Facile and Sensitive Glucose Sandwich Assay Using In Situ

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Facile and Sensitive Glucose Sandwich Assay Using in Situ Generated Raman Reporters Xiaoshuang Bi, Xuezhong Du, Jingjing Jiang, and Xuan Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504653x • Publication Date (Web): 12 Jan 2015 Downloaded from http://pubs.acs.org on January 21, 2015

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Analytical Chemistry

Facile and Sensitive Glucose Sandwich Assay Using in Situ Generated Raman Reporters

Xiaoshuang Bi, Xuezhong Du,* Jingjing Jiang, and Xuan Huang

Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, and School of Chemistry and Chemical Engineering, Nanjing University 210093, People’s Republic of China

Corresponding Author E-mail: [email protected]

1

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ABSTRACT A facile and sensitive glucose sandwich assay using surface-enhanced Raman scattering (SERS) has been developed by the use of the self-assembled p-mercaptophenylboronic acid (PMBA) monolayer on a smooth gold-coated slide and the SERS tags of Ag nanoparticles (AgNPs) modified with p-aminothiophenol (PATP) and PMBA. The photocoupling product 4,4’-dimercaptoazobenzene (DMAB), generated in situ from PATP on the AgNP surface during the SERS measurement, possessed considerably intense characteristic SERS peaks and acted as the actual Raman reporter, which improved the sensitivity of glucose detection devoid of interference of other biomolecules. The facile sandwich assay showed a high selectivity of glucose over fructose and galactose. This facile, sensitive, and selective SERS-based glucose sandwich assay can be developed into a diagnostic tool for determination of glucose levels.

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INTRODUCTION Diabetes is a widespread metabolic disease that is characterized by elevated blood glucose levels. Diabetes has become one of the most serious threats to human health worldwide. According to the World Health Organization (WHO) statistics up to Nov. 2013, there are 347 million people suffering from diabetes, and 3.8 million people will die for it every year. Electrochemical method has been the most successful analytical technique to detect glucose using glucose oxidase (GOX) or GOX/glucose dehydrogenase (GDH) based on generated hydrogen peroxide.1,2 One of the disadvantages of this method is to use enzymes, which suffer from poor storage stability and loss of biological activity as well as high cost.3 Another one is that the nanomaterials on the modified electrodes can easily fall off and the electrodes may also be inactivated by the generated hydrogen peroxide (H2O2). A facile and enzyme-free assay is highly desired. Thus, it is of great importance to develop a new strategy to detect glucose sensitively and selectively. Surface-enhanced Raman scattering (SERS) spectroscopy has recently attained considerable prominence as a powerful and nondestructive analytical technique for highly sensitive and selective detection of molecules of interest, even on the single-molecule level.4−8 Van Duyne and co-workers9,10 made pioneering researches on label-free SERS detection of glucose with the alkanethiolate tri(ethylene glycol) monolayers on a silver film over nanospheres (AgFON) as a partition layer, which preconcentrated glucose near the SERS-active substrates. However, the sensitivity and selectivity of glucose detection were limited because of inherently small Raman scattering cross section of glucose (5.6 × 10−30 cm2/molecule/sr)11 and nonspecific capture of glucose. Currently, the Raman-label detection 3



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is the mostly used method for the assays of biomolecules because strong characteristic SERS signals can be generated by the Raman reporters with large Raman scattering cross sections than by the direct detection (label-free) method.12−14 Boronic acids have long been known to form cyclic boronate esters with 1,2- or 1,3-diols, such as saccharides and others containing diols.15 The formation of stable boronic acid−diol complexes requires the presence of syn-periplanar hydroxyl groups for preferential binding of saccharides in the α-furanose form.16,17 There are two sets of syn-periplanar diol groups present in α-D-glucofuranose, thus glucose can form a 1:2 complex with two boronic acids.18 There are numerous reports that utilized the ability of glucose to form cyclic boronates with diboronic acids for the selective sensing of glucose over other saccharides.17,19,20 Recently, Olivo and co-workers demonstrated the use of a triosmium carbonyl cluster−boronic acid conjugate (Os-BA)21 or an alkyne-functionalized boronic acid (alkyne-BA)22 as a mid-IR probe in novel assays for glucose, the C≡O stretching vibration (ca. 2100 cm−1) and the C≡C stretching vibration (ca. 2000 cm−1) of which fell into the Raman-silent region (1800−2200 cm−1). p-Mercaptophenylboronic acid (PMBA) was immobilized onto bimetallic film over nanospheres (BMFON), which was prepared by deposition of polystyrene nanospheres onto a glass slide followed by sputtering of gold and silver, and its boronic acid group functioned as the carbohydrate receptor.21,22 Os-BA or alkyne-BA was used as the secondary carbohydrate receptor in the SERS-based sandwich assay.21,22 Although the Raman signals were relatively devoid of interference from any functional groups of biomolecules, the Raman signals detected in the silent region were rather weak with the detection limit of 0.1 mM in the two cases21,22 because of inherently weak 4


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Raman signals of the carbonyl and alkyne21−23 and separation of the probes from the SERS-active BMFON substrates. Herein, we reported a facile and sensitive glucose sandwich assay using self-assembled PMBA monolayers on smooth gold-coated silicon slides and SERS tags, which were composed of silver nanoparticles (AgNPs) modified with the mixture of p-aminothiophenol (PATP) and PMBA (Schemes 1 and S1 in the Supporting Information). PATP was used as an apparent Raman reporter, but its photocoupling product, 4,4’-dimercaptoazobenzene (DMAB), was generated in situ upon illumination of laser during the SERS measurements24,25 and possessed considerably strong characteristic Raman shifts completely different from PATP or PMBA. The in situ generated Raman reporter (DMAB) with remarkably large Raman scattering cross section improved the sensitivity of glucose assay devoid of the interference of relevant biomolecules. The combination of the smooth gold-coated slides and the AgNP-based SERS tags, both of which were easily prepared, were used to replace the sophisticated SERS-active substrates, and PMBA was used as both first and second carbohydrate reporters for glucose sandwich assay to improve selectivity.

EXPERIMENTAL SECTION Reagents and Chemicals. Silver nitrate (AgNO3, >99.8%) was purchased from Shanghai Shenbo Chemical Reagent Co., Ltd. (China). Trisodium citrate dehydrate (99%), anhydrous ethanol, hydrogen peroxide (30%), and sulfuric acid (98%) were purchased from Nanjing Chemical Reagent Co., Ltd. (China). PMBA and PATP were purchased from Sigma-Aldrich. D-Glucose (>99%), D-fructose (>99%), and D-galactose (>99%) were 5



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purchased from Sinopharm Chemical Reagent Co., Ltd. (analytical grade). All of the reagents were used as received. Synthesis of AgNPs. The AgNPs with an average diameter of 60 nm were synthesized according to the Lee and Meisel’s method with a slight modification.26 All glassware used were thoroughly cleaned with freshly prepared aqua regia (HNO3/HCl, 1:3, v/v) followed by washing with copious double-distilled water and drying for use. Aqueous AgNO3 solution (1 mM, 200 mL) was heated to boil, 4 mL of 1% aqueous sodium citrate solution was then rapidly added, and the solution was kept boiling for 1 h followed by cooling to room temperature under stirring to obtain AgNP colloidal solution with a color of yellow-green. The AgNP colloidal solution was used after removal of larger AgNPs from the as-synthesized AgNP colloidal solution by centrifugation at 1500 rpm for 10 min. Vulcanization of AgNPs. A total of 50 µL of aqueous Na2S solution (2 mM) was added to 10 mL of the AgNP colloidal solution under vigorous stirring for 10 min and then allowed to age without agitation for 1 h. Eventually, the solution was centrifuged at 6500 rpm for 10 min and washed with double-distilled water twice, followed by redispersing in 10 mL of water. Preparation of SERS tags (AgNP/PATP-PMBA (2:1)). The concentration of AgNPs in the colloidal solution could be estimated to be approximately 3.6 × 10−11 M using UV-vis spectroscopy with a molar extinction coefficient of 3 × 1011 M−1 cm−1.27 If the total amounts of PATP and PMBA added were smaller than the required amounts for a full monolayer of PATP and/or PMBA (average molecular area of 0.25 nm2) on the AgNP surfaces, it was conjectured that the amounts of modified PATP and/or PMBA on the AgNP surfaces were 6


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equal to the added amounts. The SERS tags were prepared by modification of AgNPs with PATP and PMBA with a molar ratio of 2:1. A total of 10 µL of the mixture of PATP (1 mM) and PMBA (0.5 mM) in ethanol was added to 10 mL of the AgNP colloidal solution under vigorous stirring for 10 min and then allowed to age without agitation for 12 h. After that, the AgNP/PATP-PMBA (2:1) colloidal solution was centrifuged at 6500 rpm for 10 min and washed with double-distilled water twice, followed by redispersing in 10 mL of water. The total surface coverage (θ) of PATP and PMBA on the AgNP surfaces was estimated on the basis of the total surface area (S) of AgNPs and the total amounts (n) of added PATP and/or PMBA,

θ = 0.25nNa/S = 0.25n/(CAgVπd2) where Na is Avogadro constant, CAg is the concentration of AgNPs, V is the volume of AgNP colloidal solution, and d is the average diameter of AgNPs. The surface coverage of PATP and PMBA on the surface of AgNP/PATP-PMBA (2:1) was calculated to be 0.92. For the control experiments, the identical amount of PMBA or PATP was added. A total of 10 µL of the mixture of PATP (0.5 mM) and PMBA (0.5 mM) in ethanol was added to 10 mL of the AgNP colloidal solution to prepared AgNP/PATP-PMBA (1:1) with a surface coverage of 0.61. 10 µL of PMBA (0.5 mM) in ethanol was added to 10 mL of the AgNP colloidal solution to prepared PMBA-modified AgNPs with a surface coverage of 0.31. Similarly, 10 µL of PATP (0.5 mM) in ethanol was added to 10 mL of the AgNP colloidal solution to prepared PATP-modified AgNPs with a surface coverage of 0.31. Fabrication of Gold-Coated Silicon Wafers. The gold substrates used in this work were prepared by evaporation of gold using an electron beam evaporation system onto clean 7



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single-crystal silicon (111) wafers (100 µm in diameter and 525 µm in thick) to obtain thin gold films of 100 nm thickness. The gold-coated silicon wafers were cut into approximately 1 × 1 cm2 and then cleaned by immersing in piranha solution (H2O2/H2SO4, 1:3, v/v) for 1 h followed by washing with water and drying with a stream of nitrogen. Self-Assembly of PMBA on Gold-Coated Silicon Wafers. The clean gold-coated silicon wafers were immersed in the anhydrous ethanol containing PMBA (5 mM) for 24 h to form self-assembled monolayers (SAMs). Afterward, the SAMs on the slides were washed thoroughly with anhydrous ethanol and dried in air. Glucose Sandwich Assay. The PMBA-functionalized gold-coated silicon wafers were incubated with aqueous glucose solutions of different concentrations for 3 h to reach binding equilibrium followed by washing with double-distilled water to remove unbound glucose. Then, the glucose-bound SAMs of PMBA were soaked in the colloidal solution of AgNP/PATP-PMBA (2:1) and unbound AgNP/PATP-PMBA (2:1) was removed by rinsing with double-distilled water followed by drying in air prior to SERS measurement. Similarly, the same procedures were used for fructose and galactose for comparison. Detection of Glucose in Urine Samples. The PMBA-functionalized gold-coated silicon wafers were incubated with the undiluted/untreated human urine samples spiked with glucose of different concentrations (0, 1, 5, and 10 mM) for 3 h followed by washing with double-distilled water and then incubated with the colloidal solution of AgNP/PATP-PMBA (2:1) followed by washing and drying in air prior to SERS measurement. Instruments and Measurements. Transmission electron microscope (TEM) images were acquired on a JEM-2100 microscope operated at 200 KV. UV-vis spectra were recorded 8


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on a UV-3600 spectrophotometer (Shimadzu) in quartz cells of 1 cm optical path. Raman spectra were measured on a LabRAM Aramis HJY Raman spectrometer equipped with a CCD detector. A 532 nm laser with 50 × objective was used for spectral measurements. Prior to spectral measurement, the instrument was calibrated with the silicon standard as its Raman signal is centered at 520 cm−1. The laser power for the spectral measurements was adjusted to 5 mW. The laser beam was focused to a 1 µm spot on the sample to excite. The exposure time was set for 10 s, and 4 scans were collected with the spectral resolution of 4 cm−1. All of the SERS spectra were acquired in air under dry conditions: (1) the self-assembled PMBA monolayers were spread with 10 µL of the colloidal solution of as-synthesized AgNPs or vulcanized AgNPs followed by drying in air; (2) 10 µL of the colloidal solutions of AgNPs modified with PMBA, PATP, and their mixtures with different molar ratios were respectively dropped onto clean glass slides followed by drying in air; (3) SERS tags were captured to the self-assembled PMBA monolayers for saccharide assay followed by washing with double-distilled water and drying in air. Three independent samples for each concentration were prepared for the SERS measurements, and five replicates were measured from five random locations on the sample surface for each independent sample. The error bar stands for the standard deviation calculated from the total of 15 measurements for each concentration. All of the Raman spectra presented were baseline-corrected but not normalized.

RESULTS AND DISCUSSION Self-Assembled PMBA Monolayers and AgNP-Based SERS Tags. PMBA was self-assembled on a smooth gold-coated silicon wafer, however no Raman signal could be 9



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detected from the SAM on the smooth gold film (Figure 1A). Upon spreading of as-synthesized AgNPs without modification (60 ± 5 nm in diameter with a localized surface plasmon resonance absorption maximum at 410 nm, Figure S1 in the Supporting Information) onto the SAM surface, strong SERS signals at 1570, 1070, 1019, and 996 cm−1 were observed owing to the characteristic vibrational modes of PMBA28−30 (Figure 1A). These signals resulted from the AgNP-enhanced Raman scattering of PMBA. To prohibit probable transfer of PMBA from the gold film to the AgNP surface, the as-synthesized AgNPs were treated with sulfides, and then the vulcanized AgNPs were spread onto the SAM of PMBA. The corresponding SERS spectrum was similar to that by spreading of as-synthesized AgNPs but with low peak intensities, owing to the isolation of the thin vulcanization layers. In comparison to the normal Raman spectrum of PMBA powder, the absence of a peak at 2561 cm−1 due to the S−H stretching vibration of PMBA in the SAM reflected that PMBA was self-assembled on the gold-coated silicon wafer via the thiol group. The SERS peaks observed by spreading of vulcanized AgNPs took small shifts relative to the normal Raman bands of the PMBA powder in comparison to the peaks observed by spreading of as-synthesized AgNPs, which further confirmed that PMBA was self-assembled on the gold film. Figure 1B show SERS spectra of AgNPs modified with PMBA, PTAP, and their mixtures. The PMBA-modified AgNPs exhibited almost identical SERS spectral features to the SAM of PMBA after spreading of the as-synthesized AgNPs. The PATP-modified AgNPs displayed considerably intense SERS peaks at 1436, 1390, 1140, and 1070 cm−1. These observed SERS peaks resulted from in situ generated DMAB from the photocoupling 10


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transformation of PATP on the AgNP surfaces under laser illumination.24,25 The Raman shifts at 1436 and 1390 cm−1 are related to the the N=N stretching vibrations of DMAB.24,31 Whereas, typical SERS peaks of PATP were observed primarily at 1582 and 1078 cm−1 using the

ultrathin

silica

shell-isolated

nanoparticle-enhanced

Raman

spectroscopy

(SHINERS)7,24,25,31 and the alkyl layer-isolated nanoparticle-enhanced Raman spectroscopy (ALINERS)32 by separation of PATP from direct contact with SERS-active substrates to avoid its photocoupling transformation. It is shown that the Raman scattering cross section of DMAB is more than three orders of magnitude higher than that of benzenethiol derivatives (such as PATP and PMBA) with synergic effect of resonance Raman and binding effect to AgNPs.31,33 For the AgNPs modified with equimolar mixture of PATP and PMBA (AgNP/PATP-PMBA (1:1)), the intensities of the SERS peaks at 1436, 1390, and 1140 cm−1 closely related to DMAB were weaker than those for the PATP-modified AgNPs when the identical amounts of PATP were added, while the peak at 1574 cm−1 contributed from both PMBA and DMAB/PATP was stronger than that for the PATP-modified AgNPs. The spectral features probably resulted from partially limited photo-induced transformation of PATP to DMAB by the possible N−B coordination interaction between PATP and PMBA. On the other hand, the development of the N−B coordination interaction would be favorable to the formation of the cyclic boronate between PMBA and glucose.17,20,34−36 Thus, the AgNPs modified with the mixture of PATP and PMBA at the molar ratio of 2:1 (AgNP/PATP-PMBA (2:1)) were selected for the SERS tags with considerably intense Raman signals as well as the second carbohydrate reporters. Glucose Sandwich Assay. The SAMs of PMBA were incubated with aqueous glucose 11



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solutions of different concentrations followed by washing to remove unbound glucose. The boronic acid of PMBA in the SAM captured glucose by the formation of a cyclic boronate between the boronic acid of PMBA and one set of syn-periplanar diol groups of glucose. Afterward, the glucose-captured SAMs of PMBA were further incubated with the colloidal solution of AgNP/PATP-PMBA (2:1) followed by washing to remove unbound SERS tags. Ultimately, the SERS tags were captured by the SAMs of PMBA via the glucose sandwich structure between them to form dual cyclic boronates. The Raman signals of the captured SERS tags mainly contributed from in situ generated DMAB by the photocoupling reaction of PATP upon illumination of laser, and these SERS peaks increased in intensity with the increase of glucose concentration (Figure 2). The intensities of the peaks at 1436 and 1140 cm−1 from DMAB as a function of glucose concentration are plotted in Figures 3 and S2 in the Supporting Information, respectively. The measured detection limit of glucose was as low as 0.01 mM because not all the SERS signals were available from random positions on the sample surface at this concentration. The detection range of glucose was 0.03−20 mM (clinically relevant glucose concentration range) with the linear response range of 0.5−10 mM (Figures S3 in the Supporting Information). The linear response range well covers the normal blood glucose levels, which will facilitate the use of blood glucose assay. The detection limit of glucose with Os-BA or alkyne-BA as a Raman probe was 0.1 mM,21,22 and the detection range of glucose was 0.1−10 mM with the Os-BA probe, wherein the C≡O stretching intensity showed a nonlinear change with the increase of glucose concentration.21 Tamer and co-workers presented a SERS-based detection method for glucose combining gold nanorod particles and a gold-coated slide surface modified with the two component SAMs, 12


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consisting of 3-mercaptophenylboronic acid and 1-decanethiol, with the detection limit of 0.5 mM and the linear concentration range of 2−16 mM.30 The tandem assay of glucose using SERS, based on the disassembly of the concanavalin A-sandwiched microstructures between the mannose-modified Fe3O4@SiO2 and Ag@SiO2 particles, could not be available at the glucose concentration of 0.02 mM even after magnetic separation and enrichment.37 Our results indicate that the glucose sandwich assay using the SERS tags with in situ generated Raman reporters was facile, sensitive, and practical. Selectivity of Glucose Assay. Fructose (~0.05 mM) and galactose (~0.05 mM) are the most abundant monosaccharides in human blood after glucose (~5 mM).17,20 However, D-fructose generally shows much stronger binding affinity to all aryl monoboronic acids in comparison to D-glucose and D-galactose in aqueous solution.20 The normal order of binding constants is D-fructose > D-galactose > D-glucose.20 Similarly, the SERS-based sandwich assays of fructose and galactose were carried out (Figure S4 in the Supporting Information), and then the intensities of the SERS peak at 1436 cm−1 as a function of concentration of the two saccharides are also plotted in Figure 3 for comparison. The weak SERS signals observed for fructose and galactose could be attributed to their lower tendency to form dual cyclic boronates.15,20 Although fructose and galactose have greater affinity for monoboronic acids to form 1:1 complexes, glucose has the ability of binding to diboronic acids to form 1:2 complexes.15,20 That is to say, all of the three saccharides could interact with the self-assembled

PMBA

monolayers,

but

there

was

preferential

binding

of

AgNP/PATP-PMBA (2:1) to the surface-captured glucose with another set of syn-periplanar diol groups. Recently, Yu and co-workers used the PMBA-modified gold surface of a 13



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quasithree-dimensional plasmonic nanostructure array (Q3D-PNA) SERS substrate to detect fructose.28 The symmetry breaking of PMBA upon binding of fructose with a high affinity led to the change of area ratio between totally symmetric 8a ring mode and nontotally symmetric 8b ring mode, and the change of area ratio depended on binding affinity and concentration of the saccharides for selective detection of fructose over other saccharides.28 Our SERS-based sandwich assay provided the selectivity of glucose over other saccharides. Considering that the concentration of glucose in human blood is almost two orders of magnitude higher than those of fructose and galactose,17,20 the SERS-based glucose sandwich assay was almost unaffected by fructose and galactose around 0.05 mM. Glucose Assay in Urine. Glucose monitoring for diabetes is usually carried out in urine or blood samples; the former is not a substitute but rather an alternative or complement, which can provide very valuable information where blood glucose monitoring is not accessible, affordable, or desirable.21 The undiluted/untreated urine samples spiked with glucose of different concentrations (0, 1, 5, and 10 mM) were determined using our SERS-based glucose sandwich assay (Figure 4) to demonstrate its potential practicality. No SERS signal could be detected for the blank urine sample. It is known that no glucose can be detectable in normal human urines except for diabetes and kidney-related diseases. It is clear that strong Raman signals from the captured SERS tags were observed without spectral interference from inherent biomolecules (such as urea, creatinine, etc.) in the urine samples even at the glucose concentration of 1 mM. The SERS intensities of the peaks at 1436 and 1140 cm−1 are presented in Figures 3 and S2 in the Supporting Information for comparison, respectively. The amounts of glucose added in the normal urine samples were in good 14


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agreement with the concentrations of standard glucose solutions. This agreement reflects that the developed method was reliable to avoid interference from other biomolecules. Therefore, this facile and sensitive SERS-based glucose sandwich assay without use of enzymes can be developed into a diagnostic tool for determination of glucose levels.

CONCLUSIONS We have demonstrated the facile and sensitive SERS-based glucose sandwich assay by the use of the self-assembled PMBA monolayers on smooth gold-coated substrates and the SERS tags of AgNPs modified with PATP and PMBA. The facile sandwich assay shows a high selectivity of glucose over other saccharides. The photocoupling product DMAB, generated in situ from PATP on the AgNP surface during the SERS measurements, possessed considerably intense characteristic SERS peaks completely different from PATP or PMBA and acted as the actual Raman reporter. The in situ generated Raman reporter improved the sensitivity of glucose detection devoid of interference of other biomolecules. This facile, sensitive, and selective non-enzymatic SERS-based glucose sandwich assay can be developed into a diagnostic tool for determination of glucose levels.

ASSOCIATED CONTENT Supporting Information TEM image of AgNPs, UV-vis spectra of AgNPs without and with the modification of the mixture of PATP and PMBA, SERS spectra of sandwich assays of galactose and fructose of different concentrations, and SERS peak intensity as a function of concentration of different 15



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saccharides together with the peak intensities of urine samples spiked with glucose. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: 86-25-83317761. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21273112) and Natural Science Foundation of Jiangsu Province (BK2012719).

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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

Scheme 1. Schematic Illustration of Glucose Sandwich Assay Using PMBA-Modified Self-Assembled Monolayer on a Smooth Gold-Coated Silicon Wafer and SERS Tags of AgNPs Modified with PATP and PMBA.

20


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Intensity

15000

10000

5000

1570

Au/PMBA + AgNPs Au/PMBA + Ag@S PMBA powder Au/PMBA

1576

20000

1592

A

2561

0 1200

1400

1600

Raman shift / cm

1436 1390 1472

20000

1304

1190

30000

1140

40000

1070

B

AgNP/PATP-PMBA (2:1) AgNP/PATP-PMBA (1:1) AgNP/PATP AgNP/PMBA

2500 2600

−1

1574

1000

Intensity

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


1002 996 1003 1019 1024 1025 1070 1077 1089

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10000

1570 0 800

1000

1200

1400

Raman shift / cm

1600

1800

−1

Figure 1. (A) SERS spectra of self-assembled PMBA monolayers on smooth gold-coated silicon wafers before and after spreading of as-synthesized and vulcanized AgNPs in comparison to normal Raman spectrum of PMBA powder. (B) SERS spectra of AgNPs modified with PMBA, PATP, and their mixtures with different molar ratios.

21


glucose

1140

14000

30 mM 20 10 5 1 0.5 0.1 0.05 0.03 0

12000 10000 Intensity

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

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1436


8000 6000 4000 2000 0 800

1000

1200

1400

Raman shift / cm

1600

1800

−1

Figure 2. SERS spectra of the PMBA-functionalized gold-coated silicon wafers incubated with glucose of different concentrations followed by incubating with SERS tags, washing, and drying.

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glucose galactose fructose urine & glucose

6000

1500

5000 SERS intensity

−1

SERS intensity (1436 cm )

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


4000 3000

1000 500 0 0.0 0.2 0.4 0.6 0.8 1.0

2000

Concentration / mM

1000 0 0

5

10

15

20

25

30

Concentration / mM

Figure 3. Intensity of the SERS peak at 1436 cm−1 as a function of concentration of saccharides (glucose, galactose, and fructose) together with the SERS peak intensities of urine samples spiked with glucose. Inset shows the magnified region in the low concentrations.

23



10000

8000

Intensity / a.u.

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

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6000

4000

10 mM 5 mM 1 mM 0 mM

2000

0 800

1000

1200

1400

Raman shift / cm

1600

1800

−1

Figure 4. SERS spectra of the PMBA-functionalized gold-coated silicon wafers incubated with the urine samples spiked with glucose of different concentrations (0, 1, 5, and 10 mM) followed by incubating with SERS tags, washing, and drying.

24


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TOC Graphic

25


Facile and Sensitive Glucose Sandwich Assay Using In Situ

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