection. In particular, as the alkyne reporter was employed here, the distinct Raman emission for quantification was narrow and stable with zero background in the Raman silent region, suffering no fluctuation from other bio-system inputs. Therefore, the percentage recovery (between 98% and 105%) was deemed acceptable using the as-prepared SERS platform (Table S1). Furthermore, clinical specimens were also finally addressed. Five fresh blood samples from hepatitis patients were obtained directly from local hospital, and a little serum of every samples were a drop added in the proposed SERS test kit to perform ALP assay according to the above-mentioned procedures after necessary deproteinization operations. The detection results are given in Table S2, which agree fairly well with the clinical testing results achieved in laboratory department of the hospital. Although the relative errors possibly caused by too simple sample pre-treatment are somewhat significant, this rapid, reliable and highly sensitive sensing system maintains the potential for further application in actual operations.

CONCLUSION In summary, we present a novel reliable SERS platform for ALP based on controllable “hot spot” amplification initiated by enzyme-mediated redox active metallisation. The efficient reducing agent produced from specific enzymatic dephosphorylation leads to both sufficient sensitivity, and selectivity, for clinical use. SERS is used to monitor the dynamic process driving the well-organised silver coating of the gold NPs and revealed the detection rationale


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underpinning the assay design. Meanwhile, the steady narrow Raman band, as well as the single signal output in the Raman silent region, with zero-background from the alkyne reporter, showed high reliability. In particular, this strategy was able to balance LOD and response time, which potentially decreased the need for time-consuming clinical trials. Hence, with the observed stability over a one month period, its simple, rapid operation, satisfactory LOD, and accuracy, we envision the “hot spot” amplification SERS platform as being valuable to those involved in biomedical diagnostics. The proposed SERS test kit could be an important point-of-care candidate for the reliable, efficacious, and sensitive detection of ALP.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available, free of charge, on the ACS Publications website. Figure S1-S9, Table S1-S2, and the Appendix cover the experimental work on the self-synthesis of the alkyne reporter (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT We acknowledge the financial support from the National Natural Science Foundation of China (Grants 21475100, 81471696, 41273093, and 21175101)

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