Dual Molecularly Imprinted Polymer-Based Plasmonic

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Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Dual Molecularly Imprinted Polymer-Based Plasmonic Immunosandwich Assay for the Specific and Sensitive Detection of Protein Biomarkers Rongrong Xing, Yanrong Wen, Yueru Dong, Yijia Wang, Qi Zhang, and Zhen Liu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China Downloaded via GUILFORD COLG on July 26, 2019 at 17:55:32 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Molecularly imprinted polymers (MIPs), which are synthesized in the presence of a template, have been widely used as antibody mimics for important applications. Through the combination with a highly sensitive detection scheme such as chemiluminescence and surface-enhanced Raman scattering (SERS), MIP-based sandwich assays have emerged as promising analytical tools for the detection of disease biomarkers. However, so far, MIPs have been used only as target-capturing probes, whereas labeling by other means was needed, which limits the application range. Herein, we present a new approach, called a dual MIP-based plasmonic immunosandwich assay (duMIPPISA), for the specific and sensitive detection of protein biomarkers in complex biological samples. A C-terminal epitope-imprinted self-assembled gold nanoparticle monolayer-coated glass slide was prepared as a plasmonic substrate for the specific extraction of target protein, while N-terminal epitope-imprinted Raman-responsive Ag@SiO2 nanoparticles were prepared as nanotags for the specific labeling of captured protein. The formed MIP−protein−MIP sandwich-like complexes could produce a significantly enhanced SERS signal. The dual MIP-based recognitions ensured high specificity of the assay, while SERS detection provided ultrahigh sensitivity. The duMIP-PISA of neuron-specific enolase (NSE) in human serums was demonstrated, which permitted the differentiation of small cell lung cancer patients from healthy individuals. As compared to regular ELISA, the duMIP-PISA exhibited multiple merits including a simpler procedure, faster speed, lower sample volume requirement, and wider linear range. The approach well demonstrated the great potentials of MIPs and can be easily modified and extended to other protein biomarkers. Therefore, the duMIP-PISA approach holds great promise in many important applications such as disease diagnosis.

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which corresponding labels or amplifying reagents such as radioactive isotopes, enzymes, and fluorescence and chemiluminescence probes are essential. However, these reagents suffer from many drawbacks. Radioactive isotopes are harmful to health, enzymes are unstable and prone to lose activity, and fluorescent and chemiluminescent probes are susceptible to environmental factors, such as solvent, temperature, pH, ionic strength, sample composition, and so on. Therefore, antibodyfree immunoassays that can overcome the above disadvantages are highly desirable. Molecularly imprinted polymers (MIPs), as synthesized receptors with antibody-like binding properties, are produced through copolymerization in the presence of a template.11−18 As compared to antibodies, MIPs exhibit several merits, such as being easy to prepare, cost-efficient, stable, and resistant to harsh environments. MIPs have been widely applied in

roteins are one of the most important classes of biomarkers. For example, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), prostate specific antigen (PSA), and carcinoma antigen 15-3 (CA 15-3) have been routinely used as tumor markers for the early diagnosis of cancer.1,2 However, many protein biomarkers in biological samples are often present at very limited concentration, and there are many high-abundance coexisting interfering species. The determination of protein biomarkers usually requires high-quality antibodies for sample enrichment and cleanup as well as highsensitivity detection techniques. Immunoassay has been an essential tool for the determination of protein biomarkers in clinical diagnostics, in which highly specific antibodies are critical. 3,4 However, the generation of high-quality antibodies is costly, time-consuming, and even impossible sometimes. Antibodies also suffer disadvantages such as instability and poor reproducibility.5,6 Meanwhile, immunoassays employ a variety of high-sensitivity detection methods, such as radiodetection,7 enzymatic amplification,8 fluorescence,9 and chemiluminescence,10 for © XXXX American Chemical Society

Received: April 15, 2019 Accepted: July 14, 2019

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DOI: 10.1021/acs.analchem.9b01826 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Schematic illustration of the duMIP-PISA for the detection of protein biomarkers.

separation,19,20 sensing,21,22 proteomics,23,24 bioimaging,25,26 and controlled drug release.27,28 Through coupling with a sensitive detection scheme, such as fluorescence,29,30 electrochemical detection31,32 and chemiluminescence,33,34 MIPbased sandwich assays have gained increasing attention. Among many ultrasensitive detection schemes, surfaceenhanced Raman scattering (SERS) has attracted great interest because of its multiple significant advantages, including high sensitivity, rapid readout speed, less susceptibility to environmental factors, and possibility for on-site or field detection.35−37 Recently, through the combination of MIPs with SERS, an antibody-free and enzyme-free analytical approach named boronate affinity sandwich assay (BASA)38−41 and its updated version termed plasmonic immunosandwich assay (PISA)42−46 have emerged as an appealing technique. In particular, PISA, by virtue of an extra amplification mechanism between an extraction substrate and SERS labeling nanotags, can provide ultrahigh sensitivity and has demonstrated great potential for real-world applications such as disease diagnosis44,46 and single-cell analysis.42 However, in all of the MIPbased sandwich assays, MIPs have been employed only as target-capturing probes whereas labeling by other means rather than MIPs have to be used. This is because the MIPs that were used were prepared using whole targets as the templates so that once they bound with the targets only a limited portion of the surface of the targets remained accessible for labeling. Therefore, new types of MIP-based sandwich assays hold great promise for wide applications. Herein, we present a new approach, called dual MIP-based PISA (duMIP-PISA), for the specific and sensitive determination of protein biomarkers in complex biological samples. This new analytical approach benefited from our newly developed imprinting approach called controllable oriented surface imprinting of a boronate affinity-anchored epitope.47 Different from whole target-templated molecular imprinting approaches,20−22,27,28 this approach permitted the facile and efficient imprinting of C-terminal epitope peptides, providing new access to the specific recognition of proteins and peptides through binding their characteristic terminal peptide fragments. In this study, we further extended this approach to Nterminal epitope peptides. Because such epitope-imprinted MIPs allow for simultaneously binding a target protein from

two different sites, they could enrich the arsenal of MIPs to create new PISA assays to cover a wider scope of applicable target proteins. Although epitope imprinting had been well developed for the specific recognition of proteins,48−50 organosiloxane-based surface imprinting was used in this study because it has been reported to be highly favorable for the maintenance of the plasmonic effect.44 The principle and procedure of the duMIP-PISA are illustrated in Figure 1. Cand N-terminal nonapeptides of a protein biomarker are selected as epitope sequences. To obtain glycated epitope templates, the C-terminal nonapeptide is first attached with a lysine in its C-terminal, and then the additional lysine is glycated with fructose while the N-terminal nonapeptide in its N-terminus is directly glycated with fructose. A boronic acidfunctionalized gold nanoparticle (AuNP) self-assembled monolayer (SAM)-coated glass substrate is prepared (Figure S1a) and immobilized with the glycated C-terminal epitope by virtue of the boronate affinity. Boronic acid and p-aminothiophenol (PATP, Raman-active)-functionalized Ag@SiO2 NPs are prepared (Figure S1b) and immobilized with the Nterminal glycated epitope by virtue of the boronate affinity. Subsequently, imprinting layers are respectively formed on the surface of the two substrates via the polycondensation of multiple silylating reagents containing functionalities capable of interacting with the epitope sequences, including aminopropyltriethoxysilane (APTES), 3-ureidopropyltriethoxysilane (UPTES), isobutyltriethoxysilane (IBTES), and tetraethyl orthosilicate (TEOS). Finally, the glycated epitopes are removed by rinsing with an acidic acetonitrile solution to disrupt noncovalent and boronate affinity interactions, leaving imprinted cavities on the substrate surface. The prepared Cterminal epitope-imprinted AuNP SAM-coated glass slide was used as a plasmonic substrate for the specific extraction of the protein biomarker from complex biological samples, while the N-terminal epitope-imprinted Ag/PATP@SiO2 NPs were used as Raman nanotags for the specific labeling of the captured protein. Because dual MIP-based recognition is involved, the established PISA is expected to provide improved specificity as compared to single MIP-based PISA. Neuron-specific enolase (NSE), which has been used or suggested as a biomarker for multiple diseases,51,52 including Alzheimer’s disease, neuroblastoma, neuroendocrine diseases, and small cell lung cancer B

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For the immobilization of the glycated N-terminal epitope, 1 mL of 1.0 mg/mL glycated N-terminal epitope dissolved in phosphate buffer (10 mM, pH 7.4) was added to 9 mL of a 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA)-functionalized Ag/PATP@SiO2 NP suspension. After incubating at room temperature for 2 h, the obtained glycated N-terminal epitope-immobilized Ag/PATP@SiO2 NPs were centrifuged at 8000 rpm for 10 min and washed with phosphate buffer (10 mM, pH 7.4) three times. For oriented surface imprinting, the glycated N-terminal epitope-bound Ag/PATP@SiO2 NPs were dispersed into 15 mL of anhydrous ethanol containing 0.45 mL of ammonium hydroxide (28%), and 1 mL of water was added to the resulting suspension and stirred for 5 min. Then monomers (APTES, UPTES, IBTES, and TEOS) of the desired ratio dissolved in 4 mL of anhydrous ethanol (the volume of APTES was kept at 2 μL) were added to the above suspension and stirred at 25 °C for a certain period of time (40, 50, 60, or 70 min). The resulting N-terminal epitope-imprinted Ag/PATP@ SiO2 NPs were collected by centrifugation at 8000 rpm for 10 min. For the removal of the glycated N-terminal epitope, the Nterminal epitope-imprinted Ag/PATP@SiO2 NPs were dispersed into 20 mL of ACN/H2O/HAc = 50:49:1 (v/v) and shaken for 20 min at room temperature. The above elution process was repeated three times. After the glycated N-terminal epitope template was removed, the prepared N-terminal epitope-imprinted Ag/PATP@SiO2NPs were centrifuged at 8000 rpm for 10 min and washed with anhydrous ethanol and water three times each. Finally, the N-terminal epitopeimprinted Ag/PATP@SiO2 NPs were redispersed in phosphate buffer (10 mM, pH 7.4). Nonimprinted Ag/PATP@ SiO2 NPs were prepared using the same procedure except for the absence of the glycated N-terminal epitope template.

(SCLC), was selected as a model protein biomarker in this study. The prepared epitope-specific MIPs exhibited excellent binding properties. A new PISA format (i.e., duMIP-PISA) was developed using the two MIPs, which showed improved specificity. The developed duMIP-PISA was successfully applied to the determination of NSE in serums from healthy individuals and SCLC patients. As compared to a commercial kit-based ELISA, the duMIP-PISA exhibited multiple advantages including a simpler procedure, shorter time consumption, a lower sample volume requirement, and a wider linear range. The approach can be easily modified and extended to other protein biomarkers. Therefore, we can foresee that the duMIPPISA approach has great promise in many important applications, particularly in disease diagnosis.

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EXPERIMENTAL SECTION

Preparation of C-Terminal Epitope-Imprinted AuNP SAM-Coated Glass Substrates. The preparation procedure included three major steps: (1) immobilization of the glycated C-terminal epitope, (2) oriented surface imprinting, and (3) removal of the glycated C-terminal epitope. For the immobilization of the glycated C-terminal epitope, 5 μL of a 1.0 mg/mL glycated C-terminal epitope dissolved in ammonium bicarbonate buffer (50 mM, pH 8.5) containing 500 mM NaCl was dripped onto each spot on the boronic acid-functionalized AuNP SAM-coated glass substrates and then incubated for 2 h in a humid environment. During this period, ammonium bicarbonate buffer (50 mM, pH 8.5) containing 500 mM NaCl was supplemented every 20 min. The obtained glycated C-terminal epitope-immobilized AuNP SAM-coated glass substrates were washed with ammonium bicarbonate buffer (50 mM, pH 8.5) three times. For oriented surface imprinting, 0.2 mL of water was added to 3 mL of anhydrous ethanol containing 0.09 mL of ammonium hydroxide (28%). Then monomers (APTES, UPTES, IBTES, and TEOS) of the desired ratio were dissolved in 40 mL of anhydrous ethanol (the volume of APTES was kept at 20 μL), and 0.8 mL of the resulting solution was added to the above solution and mixed evenly. The glycated C-terminal epitope-bound AuNP SAM-coated glass substrates were quickly immersed in the mixed solution and reacted at 25 °C for a certain period of time (50, 60, 70, or 80 min). For the removal of the glycated C-terminal epitope, the resulting C-terminal epitope-imprinted AuNP SAM-coated glass substrates were immersed in 4 mL of acetonitrile (ACN)/ H2O/acetic acid (HAc) = 50:49:1 (v/v) and shaken for 20 min at room temperature. The above elution process was repeated three times. After removing the glycated C-terminal epitope template, the prepared C-terminal epitope-imprinted AuNP SAM-coated glass substrates were washed with anhydrous ethanol and water three times each. Finally, the C-terminal epitope-imprinted AuNP SAM-coated glass substrates were dried in air and stored at 4 °C before use. Nonimprinted AuNPs SAM-coated glass substrates were prepared using the same procedure except for the absence of the glycated C-terminal epitope template. Preparation of N-Terminal Epitope-Imprinted Ag/ PATP@SiO2 NPs. The preparation procedure was composed of three steps: (1) immobilization of the glycated N-terminal epitope, (2) oriented surface imprinting, and (3) removal of the glycated N-terminal epitope.

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RESULTS AND DISCUSSION Synthesis and Characterization. According to previous works, the coupling of Au and Ag can produce a strong SERS signal,42,43 and AuNP SAM-coated glass slides as plasmonic substrate arrays are better than a Au thin layer-coated glass slide.44 Therefore, narrowly dispersed AuNPs were synthesized and used as base nanoparticles for forming a SAM on the surface of glass slides to create extraction arrays with a more effective plasmonic effect. As shown in Figure S2a, the average diameter of AuNPs was estimated to be 60 nm by transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. A scanning electron microscopy (SEM) image of a SAM of C-terminal epitope-imprinted AuNPs on a glass slide is shown in Figure S2b, with well-organized AuNPs on the slide. The uniformly dispersed size and well-formed SAM coating were highly favorable for the detection sensitivity as well as the detection reproducibility. Meanwhile, uniform sizedispersed silver nanoparticles (AgNPs) were synthesized and used as core nanoparticles to further prepare Raman nanotags. As shown in Figure S2c,d, the average diameters of the prepared AgNPs and N-terminal epitope-imprinted Ag/ PATP@SiO2 NPs were estimated by TEM and DLS to be 60 and 75 nm, respectively. The changes in the extinction spectrum of N-terminal epitope-imprinted Ag/PATP@SiO2 NPs in the preparation process were examined (Figure S2e). Ag/PATP@SiO2 NPs had a 9 nm red shift in the surface plasmon band of AgNPs because of the presence of a silica layer. After Ag/PATP@SiO2 NPs were modified by boronic C

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Figure 2. Comparison of the amounts of different proteins captured by (a) C-terminal epitope-imprinted and (b) N-terminal epitope-imprinted MNPs as well as corresponding nonimprinted MNPs prepared under optimized conditions. The error bars represent the standard deviation for three parallel experiments.

protein are different, their glycation methods were also different. For the glycation of C-terminal epitopes, as in our previous method,47 a lysine was first introduced to the Cterminus of HNFRNPSVL, and then the amino group on the lysine residue reacted with fructose through reductive amination. However, for the glycation of the N-terminal epitope, because of the presence of the amino group in the Nterminus of the epitope, additional lysine was unnecessary and the N-terminal amino acid residue was reacted directly with fructose through reductive amination. The obtained glycated C- and N-terminal epitopes were HNFRNPSVLK-Fru and Fru-SIEKIWARE, respectively, and their structures are shown in Figure S3. These synthesized glycated epitopes were used as the imprinting templates in this work. Optimization of the Monomer Ratio and Imprinting Time. To obtain high-performance imprinted materials, the monomer ratio and imprinting time were optimized in this study. For the convenience of optimization, magnetic nanoparticles (MNPs) were chosen as a substrate material because of facile magnetic separation. First, boronic acid-functionalized Fe3O4@SiO2 MNPs were prepared, and then their boronate affinities were verified. As shown in Figure S4a, the Fe3O4@SiO2@DFFPBA MNPs exhibited good selectivity for adenosine (cis-diol compound) but excluded deoxyasdenosine (non-cis-diol compound), which proved that the Fe3O4@SiO2 MNPs had been successfully modified with DFFPBA. Meanwhile, the prepared Fe3O4@SiO2@DFFPBA MNPs could selectively bind to glycated C- and N-terminal epitopes, which suggests that the C- and N-terminal epitopes were successfully glycated with fructose (Figure S4b). Thus, the glycated C- and N-terminal epitopes could function as templates in subsequent boronate affinity-based imprinting. Second, epitope-imprinted MNPs were synthesized, and the combination of monomers was selected according to the common features of amino acids of the epitope sequence.47 In this study, APTES, UPTES, and IBTES were selected as functional monomers because they can interact with epitope peptides to produce electrostatic, hydrogen bonding, and hydrophobic interactions, respectively. TEOS was used as a cross-linker to form a hydrophilic silica skeleton, which can not only cross-link other functional monomers but also help to reduce nonspecific adsorption. Meanwhile, the imprinting approach based on these monomers and cross-linker allowed for controlling the thickness of the imprinting layer through adjusting the imprinting time once the monomer ratio was determined.47 Because the amino acid composition and length of glycated C- and N-terminal epitopes are different, to obtain

acid, there was a 12 nm blue shift for Ag/PATP@SiO2@ DFFPBA NPs. The prepared N-terminal epitope-imprinted and nonimprinted Ag/PATP@SiO2 NPs also exhibited a clear blue shift, which was attributed to the presence of an imprinting layer. Meanwhile, the extinction spectra for the imprinted and nonimprinted Ag/PATP@SiO2 NPs were almost the same, indicating that the imprinting procedures were the same, so the nonimprinted Ag/PATP@SiO2 NPs can be used as a comparison to reflect the imprinting effect. Figure S2f shows the Raman spectra of related NPs in the preparation process of N-terminal epitope-imprinted Ag/PATP@SiO2 NPs, and a C-terminal epitope-imprinted AuNP SAM-coated glass slide was used as a substrates. The overall Raman intensity of the N-terminal epitope-imprinted Ag/PATP@SiO2 NPs was slightly weaker than that of Ag/PATP NPs, but the N-terminal epitope-imprinted Ag/PATP@SiO2 NPs still had strong intensity as SERS nanotags. The characteristic peak was not contributed directly from PATP, but its photocatalytic coupling reaction product, 4,4-dimercaptoazobenzene (DMAB), was generated on AgNPs upon laser irradiation during the SERS measurements. The peaks at 1390 and 1435 cm−1 are assigned to the NN relative vibrational modes of DMAB, while the peaks at 1143, 1073, and 1574 cm−1 are assigned to the vibrational modes of C−N, C−S, and C−C bonds. In this study, 1435 cm−1 was chosen as the Raman characteristic peak for PISA assay. Selection and Glycation of Epitopes. The selection of epitopes is simple. Terminal nonapeptides can be chosen as ideal epitopes because they represent nearly unique codes in unstructured domains for the identification of a specific protein.48 Practically, the length or the number of amino acid residuals of epitope peptides can be slightly shorter or longer than that of nonapeptides. Because the imprinting approach47 used herein was developed on the basis of nonapeptide epitopes, nonapeptides were solely selected as epitopes in this study. C- and N-terminal nonapeptides of a protein of interest can be selected as epitopes providing that they are not associated with posttranslational modifications. C-Terminal nonapeptides are usually not posttranslationally modified whereas N-terminal nonapeptides of some proteins are modified. Therefore, terminal nonapeptides modified with bulky groups such as glycans or a secondary structure-forming group such as a disulfide bond should be avoided to be selected as epitopes. From a database such as UniProt, C- and N-terminal nonapeptides from human NSE were found to be HNFRNPSVL and SIEKIWARE, respectively. Because the recognition directions of C- and N-terminal epitopes of a D

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further experiments. As shown in Figure S10b, the Raman intensity increased with increasing labeling time within 5−15 min but became nearly constant when the labeling time exceeded 15 min. Thus, 15 min was considered to be the optimal labeling time and used for further experiments. Such short extraction and labeling times were favorable for a fast assay. Specificity Test. The specificity of duMIP-PISA was investigated by using NSE as a target protein and RNase A, BSA, RNase B, and HRP as competing proteins. As shown in Figure 3, the duMIP-PISA approach exhibited excellent

good imprinting performance the monomer ratio and imprinting time need to be optimized separately. In the optimization, the imprinting effect was evaluated in terms of the imprinting factor (IF), which was calculated according to the ratio of the amount of epitope captured by imprinted MNPs over that by nonimprinted MNPs. The results for Cterminal epitope-imprinted MNPs and nonimprinted MNPs are shown in Figure S5a−c, which suggest that imprinting with 10:20:20:50 APTES/UPTES/IBTES/TEOS at 60 min produced the highest IF value (5.8) for the C-terminal epitope. The results for N-terminal epitope-imprinted MNPs and nonimprinted MNPs are shown in Figure S5d−f, which suggest that imprinting with a 10:10:20:60 ratio at 50 min produced the highest IF value (5.2) for the N-terminal epitope. These results indicate that the separate optimization for C- and N-terminal epitopes was necessary and effective. Final, the specificity of the prepared epitope-imprinted MNPs was investigated under optimal imprinting conditions. NSE was used as a target protein, and ribonuclease A (RNase A), bovine serum albumin (BSA), ribonuclease B (RNase B), and horseradish peroxidase (HRP) were used as competing proteins. The amounts of proteins extracted by the C- and Nterminal epitope-imprinted MNPs were measured and compared. The results are shown in Figure 2. Both C- and N-terminal epitope-imprinted MNPs exhibited high specificity toward NSE, giving maximum cross-reactivities of 17.1 and 18.7%, respectively. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF MS) analyses also confirmed the specificities of the epitope-imprinted MNPs (Figure S6 and S7), which were consistent with the UV detection results. Furthermore, the specificity of the C- and Nterminal epitope-imprinted MNPs at the peptide level was also examined. Tryptic digests of HRP and BSA were employed as competing species. A mixture of the C- or N-terminal epitope and the tryptic digests of HRP and BSA (molar ratio 1:1:1) was extracted with the epitope-imprinted and nonimprinted MNPs. Without extraction with the C-terminal epitopeimprinted MNPs, the C-terminal epitope and 10 peptides from BSA and 4 peptides and 5 glycopeptides from HRP were detected. With the extraction, only the C-terminal epitope was detected (Figure S8 and Tables S1). Meanwhile, without extraction with the N-terminal epitope-imprinted MNPs, the N-terminal epitope and 11 peptides from BSA and 3 peptides and 5 glycopeptides from HRP were detected. With the extraction, only the N-terminal epitope was detected (Figure S9 and Tables S2). In contrast, nearly nothing was extracted by corresponding nonimprinted MNPs. Clearly, these results indicated that the prepared epitope-imprinted MNPs under the optimal imprinting conditions exhibited excellent specificity at the protein and peptide levels. Therefore, the optimized imprinting conditions were employed for the preparation of the C-terminal epitope-imprinted AuNP SAM-coated glass slide and N-terminal epitope-imprinted Ag/PATP@SiO2 NPs. Optimization of Extraction Time and Labeling Time. To obtain the highest Raman signal intensity and shortest operation time, the extraction time of NSE by a C-terminal epitope-imprinted AuNPs SAM coated-glass slide, and the labeling time of NSE by N-terminal epitope-imprinted Ag/ PATP@SiO2 NPs were optimized. As shown in Figure S10a, the Raman intensity increased with increasing extraction time within 5−15 min and then remained nearly constant with further increases in the extraction time. Thus, 15 min was considered to be the optimal extraction time and was used for

Figure 3. Overall selectivity of duMIP-PISA toward different proteins. The error bars represent the standard deviation for three parallel experiments.

specificity toward NSE, yielding a maximum cross-reactivity of only 6.7%. Compared to the specificity of the C- or Nterminal epitope-imprinting MNPs in single recognition, the specificity of the duMIP-PISA was significantly improved, which benefited from the double recognition toward different target sites (i.e., the C- and N- terminal epitopes by the two MIPs used). Response−Concentration Dependence. The Raman spectra for NSE at different concentrations in standard solutions are shown in Figure 4a. The intensity of the characteristic Raman peak gradually increased with increasing concentration of NSE. Binding isotherms for the duMIP-PISA and dual nonimprinted polymer-based PISA (duNIP-PISA, both extraction array and labeling nanotags were nonimprinted) were examined by plotting the intensity against the logarithm of the NSE concentration, which is shown in Figure 4b. For duMIP-PISA, the intensity increased linearly with the logarithm of the concentration of NSE within the range from 100 pg/mL to 10 μg/mL (y = 892.4x − 1281.5, R2 = 0.983). The LOQ value was 10 pg/mL (S/N = 10). The linear relationship was used as a calibration curve for quantitative analysis. Meanwhile, fitting the data by the logistical function gave an overall dissociation constant (Kd) of 3.4 × 10−9 M, suggesting that the MIPs used herein exhibited a high binding affinity for the target. As a comparison, for duNIP-PISA, the intensity of the Raman signal was almost unchanged as the concentration of the target protein increased. Real-World Applications. A practical application of duMIP-PISA was demonstrated by the determination of NSE in human serum. For the analysis of complex real samples, where the sample matrix may apparently contribute to the analytical signal (matrix effect), the standard addition approach is often used. Although the conventional standard addition method is not suitable for PISA because the signal intensity is a E

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Figure 4. (a) Raman spectra for NSE at different concentrations obtained by duMIP-PISA and (b) dependence of the Raman intensity detected by duMIP-PISA and corresponding duNIP-PISA on the logarithm of the NSE concentration. The error bars represent the standard deviation for three parallel experiments.

linear function of the target logarithm concentrations, quantitative analysis by PISA can be as simple as or even simpler than that of other immunoassays. In this study, because the prepared MIPs were highly specific for the target protein, one can assume that the matrix effect can be effectively eliminated in the PISA assay and thereby the original concentration of target protein from the unspiked serum samples can be calculated in accordance with the linear calibration curve. For instance, according to the calibration curves shown in Figure 4b, the original concentration of NSE in a healthy human individual was calculated to be 8.3 ± 2.4 ng/mL. To verify if the above assumption is acceptable, we carried out a modified standard addition approach.43 A series of serum samples spiked with a known NSE concentration were measured by duMIP-PISA (Figure S11a), and then the signal intensity was plotted against the logarithm of the total concentration of NSE in the spiked samples. As shown in Figure S11b, the obtained plot obeyed a good linear relationship (y = 934.1x − 1512.3, R2 = 0.994), which is very close to the calibration curve. Therefore, the assumption is reasonable and the calculated concentration for an unspiked sample is acceptable. Similarly, the NSE concentrations of two SCLC patient serums were detected by duMIP-PISA and further confirmed by the modified standard addition method (Figure S12). The measured results are listed in Table 1. Interestingly, all of the linear relationships obtained by the modified standard addition method were close to the

calibration curve. This suggests that once the PISA approach has been verified, no more calibration is needed. Comparison with Commercial Kit-Based ELISA. The NSE concentrations in the above-mentioned healthy human serum and two SCLC patient serums were also quantified by commercial kit-based ELISA and compared with those obtained by duMIP-PISA. Figure S13 shows the linear calibration curve for ELISA. The logarithm of OD increased linearly with the logarithm of the NSE concentration within the range of 0.78 to 50 ng/mL (y = 0.735x − 0.804, R2 = 0.999). According to the linear relationship, the concentrations of NSE for the healthy individual and the SCLC patients were calculated, and the results are given in Table 1. The performances of duMIP-PISA and commercial kit-based ELISA are also compared in Table 1. Although the results by duMIP-PISA are all slightly higher than those by ELISA, there was no significant difference. On the basis of the analytical results, SCLC patients can be easily differentiated from healthy individuals by the two approaches. However, as compared to the ELISA approach, the method reported in this study exhibited several significant advantages, including a simpler procedure (5 as compared to 11 steps), less time-consuming (50 min as compared to 5 h), a lower sample volume requirement (5 μL as compared with 100 μL), and a wider linear range (0.1 ng/mL to 10 μg/mL as compared to 0.78−50 ng/mL).

Table 1. Comparison of the Performance of duMIP-PISA and Commercial Kit-Based ELISAa

Previously, MIPs have been employed only as target-capturing probes whereas labeling by other means rather than MIPs has to be used in MIP-based sandwich assays. Different from this, we have developed a new MIP-based sandwich assay called duMIP-PISA in which both the target-capturing probes and the labeling nanotags were MIPs. This approach benefited from the epitope imprinting methodology employed,47 which allowed for the facile and efficient imprinting of terminal epitope peptides in a controllable manner. This study well demonstrated the great potentials of MIPs for molecular recognition in disease diagnosis. Through double MIP-based recognitions, duMIP-PISA exhibited much improved specificity as compared to single MIP-based recognition. Real-world application has been demonstrated by the determination of NSE in serum samples from healthy individuals and cancer patients. duMIP-PISA exhibited significant advantages over traditional immunoassays, such as low cost, good stability, a simplified procedure, fast speed, low sample consumption, and

item measured serum NSE concentration of a healthy individual measured serum NSE concentration of SCLC patient-1 measured serum NSE concentration of SCLC patient-2 time required number of required steps sample volume required liner range

commercial kit-based ELISA

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current method

5.6 ± 0.4 ng/mL

8.3 ± 2.4 ng/mL

173.4 ± 9.6 ng/mL

189.7 ± 45.6 ng/mL

47.2 ± 3.5 ng/mL

56.1 ± 14.9 ng/mL

5h 11 100 μL 0.78−50 ng/mL

50 min 5 5 μL 0.1 ng/mL−10 μg/mL

a Reference value of NSE concentration in healthy human serum: 0− 16.3 ng/mL.

F

CONCLUSIONS

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Analytical Chemistry

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a wide detection range. Because the imprinting approach that is used is facile, versatile, and efficient, the duMIP-PISA approach can be easily extended to other protein biomarkers. Therefore, we foresee that this approach could be a promising tool for many important applications such as disease diagnosis, biochemical research, and signaling pathway studies.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b01826. Experimental details (including reagents and materials, instruments, preparation of MNPs, preparation of boronic acid-functionalized Fe3O4@SiO2 MNPs, preparation of epitope-imprinted MNPs, selectivity of boronic acid-functionalized Fe3O4@SiO2 MNPs, optimization of the monomer ratio and imprinting time, protein digestion by trypsin, selectivity test of epitopeimprinted MNPs, preparation of AuNPs, preparation of DFFPBA-functionalized AuNPs SAM-coated glass substrates, preparation of AgNPs, preparation of DFFPBAfunctionalized Ag/PATP@SiO2 NPs, optimization of extraction time and labeling time, selectivity of duMIPPISA, binding isotherm, determination of NSE in human serum, and ELISA of NSE) and supplementary figures and tables (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zhen Liu: 0000-0002-8440-2554 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support of the Key Grant (21834003) and the National Science Fund for Distinguished Young Scholars (21425520) from the National Natural Science Foundation of China, the “333” Talents Project from the Jiangsu Provincial Government, China (BRA2016351), and program A for outstanding Ph.D. candidate of Nanjing University (201801A006).

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DOI: 10.1021/acs.analchem.9b01826 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.9b01826 Anal. Chem. XXXX, XXX, XXX−XXX