Molecularly Imprinted Polymer-Based Plasmonic Immunosandwich

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Molecularly-Imprinted Polymer-based Plasmonic Immunosandwich Assay for Fast and Ultrasensitive Determination of Trace Glycoproteins in Complex Samples Xueying Tu, Pir Muhammad, Jia Liu, Yanyan Ma, Shuangshou Wang, Danyang Yin, and Zhen Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03597 • Publication Date (Web): 24 Nov 2016 Downloaded from http://pubs.acs.org on November 24, 2016

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Analytical Chemistry

Molecularly-Imprinted Polymer-based Plasmonic Immunosandwich Assay

for

Fast

and

Ultrasensitive

Determination

of

Trace

Glycoproteins in Complex Samples Xueying Tu, Pir Muhammad, Jia Liu, Yanyan Ma, Shuangshou Wang, Danyang Yin, and Zhen Liu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 163 Xianlin Avenue, Nanjing 210023, China * To whom correspondence should be addressed. E-mail: [email protected]

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Abstract Glycoproteins play significant roles in many biological processes. Assays of glycoproteins have significant biological importance and clinical values, for which immunoassay has been the workhorse tool. However, immunoassay suffers from some disadvantages, such as poor availability of high-specificity antibodies and limited stability of biological reagents. Herein, we present an antibody-free and enzyme-free approach, called molecularly-imprinted polymer (MIP)-based plasmonic immunosandwich assay (PISA), for fast and ultrasensitive detection of trace glycoproteins in complex sample. A gold-based boronate affinity MIP array was used to specifically extract the target glycoprotein from complex samples. After washing away unwanted species, the captured glycoprotein was labeled with boronate affinity silver-based Raman nanotags. Thus, sandwich-like complexes were formed on the array. Upon being shined with a laser beam, the gold-based array generated surface plasmon wave, which significantly enhanced the surface-enhanced Raman scattering (SERS) signal of the silver-based Raman nanotags. The MIP ensured the specificity of the assay, while the plasmonic detection provided ultrahigh sensitivity. Erythropoietin (EPO), a glycoprotein hormone that controls erythropoiesis or red blood cell production, was employed as a test glycoprotein in this study. Specific detection of EPO in solution down to 2.9 × 10-14 M was achieved. Using a novel strategy to accommodate the method of standard addition to logarithmic dose-response relationship, EPO in human urine was quantitatively determined by this approach. The analysis time required only 30 min in total. This approach holds promising application prospects in many areas, such as biochemical research, clinical diagnosis and anti-doping analysis.

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Introduction Glycoproteins are a large family of carbohydrate-conjugated proteins that play crucial roles in a wide variety of biological processes such as molecular recognition, signal transduction, cell adhesion, immune response and regulation of cellular development.1-3 As the abnormal structural changes and expression of glycoproteins are correlated with the occurrence and development of diverse diseases, many glycoproteins have been employed as disease biomarkers.4 Moreover, glycoprotein hormones regulate biochemical and physiological processes and thus have been used as not only therapeutic drugs but also doping agents in sports.5 Because of the limited concentration of important glycoproteins in biological samples as well as the severe interference of high-abundance coexistent species, the determination of trace glycoproteins in biological samples requires specific recognition and sensitive detection schemes. Benefited from antibodies with high specificity towards their targets, immunoassay has been a robust tool for protein analysis in many areas such as biochemical research,6 clinical diagnosis,7 and anti-doping analysis.8 However, biorecognition based on antibodies suffers from limited sources, high price and poor storage stability of antibodies. Meanwhile, sensitive detection schemes, such as colorimetry, radiometry, fluorimetry, chemiluminometry and electrochemical analysis, have been applied in immunoassays to detect trace targets, which require the use of corresponding antibody- or antigen-conjugated labels, such as enzymes, radioisotopes, DNA/RNA reporters, fluorescent, chemiluminescent and redox probes. However, these labels are associated with some limitations. For instance, enzymes, DNA and RNA reporters suffer from limited stability, radioisotopes may cause health hazards and the use of them requires special authorization and facility, while fluorescent, chemiluminescent and redox probes are disturbing and susceptible to the sample and surrounding 3



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environment. Therefore, antibody-free and enzyme-free immunosandwich assays with novel detection schemes that can overcome the above drawbacks are highly desirable. Molecularly imprinted polymers (MIPs), which are synthetic receptors prepared through templated polymerization, can exhibit antibody-like binding properties or enzyme-like activities.9-18 As MIPs are easier to prepare, more cost-efficient and more stable than antibodies, they have found promising applications in separation,19 sensing,20,21 catalysis,22 and bacteria killing.23 Recently, by using boronic acids as ligands to bind the glycans of glycoproteins, we have synthesized a series of high-performance MIPs via the photolithographic boronate affinity molecular imprinting24,25 and the boronate affinity-based controllable oriented surface imprinting approaches.26-29 These boronate affinity MIPs exhibited highly attractive binding properties, including high specificity, high affinity, bio-compatible binding pH range and superb tolerance to interferences. Due to these desirable features, boronate affinity MIPs have been used as substitutes of immobilized primary antibodies for immunoassays of glycoproteins in complex real samples such as human serum.24, 25, 27, 28 Particularly, by combining surface-enhanced Raman scattering (SERS) with boronate affinity MIPs, we have established an enzyme-free and antibody-free immunoassay called boronate affinity sandwich assay (BASA) for the determination of glycoproteins in complex samples. Compared with the detection schemes mentioned above, SERS exhibits several significant advantages, including comparable or higher sensitivity, less susceptibility to sample and experimental environments, and possibility for non-destructive analysis. BASA relied on the combination of a boronate-affinity macroporous monolithic MIP that functions similar to a capture antibody to ensure high specificity, as well as boronate-affinity silver nanoparticles (AgNPs) that function as Raman nanotags to provide high sensitive detection. BASA exhibited significant advantages over conventional immunoassays in terms 4


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of cost efficiency, stability and speed. However, the sensitivity of current BASA is not very high, with a limit of detection (LOD) of 10-11 M. Therefore, new alternatives of BASA with ultrahigh sensitivity are of significant importance for the determination of trace glycoproteins in complex samples. Interestingly, a secondary signal enhancement mechanism was unveiled during the development of the PISA approach. The macroporous monolithic structure of the MIPs induced slightly enhanced secondary Raman scattering (2.6-fold higher than that of non-porous monolithic MIPs).25 Although the enhancement factor was not attractive, such a mechanism inspired us to explore other possibility for higher sensitivity. This brought us to a newly-emerged alternative of SERS, plasmon-enhanced Raman scattering (PERS),30 which can provide ultrasensitive detection at the single-molecule level. PERS usually relies on the resonance coupling of the surface plasmons of two noble metal structures forming a nanocavity and the molecular or nanostructural vibronic transitions of a single Raman-active molecule or nanostructure located in the nanocavity. However, existing PERS detection usually requires near-field nanocavities formed by a scanning tunneling microscopic tip and a metal surface30 or tailored nanoscale electrode pairs,31 which makes PERS sophisticated and inconvenient in practical applications. Recently, we proposed a new method called plasmonic immunosandwich assay (PISA) for the analysis of low-copy number of proteins in single living cell.32 To realize ultrasensitive plasmonic detection, we turned to the combination of a gold-based extraction probe and silver-based Raman nanotags, which was expected to generate surface plasmon upon laser radiation to enhance the total Raman signal on the basis of the SERS mode. The approach allowed for the detection of a few molecules of target proteins extracted from single cells by a microprobe modified with monoclonal antibody or MIP. However, the concentration detection sensitivity was not very high; the limit of detection (LOD) value was 6.0×10-11 M for a MIP-coated extraction probe and 3.0×10-13 M for 5



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antibody-immobilized extraction probe. In this study, we explore a MIP array version of PISA for fast and ultrasensitive determination of trace glycoproteins in complex samples. The principle and procedure of this approach is shown in Figure 1. A trace target glycoprotein in microliter-scale samples is specifically captured by a gold-based boronate-affinity MIP array. After unwanted species are washed away, the captured targets are labeled with boronate-affinity silver-based Raman nanotags, thus forming sandwich-like complexes on the array. After excessive Raman nanotags are washed away, the array is subjected to Raman detection. Upon being irradiated with a laser beam, surface plasmon on the gold-based MIP array is generated, which further enhances the SERS signal of silver-based nanotags and thereby enables ultrasensitive detection of target glycoproteins. We took human erythropoietin (EPO), a glycoprotein hormone that stimulates erythropoiesis,32-34 and whose recombinant products (e.g. EPO-α, EPO-β, etc.) have created a good deal of interest due to its clinical use for the treatment of renal anemia35, 36 and abuse as a blood doping agent for short-term improvement of performance in endurance sports,37 as a test glycoprotein. Detection of EPO has brought about challenges to analytical chemists. On one hand, the normal concentration range of EPO in healthy individuals is rather low, being 10-11–10-12 M in blood38 and 10-13–10-14 M in urine,39 while wide ranges of abundant coexisting substances will cause severe interference. The detection of such trace protein requires not only an ultrasensitive detection scheme but also an effective means to remove or suppress the interference. On the other hand, some anti-EPO antibodies were reported to be cross-reactive to other glycoproteins such as Tamm Horsfall glycoprotein and alpha-2-thiol proteinase inhibitor,40,41 which makes the specific capture or enrichment of EPO high dependent on the availability of high-specificity antibodies. We first verified the proposed plasmonic enhancement effect. We then prepared 6


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EPO-imprinted assays and boronate affinity Raman nanotags and investigated their properties and the performance of the MIP-based PISA established using them. The MIP-based PISA approach showed high specificity towards EPO and enabled the detectability of down to 2.9 × 10-14 M EPO in solution, which matched well with the concentration range of EPO in human urine. The total time needed for the assay was only 30 min. Finally, we applied the MIP-based PISA to the determination of total EPO in a human urine sample. As the calibration curve of this approach was linearly dependent on the logarithm of the protein concentration, conventional data processing strategy such as reverse extrapolation failed to work. To this end, we proposed and verified a simple strategy to solve this issue and thus quantitative analysis of complex samples based on the PISA approach was achieved.

Experimental Section Reagents and Materials. Anhydrous ethanol, chloroauric acid (HAuCl4·4H2O), disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, ammonium persulfate (APS), silver nitrate, nitric acid (65-68%) and ammonia solution (28% w/v) were purchased from Nanjing Reagent Company (Nanjing, China). Sodium dodecyl sulfate (SDS), 4-mercaptophenylboronic acid (MPBA), p-aminothiophenol (PATP), β-lactoglobulin A (β-Lac A), bovine serum albumin (BSA), hemoglobin (Hemo), lysozyme (LZM), human apo-transferrin (TRF) and complete protease inhibitor cocktail (p8340) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetraethoxysilane (TEOS, 99%) was from Heowns Biochemical Technologies (Tianjin, China). Tris (hydroxymethyl) aminomethane (Tris, electrophoresis purity) was obtained from Bio-Rad (Hercules, CA, USA). 3-Aminopropyltriethoxysilane (APTES, 98%), D-(+)-glucose, sodium cyanoborohydride (95%) and dopamine hydrochloride (98%) were from J&K Scientific (Beijing, China). Horseradish peroxidase 7



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(HRP), sodium hydroxide (NaOH) and glacial acetic acid (HAc) were purchased from Sinopharm Chemical Reagent (Shanghai, China). 4-Formylphenylboronic acid (FPBA, 97%) was purchased from Aladdin Industrial Corporation (Shanghai, 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). m-Aminophenylboronic acid monohydrate (APBA) was purchased from Energy Chemical (Shanghai, China). All other reagents used were of analytical grade or higher unless otherwise specified. The recombinant human EPO produced in Chinese hamster ovary (CHO) cell line was from Kirin (Tokyo, Japan) as Espo for EPO-α, from Roche (Mannheim, Germany) as Recormon for EPO-β. EPO-α and EPO-β were obtained as clinical injection solution under concentration of 6000 IU/0.5 mL (100 µg/mL) and 5000 IU/0.3 mL (139 µg/mL), respectively. 1× Phosphate-buffered saline (1× PBS) was obtained from Keygen Biotech (Nanjing, China). Water used in all the experiments was purified by a Milli-Q Advantage A10 ultrapure water purification system (Millipore, Milford, MA). Glass slides (75 mm × 25 mm) were purchased from the Shanghai Machinery Import and Export Corporation (Shanghai, China). Ultrafiltration cartridge and Amicon Ultra-0.5 (MWCO 10,000 Da) were products of Millipore (Beverly, MA, USA).

Instruments. Transmission electron microscopic (TEM) characterization was performed on a JEM-1011 TEM instrument (JEOL, Tokyo, Japan). UV-vis absorbance characterization was carried out on a Nanodrop-2000C instrument (Thermo Fisher Scientific, Shanghai, China). Plasmonic detection was carried out on a Renishaw InVia Reflex confocal microscope (Renishaw, UK) equipped with a high-resolution grating with 1800 grooves/cm, additional band-pass filter optics and a CCD 8


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camera. All measurements were carried out using a He-Ne laser (λ0 = 633 nm; laser power at spot, 17 mW). The laser was focused onto the sample by using a × 50 objective (N.A. 0.75), providing a spatial resolution of ca. 1 µm2. Wavelength calibration was performed by measuring silicon wafers through a ×50 objective, evaluating the first-order phonon band of Si at 520 cm-1. The integration time for Raman measurement was 1 s and spectra were collected by accumulation of 3 scans. Each measurement was repeated 10 times at 10 different locations on the spot. Each spectrum was baseline corrected except noise test.

Preparation of MPBA-modified AgNPs. MPBA-modified AgNPs were prepared as Raman nanotags according to our previously reported method.25 The preparation route is shown in Figure S1A. Briefly, 8 µL of 1 mM MPBA (dissolved in 0.2 M NaOH) was added to 1 mL AgNPs stock solution prepared above and the mixed solution was stirred for 1 h at room temperature. Fresh labeling solution was prepared prior to the plasmonic immunosandwich assay.

Preparation of FPBA-modified Ag/PATP@SiO2 NPs. FPBA-modified Ag/PATP@SiO2 NPs were prepared as Raman nanotags according to our previously reported method.42 The preparation route is shown in Figure S1B. A volume of 20 µL of 1 mM PATP dissolved in ethanol was first added dropwise to 10 mL of Ag colloidal solution under rapid stirring for 40 min. To coat silica shells on the Ag/PATP NPs surfaces, a procedure described by Baida and co-workers43 was employed with slight modifications. A volume of 40 mL of ethanol was added to the obtained Ag colloidal solution with stirring. Subsequently, a volume of 0.7 mL of ammonia solution (28%) was added to the suspension, and the mixture was stirred for 5 min. Then 10 µL of 10 mM TEOS dissolved in ethanol was added to 9



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the suspension. The reaction mixture was slowly stirred at room temperature for 70 min. After that, the resultant Ag/PATP@SiO2 NPs were centrifuged at 11,000 rpm for 10 min and washed with ethanol four times followed by redispersing in 10 mL of anhydrous ethanol. For boronic acid functionalization, amino groups were introduced by injecting 100 µL of APTES to 10 mL of anhydrous ethanol solution containing freshly prepared Ag/PATP@SiO2 NPs, and the mixture was stirred at room temperature for 1 h. The resulting amino-modified Ag/PATP@SiO2 NPs were isolated by centrifugation and redispersed with 10 mL ethanol three times. The amino-modified Ag/PATP@SiO2 NPs were dispersed in 30 mL ethanol. 300 µL of 5 mg/mL FPBA and 300 µL of 5 mg/mL sodium cyanoborohydride were added into 30 mL of Ag/PATP@SiO2 NPs suspension. After reaction for 24 h, the solution was centrifuged and the FPBA-modified Ag/PATP@SiO2 NPs were collected via centrifuging, and then washed with ethanol and water for three times each. Finally, the FPBA-modified Ag/PATP@SiO2 NPs were dispersed in 9 mL water.

Preparation of EPO-imprinted arrays. The procedure for preparing EPO-imprinted arrays is shown in Figure S2, which included the following three major steps.

Preparation of Au-coated glass slides. Each glass slide (75 mm × 25 mm) was cut into three equal pieces (25 mm × 25 mm). Then the glass slides were cleaned by immersion in a freshly prepared piranha solution for 1 h and then rinsed thoroughly with water and dried in a vacuum oven at 50 °C. Glass surface was successively derivatized by soaking into an ethanolic solution of 5% v/v APTES at 50 °C for 12 h. To prepare gold film coating on the slides, a previously reported method was employed with some modification.27 Briefly, the slides were thoroughly rinsed with ethanol to remove 10


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any physically adsorbed silane and dried in a vacuum oven at 50 °C. A gold layer was prepared onto the surface of the amino-modified glass slides according to a previously reported method44 with slight modifications. Briefly, the slides were immersed in a mixed solution (12 mM HAuCl4, 0.5 M KHCO3 and 25 mM glucose) for 5-6 h at 50 °C (air bath) until an obvious gold layer appeared on the surface of each slide. Then the Au-coated slides were gently washed with water three times, and dried in an oven at 50 °C.

Preparation of boronic acid-functionalized Au-coated arrays. The Au-coated glass slides were first immersed in an ethanolic solution of 5% v/v APTES at 50 °C for 12 h, followed by rinse with ethanol to remove residual reagents. After that, the amino-modified Au-coated glass slides were immersed in an ethanolic solution containing 1 mg/mL FPBA and 1 mg/mL sodium cyanoborohydride at room temperature for 24 h, and softly vibrated during reaction. Finally, the glass slides were washed with ethanol, dried at 40 °C. The preset array pattern was printed with hydrophobic ink onto the boronic acid-functionalized Au-coated glass slides with the aid of masks with circular holes, and stored at room temperature for further use.

Preparation of EPO-imprinted MIP arrays. The molecular imprinting approach was modified from our previous method called boronate affinity-based controllable oriented surface imprinting.26 The binding pH value was set as 7.4, which is favorable for the binding between common phenylboronic acids and sialylated glycoproteins.45 A 5-µL solution of 100 µg/mL EPO-α or EPO-β dissolved in 1× PBS was added onto each spot of the boronic acid-functionalized Au-coated arrays. Then the arrays were incubated for 20 min in a humidity chamber to form a thin template layer, followed by rinsing 11



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with 1× PBS three times. Then the template-anchored arrays were immersed into an aqueous mixture containing 2.0 mg/mL dopamine, 1.6 mg/mL APBA and 1.2 mg/mL APS at room temperature for 50, 60 and 70 min for optimization of imprinting time. Finally, the arrays were rinsed with 0.1 M HAc containing 10% SDS (w/v) to remove the template. The imprinting time was then fixed to the optimal value. To prepare non-imprinted polymer (NIP) covered arrays for comparison, the processing procedure was the same except that no template was immobilized onto the boronic acid-functionalized gold-based arrays.

Results and Discussions Synthesis and characterization of Raman nanotags. Two kinds of boronic acid-functionalized silver-based Raman nanotags were synthesized and utilized in this study. The preparation of MPBA-modified AgNPs was much simpler than that of FPBA-modified Ag/PATP@SiO2 NPs, but the stability of FPBA-modified Ag/PATP@SiO2 NPs were better than those of MPBA-modified AgNPs. TEM images, UV-Vis spectra and Raman spectra of MPBA-modified AgNPs and FPBA-modified Ag/PATP@SiO2 NPs are shown in Figure S3. The average diameters of MPBA-modified AgNPs and FPBA-modified Ag/PATP@SiO2 NPs were about 60 nm and 90 nm, respectively. The silica shell thickness of the FPBA-modified Ag/PATP@SiO2 NPs was estimated to be approximately 15 nm. The effects of Raman reporters self-assembly and silica encapsulation on the UV-Vis absorption spectrum of AgNPs were examined. The modification with MPBA did not alter the LSPR spectrum of AgNPs, while the 15 nm silica capsule led to an obvious red-shift in the surface plasmon band of AgNPs. As the molar extinction coefficient of Ag@SiO2 NPs was found to be unaffected by the variation in the silica shell thickness,46 the slightly lower maximum absorption of FPBA-modified Ag/PATP@SiO2 12


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NPs than that of MPBA-modified AgNPs meant that the concentration of FPBA-modified Ag/PATP@SiO2 NPs was at a slightly lower level than that of MPBA-modified AgNPs. These two nanotags exhibited different characteristic Raman spectra. The characteristic Raman peaks of MPBA-modified AgNPs were contributed directly from MPBA,47 while those of FPBA-modified Ag/PATP@SiO2 NPs were not contributed directly from PATP but its photocatalytic coupling reaction product, 4,4-dimercaptoazobenzene (DMAB), generated on AgNPs upon laser irradiation during the measurements.48 The assignments of the major Raman bands of them are summarized in Table S1 according to literature data.41,42 Characteristic peaks at 1,072 and 1,435 cm-1 were employed in the quantitative detection when using MPBA-modified AgNPs and FPBA-modified Ag/PATP@SiO2 NPs as Raman nanotags, respectively. We expected that FPBA-modified Ag/PATP@SiO2 NPs could provide improved sensitivity for the detection of glycoproteins as compared with BASA (the LOD of BASA was ~10-11 M),25 because the overall Raman intensity level of FPBA-modified Ag/PATP@SiO2 NPs were much stronger than that of MPBA-modified AgNPs as colloidal solutions at similar concentration levels.

Effect of the combination of gold-coated glass slides and silver-based Raman nanotags on Raman signal. As shown in Figure S4, the combination of gold-coated glass slide and MPBA-modified AgNPs (PERS mode) exhibited much higher signal intensity as compared with the combination bare glass slide and MPBA-modified AgNPs (conventional SERS mode), with a 19.7-fold enhancement in Raman intensity at 1072 cm-1. This result confirmed our expectation of PERS mode through the simple combination of two noble metal materials can provide improved sensitivity. This enhancement factor is nearly the same as that of MPBA-modified AgNPs on glass rod 13



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in the previous study (19.9),32 but much higher than that generated by the macroporous monolith-induced secondary Raman scattering (2.6).25 Therefore, we employed the gold thinlayer-coated glass slide as substrates to fabricate MIP arrays.

Preparation of EPO-imprinted arrays and optimization of imprinting time. As a proof-of-concept, 12-spot (3 × 4) arrays (Figure S5A) were used in this study. The boronic acid-functionalized gold-coated array was golden yellow, while the MIP array was darker due to the presence of the poly(APBA-co-dopamine) layer on the array. It should be noted that MIP arrays can be fabricated into any format of larger numbers of spots for high-throughput assay through some slight technical modifications. The thickness of the molecularly imprinted layer is critical in boronate affinity oriented surface molecular imprinting. Favorably, the thickness generated by the imprinting procedure employed herein is adjustable through changing imprinting time. According to the thickness-imprinting time relationship established previously (y = 0.25 + 3.49 x, R2 = 0.95, where y is in nm and x is in hour),26 the imprinting time for EPO-α was optimized by setting the imprinting time at 40, 50 and 60 min. The imprinting effect was evaluated in terms of imprinting factor (IF), which was calculated according to the ratio of the Raman intensity for EPO-α detected on MIP array over that on NIP, using MPBA-modified AgNPs as Raman nanotags, because of their simpler synthesis and shorter labeling time reported previously (only 2 min).25 As shown in Figure S5B, the EPO-α-imprinted array prepared with 50 min polymerization time exhibited the best performance, giving an IF value of 15.7. Under this optimal imprinting time, the thickness of molecularly imprinted layer was calculated to be 3.16 nm. Taking the length of the adduct product of a terminal APTES residue and an FPBA ligand 14


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(1.14 nm, obtained by simulation operated on the ChemBioOffice 2014 software suite) into account, the imprinted layer occupied 58% and 43% of the larger two dimensions of an EPO molecule (molecular size, 2.5 × 3.5 × 4.7 nm (ref. 43)), which obeyed the rule proposed previously that an appropriate thickness lies within 1/3 to 2/3 of the molecular size of the template in one of the three dimensions.20 Thus, this optimized imprinting time (50 min) was also adopted for the preparation of EPO-β-imprinted array since EPO-β is almost identical with EPO-α in molecular size.

Optimization of incubation time and labeling time. Before evaluating the plasmonic immunosandwich assay of EPO using FPBA-modified Ag/PATP@SiO2 NPs as Raman nanotags, the incubation time between EPO-α and MIP arrays and the labeling time between EPO-α and FPBA-modified Ag/PATP@SiO2 NPs were optimized. As shown in Figure S6A, the Raman intensity increased with increasing incubation time between EPO-α and the MIP arrays within 5–20 min but became constant when the incubation time exceeded 20 min. Thus, 20 min was considered as the optimal incubation time and used for further experiments. As shown in Figure S6B, the Raman intensity increased with increasing labeling time between EPO-α and FPBA-modified Ag/PATP@SiO2 NPs within 1-5 min, and then kept nearly constant as further increasing the labeling time. Thus, 5 min was considered as the optimal labeling time and used for further experiments.

Imprinting efficiency. Based on the Raman spectra shown in Figure S7, the imprinting efficiency was calculated to be 42.9%, which was excellent in molecular imprinting.

Response curves for EPO-α α with different Raman nanotags. For simplicity, MPBA-modified 15



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AgNPs were first used as Raman nanotags to test the detectability for EPO-α. The Raman spectra by the EPO-α-imprinted array-based PISA for EPO-α at different concentration are shown in Figure 2A. Adsorption isotherms for the imprinted assay and non-imprinted array was established by plotting the intensity at 1072 cm-1 against the logarithm of the EPO-α concentration (see Figure 2B). The signal increased linearly with the logarithm of the concentration within the range of 100 pg/mL – 10 µg/mL (R2 = 0.990) and the limit of detection (LOD) was 100 pg/mL (2.9 × 10-12 M) with a signal to noise ratio (S/N) of 4. The response curve for the MIP was further analyzed through data fitting according to the logistic function and the apparent dissociation constants (Kd) was calculated to be 4.2 × 10-9 M (R2 = 0.996). Although the LOD value with MPBA-modified AgNPs as Raman tags has been lowered by about 1 order of magnitude as compared with that of the BASA approach,19 such a detectability did not meet the need for detecting trace EPO in human urine. So the detectability with FPBA-modified Ag/PATP@SiO2 NPs was further investigated. Similarly, the Raman spectra for EPO-α at different concentrations were investigated and corresponding adsorption isotherms were established (Figures 2C and 2D). The signal increased linearly with the logarithm of the concentration within the range of 1 pg/mL – 100 ng/mL (R2 = 0.993) while the LOD value was 1 pg/mL (2.9 × 10-14 M, S/N = 4). Such a LOD value was decreased by 3 orders of magnitude as compared with that of BASA.25 Such an ultrahigh detection sensitivity greatly benefited from the use of the PERS detection mode as well as the use of FPBA-modified Ag/PATP@SiO2 NPs. Such detection sensitivity matches well with the low-end of the normal concentration range of EPO in human urine. Interesting, the logistic function fitting of the data in the adsorption isotherm for the imprinted array yield a lower Kd value (5.3 × 10-11 M, R2 = 0.998). As compared with above logistic function fitting, the sole difference herein was that FPBA-modified Ag/PATP@SiO2 NPs were used as Raman labeling tags. Such a difference may 16


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suggest a warning that when sandwich assay is used to measure the binding constants of MIPs, the sensitivity of the labeling tags should be taken into account. The adsorption isotherms for MIP arrays and NIP arrays shown in Figures 2B and 2D clearly suggested that the imprinting approach used here was highly effective. The two types of Raman labeling tags tested in this study provided two possible choices with different sensitivity. If the concentration of the target glycoprotein in the samples to be analyzed is higher than the LOD value of MPBA-modified AgNPs, MPBA-modified AgNPs can be a good choice because their synthesis is straightforward. However, if the concentration of the target glycoprotein in the samples to be analyzed is lower than the LOD value of MPBA-modified AgNPs, FPBA-modified Ag/PATP@SiO2 NPs have to be selected, though their synthesis involved relatively tedious and complex procedure.

Cross-reactivity. The selectivity of the EPO-α-imprinted arrays was examined using FPBA-modified Ag/PATP@SiO2 NPs as Raman nanotags (see Figure 3A). The cross-reactivity ranged from 1.9 to 9.6% for 1,000-fold higher concentration for interfering glycoproteins and non-glycoproteins, and was only 0.8% for 10,000-fold higher concentration for glucose. Considering the logarithmic signal intensity-concentration dependence, the actual cross-reactivity was very limited. These data suggest that the EPO-α-imprinted arrays exhibited excellent specificity towards EPO-α, even under the presence of high-abundance interferents. However, the cross-reactivity of EPO-β (another recombinant EPO) on the EPO-α-imprinted array was about 91%. Cross-reactivity of the two recombinant EPOs on their corresponding MIP arrays was further investigated. As shown in Figure 3B, the signal intensities for EPO-α and EPO-β on EPO-α-imprinted and EPO-β-imprinted arrays 17



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were comparable. This suggests that EPO-imprinted MIPs produced by the approach employed herein failed to recognize the template isoforms against other isoforms of EPO. This is reasonable because the two recombinant EPO analogues are highly similar in structure. In fact, as far as we know, antibodies that can recognize recombinant EPO analogues have not been reported yet, and this is why a variety of electrophoretic techniques39,50-52 have been developed to differentiate EPO isoforms. Since the MIPs prepared herein failed to differentiate EPO isoforms, they can be used to directly detect native or endogenous human EPO. This is a beneficial factor because native or endogenous human EPO standard is rare and rather expensive. Thus, total EPO in a sample can be detected using the MIPs prepared in this study.

Real-world applications. The method of standard addition is a type of quantitative analysis approach often used in analytical chemistry where sample matrix also contributes to the analytical signal (matrix effect). Normal calculation approach of the method of standard addition involves a linear fitting according to the signal intensity and the spiked concentration and reverse extrapolation. The absolute value of the horizontal intercept is considered to be the target’s concentration in the unspiked sample. However, this normal approach is not suitable for quantitative analysis based on the plasmonic detection in this study as well as SERS detection,25 in which the signal intensity is linearly related to the logarithm of the total concentration of the target. To exemplify real-world applications of the MIP-based PISA approach, a novel strategy was proposed herein to provide accurate results for the plasmonic detection using the method of standard addition. Since the MIPs prepared was highly specific towards the target, we assumed that the matrix effect could be effectively eliminated, thereby we could calculate the original concentration of EPO in the unspiked urine samples according to the 18


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linear calibration curve shown in Figure 2D. Then, the total concentrations of EPO in the spiked urine samples were calculated. After that, the Raman signal intensity for the unspiked and spiked urine samples was plotted against the logarithm of the obtained concentrations. If the assumption that the matrix effect could be effectively eliminated was close to the real situations, then a good linear relationship should be obtained. Herein, we employed EPO-α-imprinted arrays and FPBA-modified Ag/PATP@SiO2 NPs to measure the EPO concentration in human urine. Human urine from a healthy male volunteer were protected by complete protease inhibitor cocktail and adjusted to a pH value of 7.4 before use. Aliquots of urine samples were spiked with different EPO concentrations (ci) and then analyzed by the PISA approach. The results are shown in Figures 4A and 4B. Using the strategy proposed above, the c0 value was first calculated to be 1.18 ± 0.33 pg/mL according to the linear equation obtained with standard solutions (y = 588.35 + 1770.64 x, R2 = 0.993, Figure 2D). Taking the signal intensities for 1 pg/mL EPO solution and urine spiked with 1 pg/mL EPO as references (Figures 4A and 4B), it can be seen that the calculated EPO concentration was quite reasonable. We further plotted the intensities at 1435 cm-1 against the logarithm of the total concentrations of total EPO in the unspiked and spiked urine samples. It gave a good linear relationship (R2 = 0.997; Figure 4C), which was nearly parallel to the standard curve shown in Figure 2D. Substituting c0 with lower and higher values drove the linear relationship bending upwards and downwards respectively (see Figure S8), which further confirmed the above result. Considering changes of volume during pretreatment, the EPO concentration in urine was calculated to be 1.34 ± 0.37 pg/mL, which fell within the normal range of healthy adult individuals. The above PISA approach permitted to quantify EPOs including endogenous EPO and its 19



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recombinant isoforms, such as EPO-α and EPO-β, in human urine within 0.5 h, without intensive labor to perform purification and enrichment steps and tedious glycoform profiling procedures. However, it should be noted that this approach is not suitable for the assay of recombinant or synthetic mimics of EPO that have distinct structures from the isoforms that were used as the templates in this study, for which the target should be directly used as the imprinting template.

Conclusions In short, a MIP-based PISA approach has been developed for fast and ultrasensitive determination of trace glycoproteins in complex real samples. Gold-based MIP arrays were constructed by the boronate affinity-based controllable oriented surface imprinting approach for specific extraction of trace targets and plamonic enhancement. Taking advantage of different boronate affinity silver-based Raman nanotags, the sensitivity of MIP-based PISA approach could be adapted to varying levels to meet detection requirements of different targets. Ultrasensitive and quantitative detection of EPO in human urine were performed with the aid of a novel standard addition method for the logarithmic dose-response relationship. In addition to EPO, targets can be easily expanded to other glycoproteins. The PISA approach can also be modified to detect other species by functionalizing the gold substrate and Raman-active AgNPs with different recognition elements such as antibodies and aptamers. Besides, it can be used for a high-throughput assay by exploiting arrays with larger numbers of spots. Therefore, this approach has good application prospects in many areas, such as biochemical research, clinical diagnosis and anti-doping analysis.

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AUTHOR INFORMATION * Corresponding Author Tel.: +86 25 8968 5639; fax: +86 25 8968 5639. E-mail address: [email protected] (Z. Liu).

ACKNOWLEDGEMENT 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 as well as the Key Grant of 973 Program (No. 2013CB911202) from the Ministry of Science and Technology of China.

ASSOCIATED CONTENT Supporting Information. Synthesis routes, additional conditional optimization, additional characterization and additional validation are provided in the supporting information.

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Figure Captions

Figure 1. Schematic illustration of the MIP-based PISA approach for the detection of target glycoprotein.

Figure 2. (A) Raman spectra for EPO-α at different concentrations (dissolved in 1× PBS) obtained on EPO-α-MIP arrays using MPBA-modified AgNPs as Raman nanotags. (B) Dependence of the Raman intensity at 1072 cm-1 detected on MIP and NIP arrays on the concentration of EPO-α, using MPBA-modified AgNPs as Raman nanotags. (C) Raman spectra for EPO-α at different concentrations (dissolved in 1× PBS) obtained on EPO-α-MIP arrays using FPBA-modified Ag/PATP@SiO2 NPs as Raman nanotags. (D) Dependence of the Raman intensity at 1435 cm-1 detected on MIP and NIP arrays on the concentration of EPO-α, using FPBA-modified Ag/PATP@SiO2 NPs as Raman nanotags. Inserts in (A) and (C) are parts of spectra around the characteristic bands of the Raman nanotags used to show LODs.

Figure 3. Interference test. (A) Cross-reactivity on EPO-α-imprinted arrays. Sample: 1 ng/mL EPO-α, EPO-β, 1 µg/mL interfering protein or 10 µg/mL glucose dissolved in 1× PBS. (B) Compatibility of EPO-α and EPO-β on EPO-α-imprinted and EPO-β-imprinted MIP arrays. Sample: 1 µg/mL EPO-α or EPO-β dissolved in 1× PBS. Raman nanotags: FPBA-modified Ag/PATP@SiO2 NPs.

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Figure 4. (A) Raman spectra for complete protease inhibitor cocktail-protected urine samples spiked with different concentrations of EPO-α. Raman nanotags: FPBA-modified Ag/PATP@SiO2 NPs. (B) Raman intensities at 1435 cm-1 for complete protease inhibitor cocktail-protected urine samples spiked with different concentration of EPO-α. (C) Linear relationship (y = 1766.80 + 556.17 x, R2 = 0.997) between the Raman intensity at 1435 cm-1 and the logarithmic value of the total concentration of EPO in urine 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 array.

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Molecularly Imprinted Polymer-Based Plasmonic Immunosandwich

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