Molecularly Imprinted Plasmonic Substrates for Specific and

Subscriber access provided by University of Newcastle, Australia

Article

Molecularly-Imprinted Plasmonic Substrates for Specific and Ultrasensitive Immunoassay of Trace Glycoproteins in Biological Samples Pir Muhammad, Xueying Tu, Jia Liu, Yijia Wang, and Zhen Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00628 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 20, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

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

ACS Applied Materials & Interfaces

Molecularly-Imprinted

Plasmonic

Substrates

for

Specific

and

Ultrasensitive Immunoassay of Trace Glycoproteins in Biological Samples Pir Muhammad, Xueying Tu, Jia Liu, Yijia Wang, and Zhen Liu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China *Corresponding author: [email protected]

1

ACS Paragon Plus Environment


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

Abstract Assays of glycoproteins hold significant biological importance and clinical values, for which immunoassay has been the workhorse tool. As immunoassays are associated with disadvantages such as poor availability of high-specificity antibodies, limited stability of biological reagents, and tedious procedure, innovative alternatives that can overcome these drawbacks of are highly desirable. Plasmonic immunosandwich assay (PISA) has been emerged as an appealing alternative of immunoassay for fast and sensitive determination of trace glycoproteins in biosamples. Plasmonic substrates play key roles in PISA, not only determining the specificity but also greatly influencing the detection sensitivity. Herein, we report a new type of molecularlyimprinted plasmonic substrates for rapid and ultrasensitive PISA assay of trace glycoproteins in complex real samples. The substrates were fabricated from glass slides, first coated with selfassembled monolayer (SAM) of gold nanoparticles (AuNPs) and then molecularly imprinted with organo-siloxane polymer in the presence of template glycoproteins. The prepared molecularly-imprinted substrates exhibited not only significant plasmonic effect but also excellent binding properties, ensuring the sensitivity as well as the specificity of the assay. Alkaline phosphatase (ALP) and α-fetoprotein (AFP), glycoproteins that are routinely used disease markers in clinical diagnosis, were used as representative targets. The limit of detection (LOD) was 3.1 × 10-12 M for ALP and 1.5 ×10-14 M for AFP, which is the best among the PISA approaches reported. The sample volume required was only 5 µL and the total time required was within 30 min for each assay. Specific and ultrasensitive determination of ALP and AFP in human serum was demonstrated. Since many disease biomarkers are glycoproteins, the developed PISA approach holds great promise in disease diagnostics.

2


Page 2 of 40

Page 3 of 40

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


Keywords: Molecularly-imprinted polymer, immunoassay, glycoprotein, plasmon-enhanced Raman scattering, self-assembled monolayer

3



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

INTRODUCTION Glycoproteins play vital role in biological processes, such as molecular recognition, inter- and intra-cellular signaling, and immune responses.1-5 Structural change and abnormal expression of glycoproteins are associated with the occurrence and progression of diseases, and therefore the assays of glycoproteins hold great values in disease diagnosis and prognosis.6 However, the limited concentration of glycoproteins in biological samples and severe interference of sample matrix often hamper accurate determination. Immunoassays have been an essential tool for the determination of proteins in biological samples, in which antibodies against target proteins play a key role in the specific isolation and enrichment of the target proteins from the sample matrix while highly sensitive detection schemes, such as radiation, fluorescence, and chemiluminescence, ensured the detection of trace target protein. However, immunoassays are usually associated with some apparent disadvantages. First, antibodies often suffer from poor specificity, reproducibility, and stability.7,8 Meanwhile, antibodies against some species such as glycans are difficult to prepare.9 Second, the labeling probes used in immunoassays are associated with some obvious drawbacks, such as harmfulness to health, photobleaching, limited stability, and sensitive to environmental changes.10-12 Moreover, immunoassays are usually tedious and time-consuming. Therefore, innovative alternatives that can overcome the drawbacks of regular immunoassays are highly desirable. Molecular imprinting has developed into an important technology for the synthesis of artificial receptors that exhibited antibody-like binding properties or enzyme-like catalytic capability.13,14 As compared with antibodies, molecularly-imprinted polymers (MIPs) show some advantages such as low cost and good stability. MIP have found a wide range of important applications, such as separation,15,16 sensing,17-19 catalysis,20-21 bioimaging,22,23 and drug

4


Page 4 of 40

Page 5 of 40

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


delivery.24,25 Recently, boronate affinity-based molecular imprinting approaches have developed as efficient and generally applicable means for the creation of MIPs specific to glycoproteins, glycans, and monosaccharides.26-35 The prepared MIPs exhibited excellent specificity, high affinity, biocompatible binding pH range, and strong resistance to interference. Due to these merits, boronate affinity MIPs have been used as substitutes of immobilized primary antibodies for immunoassays of glycoproteins in complex real samples such as human serum26-32 and urine.35 Particularly, by combining boronate affinity MIPs with surface-enhanced Raman scattering (SERS), we have established an enzyme-free and antibody-free immunoassay called boronate affinity sandwich assay (BASA) for the determination of glycoproteins in complex biosamples.29 Compared with the detection schemes mentioned above, SERS exhibits several significant advantages, including comparable or higher sensitivity, less susceptibility to sample and experimental environments.36,37 Determination of glycoprotein disease biomarkers such as αfetoprotein (AFP, a biomarker for liver cancer) in human serum has been demonstrated.29 Recently, by combining boronate affinity MIPs or monoclonal antibodies with plasmonenhanced Raman scattering (PERS), another promising approach termed as plasmonic immunosandwich assay (PISA) have been established.34,35 As in BASA, the molecularlyimprinted substrates or probes used in PISA determines the specificity of the assay. Unlike BASA, however, the substrates or probes used in PISA generated surface plasmon that further apparently enhanced the SERS signal of the Raman nanotags used. Due to the plasmongenerated signal enhancement, ultrasensitive detection of single molecule with simply-structured Raman nanotags level became possible. Determination of low-copy-number proteins (less than 1000 copies per cell) including alkaline phosphatase (ALP, a glycoprotein, for which a boronate affinity MIP was used) and survivin (a nonglycoprotein, for which a monoclonal antibody was

5



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

used) in single living cells and living animals has been achieved.34 Using molecularly-imprinted arrays, fast determination of erythropoietin (EPO), a glycoprotein hormone present in a limited concentration in human bodies, in human urine was also accomplished.35 Obviously, plasmonic substrates play key roles in PISA, not only determining the specificity of the assays but also greatly influencing the detection sensitivity. Therefore, plasmonic substrates with high specificity and high sensitivity are of great importance. In this study, we report a new type of glycoprotein-imprinted plasmonic substrates for rapid and ultrasensitive PISA assay of trace glycoproteins in complex real samples. The substrates were fabricated from glass slides, first coated with the self-assembled monolayer (SAM) of gold nanoparticles (AuNPs) and then molecularly imprinted with an organo-siloxane polymer in the presence of template glycoproteins, as illustrated in Scheme 1. The AuNPs SAM-coated substrate could generate the highest Raman signal enhancement as compared with previously reported substrates, while the organo-siloxane imprinting polymer was favorable to maintain the signal enhancement effect. Based on the molecularly-imprinted plasmonic substrates, a specific and ultrasensitive PISA approach of glycoproteins was developed. The principle and procedure of this approach is illustrated in Scheme 2. A trace target glycoprotein in microliter-scale samples is specifically extracted by a molecularly-imprinted AuNPs SAM-coated substrate. After washed the unwanted species, the bound target molecules are labeled with boronateaffinity silver-based Raman nanotags, thus forming sandwich-like complexes on the substrate. After excessive Raman nanotags are washed away, the substrate is subjected to Raman detection. Upon being irradiated with a laser beam, surface plasmon on the AuNPs SAM-coated substrate is generated, which apparently enhances the SERS signal of silver-based nanotags and thereby enables ultrasensitive detection of target glycoproteins. ALP and AFP were taken as

6


Page 6 of 40

Page 7 of 40

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


representative glycoprotein targets since both are clinically proved serological biomarkers for disease diagnostics. The abnormal level of ALP in serum is closely related to various diseases including cancer, hepatobiliary, diabetes, and bone disorder.38 Whereas AFP have been proven as a marker for hepatocellular carcinoma.39,40 By combining with easy-to-prepare but relatively less sensitive Raman nanotags, i.e., 4-mercaptophenylboronic acid (MPBA)-functionalized AgNPs, the limit of detection (LOD) was 3.1 × 10-12 M for ALP and 1.5 × 10-14 M for AFP. Such detectability is the best among the PISA approaches reported. On the other hand, the MIPs prepared by the organo-siloxane polymerization protocol exhibited excellent binding properties, including excellent affinity, specificity and high resistance to interference. Due to the highly desired properties of the molecularly-imprinted plasmonic substrates, specific and ultrasensitive determination of ALP and AFP in human serum was achieved. The sample volume required was only 5 µL and the total time required was within 30 min for each assay.

EXPERIMENTAL SECTION Reagents and materials. 4-(Aminomethyl) phenylboronic acid hydrochloride (AMPBA) and glucose were purchased from J&K Scientiﬁc (Beijing, China). ALP (EC 3.1.3.1, specific activity: 4500 U/mg) from calf intestinal mucosa was obtained from Heowns Biochemical Technologies (Tianjin, China). Human AFP and normal human serum were purchased from Shuangliu Zhenglong Biochemical Products Lab (Sichuan, China). MPBA, 3-aminopropyl trimethoxysilane (APTMS), 3-aminopropyltriethoxysilane (APTES), trimethoxypropylsilane (TMPS), bovine serum albumin (BSA), albumin from chicken egg white (OVA), apo-transferrin (Trf), and βcasein (β-Cas) were from Sigma-Aldrich (St. Louis, MO). Tris hydroxymethyl aminomethane (Tris), hydrogen tetrachloroaurate (HAuCl4 3H2O, 99.9%), glacial acetic acid (HAc), trisodium

7



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

citrate, glycine, acetonitrile (ACN), glycerol, sodium chloride (NaCl), magnesium chloride (MgCl2), zinc chloride (ZnCl2), NaH2PO4, Na2HPO4 and anhydrous ethanol/methanol, were of analytical grade and purchased from the Nanjing Reagent Company (Nanjing, China). Sulfuric acid (98%), hydrogen peroxide (30%), potassium bicarbonate (KHCO3), trisodium citrate and hydrochloric acid (36%) were purchased from Shanghai Lingfeng Chemical Reagent (Shanghai, China). Sodium dodecyl sulfate (SDS) was obtained from Bio-Rad (Hercules, CA). All these reagents were used without further purification. Other chemicals were of analytical grade or higher. Water was purified with a Milli-Q Advantage A10 (Millipore, Milford, MA), and was used to prepare all the solutions. Glass slides (75 mm × 25 mm) were from the Shanghai Glass Factory (Shanghai, China).

Instruments. Plasmonic detection was conducted on a Renishaw InVia Reflex confocal microscope (Renishaw, UK) equipped with a high-resolution grating with 1,800 grooves/mm, additional band-pass filter optics, and a CCD camera. All measurements were carried out using a He-Ne laser (λ0 = 633 nm; laser power at spot, 17 mW and excitation laser line (1 s integration time and 1 accumulation. The laser was focused onto the sample by using a × 50 objective lens (N.A. 0.75), providing a spatial resolution of ca. 1 µm. Wavelength calibration was performed by measuring silicon wafers through a ×50 objective, assessing the first-order phonon band of Si at 520 cm-1. The spectra were recorded using the Renishaw WiRE software and analyzed with Origin Pro 9 software. Each spot was detected for 5 to 6 times at different locations. Each spectrum baseline was corrected except noise test. UV-Vis absorption spectra were recorded on a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). The AuNPs SAM on glass slide was placed perpendicular to the light beam inside the cuvette. Scanning electron microscopic (SEM)

8


Page 8 of 40

Page 9 of 40

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


characterization was carried out on a FE-SEM S-4800 system (Hitachi, Tokyo, Japan). Transmission electron microscopy (TEM) characterization was performed on a JEM-1011 system (JEOL, Tokyo, Japan). Characterization of particle size was carried out on a BI-200SM dynamic light scattering (DLS) instrument (Brookhaven Instrument, Holtsville).

Synthesis of gold nanoparticles. Gold nanoparticles (AuNPs) with an average diameter of 13 to 15 mm were synthesized by the citrate reduction method.41 Briefly, a volume of 250 mL solution of 1 mM HAuCl4 added in a round bottom flask was brought to boil with vigorous stirring, and 25 mL of sodium citrate solution (38.8 mM) was rapidly added to the vortex of the solution. The solution was maintained at the boiling point with continuous stirring for about 8-10 min and underwent a series of color changes before finally turning to wine red. The nanoparticles were then cool down at room temperature under constant stirring conditions and stored at 4 °C in refrigerator.

Preparation of the AuNPs SAM-coated glass slides. The procedure is illustrated in Scheme 1A, which includes two major steps: amino-functionalization and self-assembly of AuNPs. Each glass slide (75 mm × 25 mm) was first cut into equal pieces (25 mm × 10 mm). Then the obtained uniform-sized pieces were immersed in a boiling piranha solution (H2O2: H2SO4 = 2:8, v/v) for 1 h. After cooling to room temperature, the glass surface was rinsed repeatedly with water, followed by ethanol, by ultrasonic oscillation in water and dried under a nitrogen stream. To functionalize with amino groups, the cleaned glass slide was soaked into a 4% (v/v) ethanolic solution of APTES for 8 to 12 h and then rinsed repeatedly with ethanol and water to remove the un-adsorbed APTES from the surface. Then, the amino-modified glass slide was soaked in 4 mL

9



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

Page 10 of 40

of the Au colloidal solution for 12 h and rinsed with a water to remove the non-functionalized nanoparticles. Thus a self-assembled monolayer of AuNPs covered on the slide surface was formed. Finally, the AuNPs-coated glass slides were stored in water for later use.

Fabrication of molecularly-imprinted AuNPs SAM-coated glass substrates. The molecular imprinting procedure is illustrated in Scheme 1B, which includes four major steps: 1) boronic acid functionalization, 2) template immobilization, 3) molecular imprinting, and 4) template removal. These steps are described as below. The AuNPs SAM-coated glass slides were first modified with boronic acid by immersing in an ethanolic solution containing 1 mg/mL AMPBA at room temperature for 12 h, and softly vibrated during reaction. The glass slides were then washed with anhydrous ethanol for 2-3 times or 50 mM glycine-NaOH buffer (pH 9.5) and dried at room temperature. The AMPBA molecule that consists of nitrogen lone-pair was immobilized on AuNPs surface via electrostatic interaction to the AuNPs surface (amine-Au bond). To immobilize the template onto the substrates, the boronic acid-immobilized AuNPs substrate was immersed in a template solution of certain concentration dissolved in an appropriate buffer for 1 h to form a thin template layer, followed by rinsing with the buffer. The concentration of the template was 15,000 U/L (5.95×10-8 M) ALP or AFP (100 µg/mL). The buffer used to dissolve the template was 50 mM glycine-NaOH buffer (pH 9.5) for ALP or 100 mM phosphate buffer (pH 7.5) for AFP. The imprinting process was carried out according to the boronate affinity oriented surface imprinting approach27 with some modifications. Briefly, the template-modified substrate was immersed in 3ml of glycine-NaOH aqueous buffer (pH 8.0) containing 15 µl of TMPS and 15 µl 10


Page 11 of 40

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


of TMPS (1:1 v/v) under a constant shaking at room temperature for a certain period. The polymerization reaction generated a uniform organo-siloxane polymer imprinting layer on the substrates. Finally, the template was removed by washing the imprinted substrates using a 100 mM HAc solution containing 5% (w/v) SDS for 60 min. The removal of the template molecules left behind well-defined imprinted cavities to rebind the target molecules specifically. To ensure the complete removal of any surfactant, the imprinted substrates were washed with 50 mM glycineNaOH buffer (pH 9.5) and air dried until further used. When the imprinted substrates were used as microarrays for multiple-spot detection, detection spots were defined by printing a cycle array with hydrophobic ink on the target-imprinted slides and non-imprinted slides under investigation, to prevent solutions added to the detection spots from dispersing and cross-contaminating. In such a way, solutions added to each spot were confined within the predefined area. However, when the substrates were used for single detection or property characterization, such a printing processing was not applied. To prepare non-imprinted polymer (NIP)-coated substrates for comparison, the procedure was the same as above except that no template was immobilized onto the boronic acidfunctionalized substrate.

Effect of substrate nature on Raman signal intensity. A volume of 2 µL of MPBA-modified AgNPs colloidal solution was added on the surface of bare glass, Au-plated, AuNPs SAM-coated, poly(APBA-co-dopamine) thin layer-coated and organo-siloxane polymer thin layer-coated glass slides, dried at room temperature and then detected by Raman spectrograph.

11



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

Page 12 of 40

Optimization of the imprinting conditions. The imprinting conditions, including the ratio between the two monomers (APTMS and TMPS), the buffer volume and the polymerization time for ALP and AFP, were optimized. To optimize the volumetric ratio of APTMS vs TMPS, imprinting of ALP was carried out by varying the ratio among 1:0, 2:1, 3:2, 1:1, 1:2 and 2:3, while keeping the buffer volume at 2 mL and the imprinting time at 40 min. Once the optimal ratio was obtained, it was used throughout in later experiments for the imprinting of both ALP and AFP. Afterward, imprinting of ALP at the use of different buffer volumes (0.5, 1, 2, 3, 4, and 5 mL) was investigated while the imprinting time was still kept at 40 min. Once the optimal buffer volume was found, it was used throughout in later experiments. Similarly, to optimize the imprinting time, the imprinting time varied among 20-60 min for the imprinting of ALP whereas it varied among 30-70 min for the imprinting of AFP. All these optimized parameters were kept constant in later experiments. NIP-coated substrates were prepared under otherwise identical conditions. To evaluate the effects of imprinting conditions on the properties of the imprinted substrates, aliquots 10 µL of 100 U/L ALP were dropped on the ALP-imprinted and non-imprinted substrates and incubated in a humidified chamber for 20 min each and washed with 50 mM glycine-NaOH-acetonitrile solution (pH 9.5, 80:20, v/v) for three times, while aliquots 10 µL of 10 µg/mL AFP were dropped on AFP-imprinted and non-imprinted substrates and incubated in a humidified chamber for 20 mins each and washed with 100 mM phosphate (pH 7.4)-acetonitrile solution (80:20, v/v) for three times. After that, the extracted glycoproteins by the substrates were labeled with 5 µL of MPBA-modified AgNPs for 2 min. Then, the substrates were washed with the same washing buffers, dried at room temperature and detected by the Raman spectrograph.

12


Page 13 of 40

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


Optimization of the labeling time. The labeling time was optimized using ALP as the target. Each spot of the ALP-imprinted substrate was exposed to 5 µL of 1000 U/L ALP solution containing 50 mM glycine-NaOH (pH 9.5) for 20 min in a humidity chamber. After washing with acetonitrile-water solution (20:80, v/v) for 5 min, glycoprotein captured on each spot was incubated with 5 µL of MPBA-modified AgNPs for 0.5, 1, 2, 3 and 4 min, respectively. The spots were washed with 20:80 v/v acetonitrile-10 mM phosphate buffers (pH 8.0) for 3 times, dried and then detected by the Raman spectrograph.

Procedure of the PISA approach. A volume of 5 µL of the sample solution was added to the imprinted substrate and incubated for 20 min under humidified chamber, followed by washing with a washing buffer 50 mM glycine-NaOH-acetonitrile solution (pH 9.5, 80:20, v/v) for the detection of ALP of with 100 mM phosphate buffer-acetonitrile solution (pH 7.4, 80:20, v/v) for the detection of AFP, for 3 times each. Then, 5 µL of the Raman nanotags was dropped on same spots and incubated for 2 min. Finally, the substrate was washed with same washing buffer, dried at room temperature and detected by the Raman spectrograph.

Specificity test. The selectivity of the ALP and AFP-imprinted substrates toward a variety of interfering species, including BSA, OVA, Hb, and glucose, was investigated. Aliquots 5 µL solution of 1000 U/L ALP (dissolved in 50 mM glycine-NaOH buffer, pH 9.5) or 1 µg/mL of AFP or 1 mg/mL (dissolved in 100 mM phosphate buffer, pH 7.4) of the interferants were directly added onto each spot of the imprinted substrates and incubated for 20 min in a humidity chamber, followed by rinsing with 10 mM phosphate buffer (pH 7.4) for 3 times. After washing,

13



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

Page 14 of 40

the captured species were labeled by incubating with 5 µL MPBA-modified AgNPs for 2 min. After removing free Raman nanotags by washing with 100 mM phosphate buffer (pH 7.4) for 3 times, dried at room temperature and then detected by the Raman spectrograph.

Determination of ALP and AFP in human serum. The human serum was 20-fold diluted with freshly prepared 50 mM Tris-HCl buffer (pH 9.5). Human serum analysis was processed according to our previously reported method30 with slight modification. Briefly, serum (10 µL) was diluted with 200 µL of 50 mM Tris-HCl buffer (pH 9.5) and then different amount of ALP and AFP were added to the pretreated human serum, making the spiked concentrations to be 1, 5 and 10 U/L of ALP and 1, 5 and 10 pg/mL for AFP, respectively. Then, aliquots of 5 µL of the spiked and unspiked serum samples were subjected to the PISA assay.

RESULTS AND DISCUSSION Basic considerations. SAM of AuNPs was considered as new option for fabrication of plasmongenerating substrates because of two reasons. One is that the self-assembly procedure is simple and the thickness of the formed layer is uniform. More importantly, AuNPs are typical plasmonic nanomaterials and exhibit apparent plasmonic effects.42 On the other hand, an organo-siloxane based sol-gel approach was developed for the imprinting because of three aspects of reasons. First, organo-siloxane polymers can form in aqueous phase, which favors the conformation of the template glycoproteins. Second, when the monomers are appropriately selected, the resultant organo-siloxane polymers can contain desirable functionalities such as amine, hydroxyl and methyl groups that can provide multiple noncovalent interactions including hydrogen bonding, electrostatic and hydrophobic interactions.43-45 In addition to the boronate affinity interaction 14


Page 15 of 40

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


between the boronic acid ligand on the substrate and the glycans of the target glycoproteins, these interactions can jointly provide high binding affinity toward the target glycoproteins. Third, organo-siloxane polymers are electrically non-conductive, which may favor the maintenance of the signal enhancement effect of the plasmonic substrates.

Synthesis and characterization of the nanoparticles. In this study, uniform size-dispersed AuNPs were synthesized and used as base nanoparticles to form self-assembled monolayer on the surface of the substrates that generate plasmonic effect, while uniform size-dispersed AgNPs were synthesized and used as core nanoparticles to further prepare Raman nanotags. Figure S1 A-D shows the TEM images and the DLS size distribution of the prepared AuNPs and AgNPs. From these results, the average diameters of the AuNPs and AgNPs were estimated to be 13±2 nm and 56±5 nm, respectively. These uniformly dispersed sizes highly favored for uniform thickness of the SAM coating as well as the detection reproducibility. The localized surface plasmon resonance (LSPR) of AgNPs before and after boronic acidmodification was characterized by UV/Vis extinction spectrometry. The modification with MPBA did not apparently alter the LSPR spectrum of AgNPs (Figure S2), which suggests a good dispersity of the MPBA-modified AgNPs. The transverse LSPR spectra of the AuNPs in colloidal solution and AuNPs SAM-coated glass slide were also measured. The spectrum was slightly shifted after AuNPs self-assembled onto the glass slide. The maximum extinction was 521 nm in the colloidal solution, but shifted to 546 nm on the AuNPs SAM-coated glass slide (Figure S3). More importantly, the absorbance around the excitation wavelength (633 nm) was much enhanced on the AuNPs SAM-coated glass. It is supported by the literature finding that upon being shined by light, neighboring AuNPs within AuNPs SAM can induce strong

15



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

Page 16 of 40

electromagnetic coupling effect so that longitudinal plasmons are generated on AuNPs SAM.46 Such surface plasmons can favors for the PERS detection used in this work, because the SERS signal of Raman nanotags attached on AuNPs SAM will be apparently enhanced by the plasmons. The Raman spectra of bare, MPBA powder and MPBA-modified AgNPs are shown in Figure S4. MPBA-modified AgNPs exhibited intense Raman peaks while bare AgNPs show no visible peaks. Peaks in the Raman spectra are assigned and listed in Table S1. The characteristic Raman peaks of MPBA-modified AgNPs are in good agreement with the literature.47 Compared with the Raman spectrum of MPBA powder, the Raman spectrum of the MPBA-modified AgNPs shifted downwards, the in-plane ring breathing mode coupled with thiol (-SH) bending mode (1001 cm1

), in-plane ring breathing mode (1025 cm-1), in-plane ring breathing mode coupled with carbon-

sulfur (C–S) and B–OH stretching mode (1072 cm-1) in the SERS spectrum for the MBPAmodified AgNPs were apparently enhanced while the carbon-carbon (C–C) stretching mode (1573 cm-1) was slightly reduced. The Raman peak at 1072 cm-1 was used for property characterization and quantitative analysis.

Effect of the structure and nature of substrate on the Raman signal intensity. The nature and structure of the substrate may significantly influence the plasmonic effect and the detection sensitivity of PISA. Au-plated glass has been verified as an effective surface plasmon-generating substrate and ultrasensitive detection on poly(APBA-co-dopamine) imprinting layer-coated substrate has been achieved previously.34,35 To further enhance the detection sensitivity, new possibilities were explored in this study. AuNPs SAM-coated glass was prepared as new surface plasmon-generating substrate and the organo-siloxane polymer was employed as new imprinting layer, while Au-plated glass and poly(APBA-co-dopamine) imprinting layer were used for

16


Page 17 of 40

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


comparison. Thus, six kind of substrate-imprinting layer combinations were involved. Figure 1 shows the effects of these combinations on the Raman signal intensity with bare glass as a control. As compared with bare glass, both bare Au-plated and AuNPs SAM-coated substrates could generate apparent surface plasmon, which enhanced the intensity by 17.5- and 24.8-fold, respectively. Clearly, AuNPs SAM-coated glass generated stronger plasmonic effect. After the plasmon-generating substrates were covered with an imprinting layer, the Raman signal intensity was reduced by different extents. On the Au-plated glass, the signal intensity was reduced to 22% and 80% by poly(APBA-co-dopamine) and organo-siloxane polymer, respectively. While on the AuNPs SAM-coated glass, the signal intensity was decreased to 36% and 94% by poly(APBAco-dopamine) and organo-siloxane polymer, respectively. Clearly, the use of an organo-siloxane polymer as the imprinting layer was favorable for maintaining the PERS signal. The combination of AuNPs SAM-coated glass and organo-siloxane polymer provided the highest signal enhancement. It was therefore adopted to prepare molecularly-imprinted substrates for the PISA assay. Using such a combination, the finally obtained imprinted substrate exhibited dark purple color (Figure S5), different from the dark gold color of the combination of Au-plated glass and poly(APBA-co-dopamine).35 The higher plasmonic effect of AuNPs-SAM over Au-plated layer was probably due to their different structures. In AuNPs-SAM, AuNPs were nearly uniformly distributed and isolated each other for about 10-50 nm, while gold layer obtained by chemical plating was composed of continuously and closely attached larger gold nanoparticles (Figure S6). The different effect of the organo-siloxane polymer and poly(APBA-co-dopamine) thinlayers on the plasmon enhanced Raman signal was probably due to their different nature. The poly(APBA-co-dopamine) polymer

17



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

Page 18 of 40

was somehow electrically conductive while the organo-siloxane polymer was completely nonconductive electrically.

Optimization of the imprinting conditions. Molecular imprinting conditions that influence the binding properties of the obtained MIPs were optimized according to imprinting factor (IF), which was the ratio of a signal generated by template molecules captured by a MIP over that by a NIP prepared under otherwise identical conditions. With ALP as the template, the volumetric ratio of APTMS over TMPS and the buffer volume were first optimized. Since the monomers have different properties, their ratio will influence the binding properties of the prepared MIP. While the buffer volume used will affect the polymerization speed. As shown in Figure 2A and 2B, the optimal monomeric-volumetric ratio was found to be 1:1 while the optimal buffer volume was 3 mL. The thickness of the imprinting layer is critical in the boronate affinity oriented surface imprinting27, 31 and thus the imprinting times for the imprinting of ALP and AFP were optimized separately. As shown in Figure 2C and 2D, the optimal imprinting time was found to be 40 and 50 min for ALP and AFP, respectively. Such optimal imprinting time is in good agreement with the general trend in the boronate affinity oriented surface imprinting that a larger molecule needs a longer imprinting time. The molecular weight is 56 and 67 kDa for ALP and AFP, respectively. Under the optimized conditions, the IF values were 11.6 and 11.2 for ALP and AFP-imprinted substrate, respectively, which are excellent in molecular imprinting.

Optimization of the labeling time. To obtain the highest Raman signal intensity and shortest labeling time, the incubation time of the Raman nanotags were optimized. As shown in Figure S7, the Raman signal intensity increased as increasing the reaction time from 0.5 to 2 min and

18


Page 19 of 40

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


then kept nearly constant as further increasing the reaction time. Thus, 2 min was considered as the optimal incubation time and used for further experiments. Such a short labeling time was favorable for fast assay speed.

Imprinting efficiency. In addition to imprinting factor, imprinting efficiency (IE) is another critical parameter that describes the performance of the molecular imprinting approach. The molecular imprinting efficiency for ALP and AFP-imprinted substrates were shown in Figure S8A and S8B, respectively. The IE values were measured to be 37.7 and 42.1 % for ALP and AFP-imprinted substrate, respectively, which are excellent in the field of molecular imprinting. These data suggest the high performance of the newly developed organo-siloxane-based imprinting protocol.

Response-dose dependence. The Raman spectra of ALP and AFP at different concentrations are shown in Figure 3A and 3C, respectively. The intensity of Raman peak at 1072 cm-1 are gradually increased as increasing the concentration. Adsorption isotherms for the imprinted and non-imprinted substrates were established by plotting the intensity at 1072 cm-1 against the logarithm of the ALP and AFP concentration, which are shown in Figure 3B and 3D, respectively. For ALP-imprinted substrates, the intensity increased linearly with the logarithm of the concentration of ALP within the range of 1-10000 U/L (y = 597.96 +1689.94x, R2 = 0.991). For AFP-imprinted substrates, the intensity increased linearly with the logarithm of the concentration of AFP within the range of 1 pg/mL–100 ng/mL (y = 390.08 + 924.73x, R2 = 0.990). These linear relationships were used as calibration curves for quantitative analysis. As comparison, the non-imprinted substrates did not showed apparently increased Raman signal as

19



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

Page 20 of 40

increasing the concentration of the target glycoproteins. This indicates that the NIP exhibited limited non-specific adsorption and that the imprinting procedure was effective. The LOD value of ALP was 1 U/L (3.1×10-12 M) (S/N = 4), which is one order of magnitude lower than that based on Au-plated glass substrate and the imprinting layer of poly(APBA-co-dopamine).34 The LOD of AFP was 1 pg/mL (1.5 ×10-14 M) (S/N = 4). Such detectability is the best as compared with the PISA approach reported previously.34, 35 From the response-dose curve (Figure 3B), the dissociation constant (Kd) for ALP-imprinted substrate was estimated by the logistic function fitting to be 2.4 ×10-9 M (R2 = 0.99), which is almost equivalent to the previously reported boronate affinity-based MIPs,30,34 and one order of magnitude lower than recently reported ALP-binding aptamers.48 Similarly, from the responsedose curve (Figure 3D), the Kd value for the AFP-imprinted substrate was estimated to be 4.7×10-10 M (R2 = 0.998), which was 1-2 order of magnitude lower than that for boronate affinity-based MIPs reported by our group.28,29 The lower binding constant for AFP was probably due to higher glycosylation level of AFP ( ̴ 9% ref 26, as compared with 5% for ALP, ref. 49). The generally low Kd values indicate that efficient organo-siloxane polymer-based imprinting provided well-formed cavities with shape complementary and functionality complementary toward the target glycoproteins.

Signal repeatability test. Variation in the Raman signal for ALP and AFP of different concentrations on the ALP and AFP-imprinted substrates were investigated in terms of relative standard deviation (RSD) of detection on 10 random points on one sample spot. As shown in Figure S9, at relatively high concentration (10000 U/L ALP or 10 µg/mL AFP), the RSD was 5.1 and 6.8 % for ALP and AFP, respectively. At lower concentration of ALP (1 U/L) and AFP (1

20


Page 21 of 40

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


pg/mL), the RSD value was 10.7 and 7.1 %, respectively. For blank samples, the RSD value was 4.5 and 6.1 % for ALP- and AFP-imprinted substrates, respectively. Such low variability in signal intensity is well acceptable for trace analysis.

Stability test. Stability of MIPs is a characteristic advantageous feature over natural antibodies. The stability of the glycoprotein-imprinted substrates was investigated. Figure S10 shows the Raman spectra and signal intensity for the PISA detection of ALP at different storage time of the imprinted substrates. The imprinted substrates were stored in the 50 mM glycine-NaOH buffer (pH 9.5) at 4 °C. The material exhibited high storage stability over 2 months. Even after storage for 2 months, the signal intensity generated on the substrates reduced only by 14%.

Specificity test. Specificity is a crucial parameter, which determines the usefulness of the prepared MIPs in real-world samples. The specificity of the ALP and AFP-imprinted substrates was investigated against the different interfering proteins including BSA (nonglycopeotein), OVA (glycoprotein), Trf (glycoprotein), β-Cas (nonglycopeotein), Hemo (nonglycopeotein) and glucose. The concentration of ALP and AFP were 1,000 and 10,000-folds lower than that of the interfering proteins and glucose, respectively. As shown in Figure 4, both ALP and AFPimprinted substrates exhibited the highest Raman signal intensity toward the target glycoproteins. As a contrast, the imprinted substrates showed very limited cross-reactivity toward the interfering species. The maximum cross-reactivity was caused by OVA and Trf, which are glycoproteins. Even though, the signal intensity for the interfering glycoproteins was only about 5-8% of that for the target glycoproteins. Considering the 1,000-fold higher concentrations of the interfering glycoproteins over the target glycoproteins and the logarithmic response-does

21



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

Page 22 of 40

dependence, the actual cross-reactivity should be very limited. These results suggested excellent specificity of the MIPs prepared with the new imprinting protocol. In fact, the cross-reactivity observed in this work was the lowest as compared with our previous works. 28-30, 35

Determination of ALP and AFP in human serum. A practical application of the MIP-based PISA approach was demonstrated by determining ALP and AFP in human serum. A normal standard addition is a typical analytical method used for the quantitative analysis where sample matrix also contributes to the analytical signal (matrix effect). However, for plasmonic detection in the current study as well as SERS-based assay, 29 the conventional standard addition method is not suitable for quantitative analysis because the signal intensity is linear function of the target logarithm concentrations. To solve this issue, a new strategy of standard addition method for the plasmonic detection have been proposed.35 Since the prepared MIPs were highly specific toward their targets, we could assume that the matrix effect could be effectively eliminated, and thereby we could calculate the original concentration of ALP and AFP from the unspiked serum samples in accordance to linear calibration curves of Figure 3B and 3D, respectively. Using PISA with ALP-imprinted and AFP-imprinted substrates, human serum samples spiked with varying ALP and AFP concentrations (ci) were first detected (Figures 5A and 5C). Using the calibration curves shown in Figures 3B and 3D, the concentrations of ALP and AFP in unspiked serum (c0) were calculated, being 1.34 ±0.13 U/L for ALP and 2.2±0.23 pg/mL for AFP. Then, by plotting the intensity at 1072 cm-1 against the logarithm of the total concentration of ALP or AFP in the spiked samples [log (c0 + ci)], where ci is the spiked concentration. Since the obtained plots (Figures 5B and 5D) obeyed good linear relationships (for ALP, y = 425.48 +3045.97x, R2 = 0.999; for AFP, y= 141.7+1883.24x, R2 = 0.994), which are very close the

22


Page 23 of 40

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


calibration curves, the assumption was acceptable and about calculated concentration for unspiked samples are reasonable. Finally, considering changes of volume during pretreatment, the concentration of ALP and AFP in serum was adjusted to be 2.01±0.1 U/L and 2±0.42 pg/mL, respectively. These results fell within the normal range of healthy adult individuals.

CONCLUSION In this study, we have prepared a new type of molecularly-imprinted plasmonic substrates for the specific and ultrasensitive PISA assay of traces glycoproteins in biological samples. By using AuNPs SAM-coated glass slides as the base substrates and the organo-siloxane based protocol for the imprinting, the prepared MIP substrates exhibited not only significant plasmonic effect but also excellent binding properties. The detection sensitivity of this approach was the best among the PISA approaches reported. The specific and ultrasensitive determination of ALP and AFP in human serum well demonstrated the feasibility of this approach for real-world applications. The sample volume required was only 5 µL and the total time required was within 30 min for each assay. Since many FDA-approved disease biomarkers are glycoproteins and the imprinting approach is generally applicable to glycoproteins, this approach holds great promise in disease diagnostics.

23



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

Page 24 of 40

AUTHOR INFORMATION Corresponding Author *Tel.: +86 25 8968 5639. Fax: +86 25 8968 5639. E-mail address: [email protected] (Z. Liu). Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We acknowledge the financial support of the National Science Fund for Distinguished Young Scholars (No. 21425520) and the general grant (No. 21275073) from the National Natural Science Foundation of China, the National Basic Research Program of China (No. 2013CB911202) from the Ministry of Science and Technology of China, as well as the “333” Talents Program from Jiangsu Provincial Government (No. BRA2016351).

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Synthesis routes, additional conditional optimization, additional characterization and additional validation are provided in the supporting information.

24


Page 25 of 40

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


REFERENCES (1) Wu, L.; Qu, X. Cancer Biomarker Detection: Recent Achievements and Challenges. Chem.

Soc. Rev. 2015, 44, 2963-2997. (2) Dwek, R. A., Glycobiology: Toward Understanding the Function of Sugars. Chem. Rev.

1996, 96, 683-720 (3) Ohtsubo, K.; Marth, J. D. Glycosylation in Cellular Mechanisms of Health and Disease. Cell

2006, 126, 855-867. (4) Chandler, K.; Goldman, R. Glycoprotein Disease Markers and Single Protein-Omics. Mol.

Cell. Proteomics 2013, 12, 836-845. (5) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Glycosylation and the

Immune System. Science 2001, 291, 2370-2376. (6) Tian, Y.; Zhang, H. Characterization of Disease-Associated N-linked Glycoproteins.

Proteomics 2013, 13, 504-511. (7) Baker, M. Blame it on the Antibodies. Nature 2015, 521, 274-276. (8) Baker, M. Antibody Anarchy: A Call to Order. Nature 2015, 527, 545-551. (9) Sterner, E.; Flanagan, N.; Gildersleeve, J C. Perspectives on Anti-Glycan Antibodies

Gleaned from Development of a Community Resource Database. ACS Chem. Biol. 2016, 11, 1773-1783. (10) Gosling, J.; Basso, L. Immunoassays: Laboratory Analysis and Clinical Applications;

Taylor & Francis, 1994 (11) Schweitzer, B.; Wiltshire, S.; Lambert, J.; O'Malley, S.; Kukanskis, K.; Zhu, Z.;

Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Immunoassays with Rolling Circle DNA

25



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

Page 26 of 40

Amplification: a Versatile Platform for Ultrasensitive Antigen Detection. Proc. Natl. Acad. Sci. U.S.A 2000, 97, 10113-10119. (12) Diamandis E. P.; Christopoulos T. K. Immunoassay, Academic Press, New York, 1996. (13) Vlatakis, G.;

Andersson, L. I.; Müller, R.; Mosbach, K. Drug Assay using Antibody

Mimics Made by Molecular Imprinting. Nature 1993, 361, 645-647. (14) Whitcombe, M. J.; Chianella, I.; Larcombe, L.; Piletsky, S. A.; Noble, J.; Porter, R.;

Horgan, A. The Rational Development of Molecularly Imprinted Polymer-Based Sensors for Protein Detection. Chem. Soc. Rev. 2011, 40, 1547-1571. (15) Lin, Z.; Sun, L.; Liu, W.; Xia, Z.; Yang, H.; Chen, G., Synthesis of Boronic Acid-

Functionalized Molecularly Imprinted Silica Nanoparticles for Glycoprotein Recognition and Enrichment. J. Mater. Chem. B 2014, 2, 637-643. (16) Liu, X.J.; Ouyan, C.B.; Zhao, R.; Shangguan, D.H.; Chen, Y.; Liu, G.Q. Monolithic

Molecularly Imprinted Polymer for Sulfamethoxazole and Molecular Recognition Properties in Aqueous Mobile Phase. Anal. Chim. Acta 2006, 571, 235-241. (17) Stephenson-Brown, A.; Acton, A. L.; Preece, J. A.; Fossey, J. S.; Mendes, P. M., Selective

Glycoprotein Detection Through Covalent Templating and Allosteric Click-Imprinting. Chem. Sci. 2015, 6, 5114-5119. (18) Bai, W.; Gariano, N. A.; Spivak, D. A. Macromolecular Amplification of Binding

Response in Superaptamer Hydrogels. J. Am. Chem. Soc. 2013, 135, 6977-6984. (19) Zhang, W.; Liu, W.; Li, P.; Xiao, H.; Wang, H.; Tang, B., A Fluorescence Nanosensor for

Glycoproteins with Activity Based on the Molecularly Imprinted Spatial Structure of the Target and Boronate Affinity. Angew. Chem. Int. Ed. 2014, 53, 12489-12493.

26


Page 27 of 40

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


(20) Lettau, K.; Warsinke, A.; Katterle, M.; Danielsson, B.; Scheller, F. W. A Bifunctional

Molecularly Imprinted Polymer (MIP): Analysis of Binding and Catalysis by a Thermistor. Angew. Chem. Int. Ed. 2006, 45, 6986-6990. (21) Zhang, H.Q.; Piacham, T.; Drew, M.; Patek, M.; Mosbach, K.; Ye, L. Molecularly

Imprinted Nanoreactors for Regioselective Huisgen 1,3-Dipolar Cycloaddition Reaction. J. Am. Chem. Soc. 2006, 128, 4178-4189. (22) Kunath, S.; Panagiotopoulou, M.; Maximilien, J.; Marchyk, N.; Sanger, J.; Haupt, K. Cell

and Tissue Imaging with Molecularly Imprinted Polymers as Plastic Antibody Mimics. Adv. Healthcare Mater. 2015, 4, 1322-1326. (23) Shinde, S.; El-Schich, Z.; Malakpour, A.; Wan, W.; Dizeyi, N.; Mohammadi, R.; Rurack,

K.; Gjörloff Wingren, A.; Sellergren, B. Sialic Acid-Imprinted Fluorescent Core–Shell Particles for Selective Labeling of Cell Surface Glycans. J. Am. Chem. Soc. 2015, 137, 13908-13912. (24)

Sellergren, B.; Allender, C. J. Molecularly Imprinted Polymers: A Bridge to Advanced

Drug Delivery. Adv. Drug Delivery Rev.2005, 57, 1733-1741. (25)

Puoci, F.; Cirillo, G.; Curcio, M.; Parisi, O. I.; Iemma, F.; Picci, N. Molecularly

Imprinted Polymers in Drug Delivery: State of Art and Future Perspectives. Expert Opin. Drug Delivery 2011, 8, 1379-1393. (26) Li, L.; Lu, Y.; Bie, Z.; Chen, H. Y.; Liu, Z. Photolithographic Boronate Affinity Molecular

Imprinting: A General and Facile Approach for Glycoprotein Imprinting. Angew. Chem. Int. Ed. 2013, 52, 7451-7454.

27



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

Page 28 of 40

(27) Wang, S.; Ye, J.; Bie, Z. J.; Liu, Z. Affinity-Tunable Specific Recognition of

Glycoproteins via Boronate Affinity-based Controllable Oriented Surface Imprinting. Chem. Sci. 2014, 5, 1135-1140. (28) Bi, X.; Liu, Z. Facile Preparation of Glycoprotein-Imprinted 96-well Microplates for

Enzyme-Linked Immunosorbent Assay by Boronate Affinity-based Oriented Surface Imprinting. Anal. Chem. 2014, 86, 959-966. (29) Ye, J.; Chen, Y.; Liu, Z. A Boronate Affinity Sandwich Assay: An Appealing Alternative

to Immunoassays for the Determination of Glycoproteins. Angew. Chem. Int. Ed. 2014, 53, 10386-10389. (30) Bi, X.; Liu, Z. Enzyme Activity Assay of Glycoprotein Enzymes based on a Boronate

Affinity Molecularly Imprinted 96-well Microplate. Anal. Chem. 2014, 86, 12382-12389. (31) Bie, Z.; Chen, Y.; Ye, J.; Wang, S.; Liu, Z. Boronate Affinity Glycan Oriented Surface

Imprinting: A New Strategy to Mimic Lectins for the Recognition of an Intact Glycoprotein and Its Characteristic Fragments. Angew. Chem. Int. Ed. 2015, 54, 1021110215. (32) Yin, D.; Wang, S.; He, Y.; Liu, J.; Zhou, M.; Ouyang, J.; Liu, B.; Chen, H. Y.; Liu, Z.

Surface-Enhanced Raman Scattering Imaging of Cancer Cells and Tissues via Sialic AcidImprinted Nanotags. Chem. Commun. 2015, 51, 17696-17699. (33) Wang, S.; Yin, D.; Wang, W.; Shen, X.; Zhu, J. J.; Chen, H. Y.; Liu, Z. Targeting and

Imaging of Cancer Cells via Monosaccharide-Imprinted Fluorescent Nanoparticles. Sci. Rep. 2016, 6, 22757. (34) Liu, J.; Yin, D.; Wang, S.; Chen, H. Y.; Liu, Z. Probing Low Copy Number Proteins in a

Single Living Cell. Angew. Chem. Int. Ed. 2016, 55, 13215-13218.

28


Page 29 of 40

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


(35) Tu, X.; Muhammad, P.; Liu, J.; Ma, Y.; Wang, S.; Yin, D.; Liu, Z. Molecularly Imprinted

Polymer-Based Plasmonic Immunosandwich Assay for Fast and Ultrasensitive Determination of Trace Glycoproteins in Complex Samples. Anal. Chem. 2016, 88, 1236312370. (36) Porter, M. D.; Lipert, R. J.; Siperko, L. M.; Wang, G.; Narayanan, R. SERS as a Bioassay

Platform: Fundamentals, Design, and Applications. Chem. Soc. Rev. 2008, 37, 1001-1011. (37) Lane, L. A.; Qian, X.; Nie, S. SERS Nanoparticles in Medicine: From Label-Free

Detection to Spectroscopic Tagging. Chem. Rev. 2015, 115, 10489-10529. (38) Stigbrand, T.; Wahren, B. In Serological Cancer Markers, Sell, S., Ed.; Humana Press:

Totowa, NJ, 1992, 135-149. (39) Peto, J. Cancer Epidemiology in the Last Century and the Next Decade. Nature 2001, 411,

390-395. (40)

Yao, D. F.; Dong, Z. Z.; Yao, M. Specific Molecular Markers in Hepatocellular

Carcinoma. Hepatobiliary Pancreatic Dis. Int. 2007, 6, 241-247. (41) Liao, Y. J.; Shiang, Y. C.; Huang, C. C.; Chang, H. T. Molecularly Imprinted Aptamers of

Gold Nanoparticles for the Enzymatic Inhibition and Detection of Thrombin. Langmuir 2012, 28, 8944-8951. (42) Ding, S. Y.; Yi, J.; Li, J. F.; Ren, B.; Wu, D. Y.; Panneerselvam, R.; Tian, Z. Q.

Nanostructure-based Plasmon-Enhanced Raman Spectroscopy for Surface Analysis of Materials. Nat. Rev. Mater. 2016, 1, 16021. (43) Shiomi, T.; Matsui, M.; Mizukami, F.; Sakaguchi, K. A Method for the Molecular

Imprinting of Hemoglobin on Silica Surfaces using Silanes. Biomaterials 2005, 26, 55645571.

29



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

Page 30 of 40

(44) Abbas, A.; Tian, L.; Morrissey, J. J.; Kharasch, E. D.; Singamaneni, S. Hot Spot ‐

Localized Artificial Antibodies for Label-Free Plasmonic Biosensing. Adv. Funct. Mater. 2013, 23, 1789-1797. (45) Tian, L.; Liu, K.-K.; Morrissey, J. J.; Gandra, N.; Kharasch, E. D.; Singamaneni, S. Gold

Nanocages with Built-in Artificial Antibodies for Label-Free Plasmonic Biosensing. J. Mater. Chem. B 2014, 2, 167-170. (46)

Haynes, C. L.; Van Duyne, R. P., Plasmon-Sampled Surface-Enhanced Raman Excitation

Spectroscopy. J. Phys. Chem. B 2003, 107, 7426-7433. (47) Sun, F.; Bai, T.; Zhang, L.; Ella-Menye, J.-R.; Liu, S.; Nowinski, A. K.; Jiang, S.; Yu, Q.

Sensitive and Fast Detection of Fructose in Complex Media via Symmetry Breaking and Signal Amplification using Surface-Enhanced Raman Spectroscopy. Anal. Chem. 2014, 86, 2387-2394. (48) Li, X.; He, Y.; Ma, Y.; Bie, Z.; Liu, B.; Liu, Z. Hybrid Approach Combining Boronate

Affinity Magnetic Nanoparticles and Capillary Electrophoresis for Efficient Selection of Glycoprotein-Binding Aptamers. Anal. Chem. 2016, 88, 9805-9812. (49) Weiss, M. J.; Henthorn, P. S.; Lafferty, M. A.; Slaughter, C.; Raducha, M.; Harris, H.

Isolation and Characterization of a cDNA Encoding a Human Liver/Bone/Kidney-type Alkaline Phosphatase. Proc. Natl. Acad. Sci.U.S.A.1986, 83, 7182-7186.

30


Page 31 of 40

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


Figure Captions Scheme 1. Schematic of the procedure for (A) the preparation of AuNPs SAM-coated substrate and (B) the preparation of glycoprotein-imprinted substrate. Scheme 2. Schematic representation of the MIP-based PISA approach for the detection of target glycoproteins. Figure 1. Effect of the substrate nature on the Raman signal intensity. A certain volume of the Raman nanotags was added on the surface of bare (S0), Au-plated (S1), AuNPs SAM-coated (S2), poly(APBA-co-dopamine) thin layer-covered Au-plated (P1-S1), organo-siloxane polymer thin layer-covered Au-plated (P2-S1), poly(APBA-co-dopamine) thin layer-covered AuNPs SAM-coated (P1-S2), organo-siloxane polymer thin layer-covered AuNPs SAM-coated (P2-S2) glass slides. Error bars represent standard deviations of the three replicate measurements. Figure 2. Optimization of imprinting conditions: (A) ratio between the two monomers, (B) buffer volume, (C) imprinting time for ALP-imprinted substrate, (D) imprinting time for AFPimprinted substrate. Sample: 100 U/L ALP dissolved in 50 mM glycine-NaOH (pH 9.5) or 10 µg/mL AFP dissolved in 100 mM phosphate buffer (pH 7.4). Raman nanotag: 5 µL of MPBAmodified AgNPs. Figure 3. (A) Raman spectra for ALP extracted from ALP standard solutions of different concentrations by ALP-imprinted substrate. (B) Dependence of the Raman intensity at 1072 cm-1 for ALP extracted from ALP standard solutions of different concentrations by ALP-imprinted substrate (MIP) and non-imprinted substrate (NIP). (C) Raman spectra for AFP extracted from AFP standard solutions of different concentrations by AFP-imprinted substrate. (D) Dependence

31



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

Page 32 of 40

of the Raman intensity at 1072 cm-1 for AFP extracted from AFP standard solutions of different concentrations by AFP-imprinted substrate (MIP) and non-imprinted substrate (NIP). Logistic function fitting and linear fitting for the dose-response dependence on the MIP substrate. Error bars were estimated from three replicate measurements. Figure 4. Cross-reactivity test for ALP-imprinted substrate (A) and AFP-imprinted substrate (B). Sample: ALP 1000 U/L dissolved in 50 mM glycine-NaOH buffer (pH 9.5), AFP 1 µg/mL dissolved in phosphate buffer (100 mM, pH 7.4) while the concentration of interfering proteins was 1 mg/mL or 10 mg/mL glucose dissolved in 100 mM phosphate buffer (pH 7.4); blank sample: 100 mM phosphate buffer (pH 7.4). Error bars were estimated from three replicate measurements. Figure 5. (A) Raman intensities at 1072 cm-1 for serum samples spiked with different concentration of ALP. (B) Linear dependence of the Raman intensity at 1072 cm-1 on the logarithmic value of the total concentration of ALP in the urine samples (Log [(c0 + ci) (U/L)]), where c0 was calculated according to the linear equation obtained with standard solutions on the assumption that the matrix effect was eliminated by the MIP substrate. (C) Raman intensities at 1072 cm-1 for serum samples spiked with different concentration of AFP. (D) Linear dependence of the Raman intensity at 1072 cm-1 on the logarithm of the total concentration of AFP in serum samples (Log [(c0 + ci) (pg/mL)]), where c0 was calculated according to the linear equation obtained with standard solutions on the assumption that the matrix effect was eliminated by the MIP substrate.

32


Page 33 of 40

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.

33



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 2.

34


Page 34 of 40

Page 35 of 40

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


Figure 2.

Figure 1.

35



10 8

4000 6 4

2000

14

MIP NIP IF

6000

12 10 8

4000 6 4

2000

2

2 0 2:1

3:2

1:1

1:2

2:3

0

0

0

0.5

1

2

Monomer ratio (APTMS :TMPS)

14

C 8000 MIP NIP

D 8000

10 8

4000 6 4

2000

Intensity at 1072 cm-1

12

IF

Imprinting factor

6000

4

3

5

Buffer volume (mL)

14

MIP NIP

12

IF

6000

10 8

4000 6 4

2000

2

2

0

0 20

30

40

50

0

60

30

Imprinting time (min)

40

50

60

Imprinting time (min)

Figure 2.

36


70

0

Imprinting factor

1:0

Imprinting factor

12

MIP NIP IF

6000


B 8000

14

Imprinting factor


A 8000


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

Page 36 of 40

Page 37 of 40

A18000

B 10000

MIP NIP Logistic function fitting Linear fitting


Raman Intensity

15000 100000 U/L

12000

10000 U/L 1000 U/L

9000

100 U/L

6000

10 U/L 1 U/L

3000

8000

6000

4000

2000

0.1 U/L Blank

0 600

0

800

1000

1200

1400

1600

0

1

2

Raman Scattering (cm-1)

D 8000 Intensity at 1072 cm

1 µg/mL

40000

100 ng/mL 10 ng/mL

30000

1 ng/mL

20000

100 pg/mL 10 pg/mL

10000

5

6

Logistic curve fitting Linear fitting

6000

4000

2000

1 pg/mL Blank

0 600

4

MIP NIP

-1

10 µg/mL

50000

3

Log [ALP (U/L)]

C 60000 Raman 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


800

1000

1200

0

1400

1600

0

1

2

3

4

5

Log [AFP (pg/mL)]

Raman Scattering (cm-1)

Figure 3.

37


6

7


Intensity at 1072 cm

-1

A8000 6000

4000

2000

0 P AL

A OV

Trf

u Gl

BS

P

A OV

Trf

u Gl

s k o A BS Hem ß-Ca Blan

A

He

mo

ß-C

as

nk Bla

-1

B 8000 Intensity at 1072 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

6000

4000

2000

0 AF

Figure 4.

38


Page 38 of 40

Page 39 of 40

A 5000

B 5000

Intensity at 1072 cm

-1

C0= 1.34


4000

3000

2000

1000

0

3000

2000

1000

0 0.0

um um um ser ser ser ked ked ked spi spi spi P P P L L AL /L A /L A U/L 1U 5U 10 rum Se

D 2500

2000

1500

1000

0.4

0.6

0.8

1.0

1.2

2000

C0 = 1.9 2

R = 0.991

1500

1000

500

500

0 0.2

0 L g/m 1p

0.2

Log [(c0 + ci) (U/L)]

C 2500

n tio olu Ps AF

2

R = 0.999


1

U/L

n tio olu Ps AL

4000


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


rum Se

um um um ser ser ser ed ed ed pik pik pik s s s P FP FP AF LA LA mL g/m g/m pg/ 1p 5p 10

0.4

0.6

0.8

Log [(c0 + ci) (pg/mL)]

Figure 5.

39


1.0

1.2


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

TOC graphic:

40


Page 40 of 40

Molecularly Imprinted Plasmonic Substrates for Specific and

Recommend Documents