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Biological and Medical Applications of Materials and Interfaces

Quantitative SERS Detection of Dopamine in Cerebrospinal Fluid by Dual-Recognition Induced Hot Spot Generation Kun Zhang, Yu Liu, Yuning Wang, Ren Zhang, Jiangang Liu, Jia Wei, Hufei Qian, Kun Qian, Ruoping Chen, and Baohong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01063 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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ACS Applied Materials & Interfaces

Quantitative SERS Detection of Dopamine in Cerebrospinal Fluid by Dual-Recognition Induced Hot Spot Generation Kun Zhang,† Yu Liu,† Yuning Wang,‡ Ren Zhang,‡ Jiangang Liu,† Jia Wei,† Hufei Qian,† Kun Qian,§ Ruoping Chen,* † and Baohong Liu*‡ †

Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200062, China Department of Chemistry, Institutes of Biomedical Sciences, State Key Lab of Molecular Engineering of Polymers and Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai 200433, China § School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, China ‡

ABSTRACT: Reliable profiling of extracellular dopamine (DA) concentration in the central nervous system is essential for a deep understanding of its biological and pathological functions. However, quantitative determination of this neurotransmitter remains a challenge due to the extremely low concentration of DA in the cerebrospinal fluid of patients. Herein, based on the specific recognition of boronate toward diol, and N-hydroxysuccinimide ester toward amine group, a simple and highly sensitive strategy was presented for DA detection by using surface-enhanced Raman scattering (SERS) as a signal readout. This was realized by first immobilizing 3,3′-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP) on gold thin film surfaces to capture DA, followed by introducing 3-mercaptophenylboronic acid (3-MPBA)-functionalized silver nanoparticles (SNPs) to generate numerous plasmonic "hot spots" with the nanoparticle-on-mirror geometry. Such a dual-recognition mechanism not only avoids complicated bioelementbased manipulations but also efficiently decreases the background signal. With the direct use of the recognition probe 3-MPBA as a Raman reporter, the "signal on" SERS method was employed to quantify the concentration of DA from 1 pM to 1 µM with a detection limit of 0.3 pM. Moreover, our dual-recognition-directed SERS assay exhibited a high resistance to cerebral interference and was successfully applied to monitoring of dopamine in cerebrospinal fluid samples of patients. KEYWORDS: surface-enhanced Raman scattering (SERS), quantitative detection, hot spot assembly, dual-molecule recognition, dopamine

Dopamine (DA) is a catecholamine neurotransmitter that plays a significant role in the functioning of central nervous, vascular, and hormonal systems.1,2 It is widely distributed in the brain tissues and body fluids of mammals. The abnormal variation of DA concentration in vivo has been linked to serious neurological, renal, cardio disorders such as schizophrenia, Huntington's disease, Alzheimer's disease, and Parkinson's disease.3-6 Sensitive and accurate detection of DA in biological samples is highly desirable for understanding of the biological function of this compound and for the diagnosis and treatment monitoring of DArelated clinical diseases. Multiple techniques, including electrochemistry,7,8 fluorescence,9,10 colorimetry and chromatography,1114 have been developed for DA detection. These methods, however, are associated with some limitations. The coexistence of ascorbic acid (AA), uric acid (UA), and other analogs in the central nervous system renders electrochemical analysis challenging because the oxidation potential of these substances are close to that of DA at solid electrodes, leading to overlapping voltammetric response. The latter ones, such as chromatographic and fluorescent analysis, are limited by tedious sample processing, complicated probe synthesis and/or the requirement of expensive instrumentation. It is still essential to develop simple and rapid methods for the detection of dopamine in complex biological samples with high sensitivity and selectivity. Surface-enhanced Raman spectroscopy (SERS) is an extremely powerful analytical technique that can provide non-destructive measurements down to the single-molecule level.15-18 The re-

markable signal amplification (typically 106 to 1014) in SERS mainly comes from interactions of molecules adsorbed on a nanostructured metal surface with the strong electromagnetic field near the surface (so-called hot spot).19-21 In comparison with the time-consuming synthesis of fluorescent probes, most SERS reporters are commercially available. Moreover, the excitation of samples using visible (638 nm) or near-infrared (785 nm) laser radiations in SERS detections can effectively avoid interference of biological intrinsic fluorescence. SERS-based detection schemes have been successfully applied to probe various biological molecules including metabolites,22,23 nucleic acids,24-26 lipids,27 and proteins28-30 by rationally engineering hot spots of the enhancing substrates. However, direct quantification of DA in biological fluids by SERS remains a great challenge due to the following aspects: (1) the low concentration (< 10-10 M) of DA in the cerebral extracellular fluid of Parkinson’s disease patients,31 (2) the relatively small Raman cross section of the molecule, (3) high complexity of the sample matrix such as cerebrospinal fluid (CSF). Till now, the SERS-based detection scheme has only been developed to determine DA in very a few studies: aptamer bindinginduced formation of nanorod dimers,32 antibody recognitionbased bio-barcode amplification,33 and molecular chelation at the surface of colloidal silver nanoparticles (SNPs).34,35 In all these studies, sensitive and selective detection of DA was aimed, but few of them were applied to measure physiological DA levels in real CSF samples. In addition, the laborious modification of colloidal NP surfaces with bioelements such as DNA aptamer and the signal fluctuation due to dynamic variation of hot spots for

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colloid aggregation/de-aggregation based SERS assays present technical barriers preventing broad applications of these methods in clinics and routine biological laboratories. The question thus arises that if we could find a simple and efficient way to analyze physiological DA concentration in complex biofluids by rationally engineer the SERS hot spots in an antibody and/or aptamer-free manner. To solve this problem, we report here the use of a dual-molecular recognition strategy in which the target molecule is consecutively recognized by two molecules at different sites.36 The combination of such a detection scheme with SERS has been exploited for sensing of various analytes from glucose to glycoprotein in a sandwich assay format.37-43 However, to date, no studies have reported the use of dual recognition-based SERS for DA detection in biofluids. In this work, we demonstrate the first design and fabrication of a dual-recognition-based sensing platform capable of sensitively detecting DA in human CSF samples. We use 3,3′-dithiodipropionic acid di(Nhydroxysuccinimide ester) (DSP) immobilized on a gold thin film (GTF) to capture DA via the reaction between Nhydroxysuccinimide ester and the amine group, followed by binding of the 3-mercaptophenylboronic acid (3-MPBA)functionalized silver nanoparticle (SNP) at the diol terminal group. This results in the formation of a NP-on-mirror (NPoM) geometry with the molecular conjugates located precisely at the SNP-GTF junction, namely the hot spot location upon laser excitation. Our results demonstrated that this strategy was very helpful to improve the detection performance because the hot spot generation and the resulted signal amplification are closely associated with the binding of DA. In addition, the use of such a dual-recognition sensing strategy is also beneficial to the improvement of selectivity of the assay. The "signal-on" plasmonic method was applied successfully to detect the DA level in human CSF samples, demonstrating its potential applicability in biomedical diagnosis.

Scheme 1. Schematic of dual-recognition-directed hot spot assembly and its application for DA screening using DSP adsorbed on a GTF and 3-MPBA-modified SNPs.

EXPERIMENTAL SECTION Materials and Reagents. Dopamine hydrochloride (DA), 3mercaptophenylboronic acid (3-MPBA) and 3,3′dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP) were purchased from Sigma-Aldrich. Silver nitrate (AgNO3), trisodium citrate dihydrate, glucose (Glu), lactate (Lac), catechol, epinephrine, ascorbic acid (AA), uric acid (UA), tyrosine (Tyr) and phenylalanine (Phe) were obtained from Sinopharm Chemical Reagent Co., Ltd. All the reagents were used as received without further purification. Ultrapure water (≥18 MΩ cm) purified with a Milli-Q system was used throughout the work. Apparatus. UV-vis absorption spectra were measured using an Agilent 8453 UV-visible spectrometer with a standard 10-mm path length quartz cuvette. Scanning electron microscopic (SEM) characterization was carried out on a Hitachi S-4800 microscope at an acceleration voltage of 1.0 kV. Transmission electron micro-

scopic (TEM) characterization was carried out using a JEOL JEM-1011 microscope at an acceleration voltage of 100 kV. Atomic force microscopic (AFM) analysis was conducted on a Bruker MultiMode 8 AFM system. The SNP size distribution and zeta potential were analyzed by dynamic light scattering (DLS) with a Malvern Zetasizer NanoZ instrument following dispersion in water. A traditional three-electrode system controlled by a CHI630B electrochemical workstation (Shanghai CH Instruments Co. Ltd, China) was used for the cyclic voltammetric (CV) measurements at scan rate of 50 mV s-1. The home-made gold thin film (5 mm × 5 mm), Pt wire electrode, and saturated calomel electrode (SCE) were used as the working electrode, auxiliary electrode, and reference electrode, respectively. Electrochemical impedance spectroscopic (EIS) measurements were conducted on a PGSTAT 302N system (Metrohm Autolab, Switzerland) with a frequency range of 0.1 Hz to 105 Hz by applying an alternating current signal of 5 mV in amplitude at 0.25 V vs SCE. All electrochemical measurements were carried out in 5 mM solution of K3Fe(CN)6/K4Fe(CN)6 (1:1) mixture containing 0.1 M of KCl. SERS detection was conducted on a Horiba Xplora confocal Raman microscope equipped with a 1200 gr/mm grating, a 638 nm laser source and a charged coupled device (CCD) detector. The laser was focused onto the sample through a 50× objective (NA 0.5), providing a spot size of about 1.5 µm. The integration time per spectrum was 1 s and 121 SERS spectra were recorded over an area of 2500 (50 × 50) µm2 with a 5 µm interval for each sample. Baseline correction was performed for all measurements. The Raman band of silicon at 520 cm-1 was used to calibrate the spectrometer before detection. Preparation of SNPs and 3-MPBA-SNPs. SNPs with an average diameter of 60 nm were synthesized according to the classical Lee and Meisel method,44 that is, the reduction of AgNO3 by citrate in aqueous phase. Before the synthesis, all glassware was fully cleaned with aqua regia (HNO3/HCl, 1:3, v/v) and washed with copious water for use. Briefly, 1 mL of citrate solution (1% by weight) was added into 50 mL of AgNO3 boiling solution (0.18 mg/mL) under strong stirring. The mixture was allowed to keep boiling for 1 h followed by cooling to room temperature under stirring to generate the nanoparticle colloids. The surfaces of the as-synthesized SNPs were functionalized with 3-MPBA using the method reported by Liu′ group with slight modification.39 A certain amount (1-5 µL) of 1 mM 3-MPBA (dissolved in 0.2 M NaOH) was added to 1 mL of SNP dispersion and the mixed solution was stirred for a desired period (10-90 min) at room temperature. Preparation of DSP modified Gold Thin Film. DSP was immobilized onto the film via thiol-gold conjugation. Gold thin films (thickness: 100 nm) with a titanium adhesion layer (thickness: 10 nm) were prepared by electron beam evaporation on a single-crystal silicon wafer (111) (diameter: 4.2 inch, thickness: 525 µm). The gold-coated wafer was diced into 3 mm × 3 mm squares for use. Immediately before the modification, the fold films were cleaned by immersing in a hot piranha solution (H2O2/H2SO4, 1:3, v/v) for 1 h followed by washing with water and drying in nitrogen. For each substrate prepared, 100 µL of DSP solution (5 mM) in dimethylsulfoxide (DMSO) was added to the cleaned gold substrates in individual 96-well plates (Corning), and incubated for 100 min to form a self-assembled monolayer of DSP on top of the gold films. After that, the gold substrates were washed thoroughly with DMSO and deionized water to remove nonspecifically adhered DSP molecules. For each wash step, every substrate was moved to a new well. Detection of Dopamine. A volume of 100 µL of dopamine in phosphate buffer solution (pH 7.4, 10 mM) was added to the DSPfunctionalized gold film and incubated for 45 min at room temperature, followed by washing with deionized water for 5 min to


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ACS Applied Materials & Interfaces remove unbound dopamine. Next, 100 µL of the 3-MPBA-SNP dispersion was introduced and incubated for 40 min. Finally, the substrate was washed with deionized water for 5 min, dried in nitrogen and detected by the Raman spectrograph. Detection of Dopamine in CSF. Human CSF samples used for the study were obtained from clinical specimens stored at -20 °C. The samples were used in accordance to procedures approved by the institutional ethics committees of Shanghai Children’s Hospital and informed consent was given by all patients. Prior to analysis, CSF samples were pretreated according to the literature procedure.7 The samples were first centrifuged at 12000 rpm for 15 min and the supernates were collected. After precipiting the proteins in the samples by acetonitrile, the resulting supernates were further centrifuged at 12000 rpm for 15 min to obtain the final samples for SERS assay. RESULTS AND DISCUSSION Principle of DA detection by SERS. Scheme 1 shows the workflow of our SERS-based sandwich assay for DA. First, a self-assembled monolayer of DSP was produced on the surface of a smooth gold thin film to capture DA molecules. The formed DSP-DA conjugates then will be recognized and labeled by 3MPBA-functionalized SNPs, resulting in the formation of numerous nanoparticle-molecule-film junctions that can greatly enhance the Raman intensities under 638 nm laser excitation (Figure 1A, S1 and S2). To elucidate the feasibility of this zero-background assay concept, we examined the spectral changes during each step of the assay process. As depicted in Figure 1B, neither DSP molecules nor DSP-DA complexes exhibited apparent SERS signatures on the GTF surface. After introducing 3-MPBA-SNPs, strong SERS response, however, was observed due to the formation of the NPoM hot spots, which could be directly revealed be the SEM measurement (Figure 1C). To exclude the false-positive signal caused by the non-specific adsorption of SNPs on the gold film, we performed a control experiment by adding 3-MPBA-SNPs to the film in the absence of DA, as shown in Figure S3, no apparent SERS signal was obtained, further indicating the role of DA mediated molecule recognition in the generation of NP-film hot spots, which was also confirmed by the SEM result (Figure 1C). In addition, no SERS response was observed in the absence of the gold film, revealing again the essential role of the NPoM gap in SERS amplification (Figure S4). Notably, the small Raman cross section of DA makes it difficult to directly detect this compound by SERS in complex biological samples with a high sensitivity and specificity. We solve this problem by choosing 3-MPBA as a bifunctional probe: first, it serves as the second recognition element to link the DSP-DA complex and silver NPs, forming NPmolecule-film junctions; on the other hand, 3-MPBA shows a superior adsorption ability on silver surface and has relatively high Raman activity, which to a certain extent guarantees the assay sensitivity.45

Figure 1. (A) SNP probes functionalized with 3-MPBA bind to the catechol moiety of DA that is captured by a GTF modified with DSP to form SERS hot spots. Spectral data are obtained within a designated area of the film by raster scanning under a

Raman microscope with a 638 nm laser source. (B) SERS spectra acquired from the surfaces of GTF-DSP, GTF-DSP-DA and GTFDSP-DA-SNP probes. (C) SEM images showing the DA recognition-mediated assembly of SNP probes on the GTF surface. Characterization of DSP-GTF. A self-assembled monolayer of DSP on the surface of GTF was generated by soaking the film in 5 mM of DSP solution in DMSO. This is based on the fact that disulfides undergo dissociation on gold surfaces to give rise to the corresponding thiolates.46 CV and EIS measurements were carried out to characterize the adsorption of DSP. As shown in Figure 2A, the electrochemical probe [Fe(CN)6]3−/4− gave a pair of unambiguous peaks on the bare gold film. After incubation with the DSP solution, an increase in the peak separation and a remarkable decline in the peak current could be clearly observed, demonstrating that the gold film surface was covered with a low dielectric layer. Figure 2B shows the EIS measurement result. The significantly enlarged electron transfer resistance after submerging GTF into the DSP solution also implied a successful modification of the metal surface with DSP. To obtain a dense monolayer and finally a wide dynamic range for DA assay, the adsorption time of DSP was optimized. As depicted in Figure S5, in the presence of 1 µM DA, the SERS intensity increased with extending the incubation time of DSP from 20 to 100 min and then kept nearly constant with further increasing the reaction time. Thus, 100 min was considered as the optimal incubation time and used for further experiments.

Figure 2. (A) CV and (B) EIS characterization of the gold thin film before and after functionalization with DSP.

Figure 3. Characterization of the 3-MPBA-modified SNPs: (A) TEM image of 3-MPBA-SNPs. (B) UV/vis, (C) DLS and (D) SERS spectra of SNPs before and after functionalization with 3MPBA. Characterization of 3-MPBA-SNPs. Citrate-stabilized SNPs were used as core particles to further prepare the 3-MPBA-SNP



nano-conjugates due to their ease of synthesis and strong plasmonic activity. Figure 3A shows the TEM image of the 3-MPBA functionalized SNPs. The size of the particles was estimated to be about 60 nm. The localized surface plasmon resonance (LSPR) of SNPs before and after boronate-modification was characterized by UV/vis extinction spectrometry. The SNPs had a characteristic peak centered at 428 nm. Functionalization with 3-MPBA resulted in a 2-nm red shift of the particles to 530 nm (Figure 3B). Figure S6 shows the surface charge of the silver particles. The asprepared SNPs had a zeta potential of -23 mV. After modification with 4-MBA, a positive shift of 14 mV was observed because the colloid's charge balance was partially changed. The modification with 3-MPBA caused neither a decrease of the LSPR peak nor an increase in the near-Infrared region of the extinction spectrum (Figure 3B), suggesting a good dispersibility of the MPBAmodified SNPs. This was also confirmed by the DLS measurement (Figure 3C). The hydration diameter of SNPs showed no apparent change after the functionalization with MPBA. To further confirm to the successful modification of SNPs by 3-MPBA, we measured the SERS spectra of bare SNPs and 3-MPBAmodified SNPs. As can be seen from Figure 3D, 3-MPBAmodified SNPs showed intense SERS signatures, whereas bare SNPs exhibited no visible peaks. The characteristic SERS peaks of 3-MPBA modified SNPs were in good agreement with the result reported in previous work.45 The bands at 416, 998, 1021, 1070, and 1572 cm-1 were attributed to C-S stretching, C-C inplane bending, C-H in-plane bending, C-C in-plane bending coupled with C-S stretching, and totally symmetric ring stretching, respectively. We also observed that the SERS spectrum of 3MPBA was similar to that recorded at the assembled NPoM junction. This suggested that the SERS signal in this study was mainly contributed by 3-MPBA adsorbed on SNPs (Figure S7), probably because of its larger Raman cross section compared with DA and the higher local molecular concentration at the gap. Thereby, the peak intensity of 3-MPBA at 998 cm-1 was used for property characterization and quantitative analysis. To obtain a dense monolayer and finally a wide dynamic range for DA assay, the adsorption time of 3-MPBA was optimized. As depicted in Figure S8, the Raman signal intensity increased with extending the incubation time from 10 to 60 min and then kept nearly constant with further increasing the reaction time. Thus, 60 min was considered as the optimal incubation time and used for further experiments. In addition, we also optimized the amount of 3-MPBA and found that the use of 4 µL 1 mM of 3-MPBA was appropriate for the surface modification (Figure S9). Optimization of Detection Conditions. We further optimized the experimental conditions for DA detection by SERS. The design feature of our DA assay is the stepwise reaction of DA with DSP and 3-MPBA-SNPs to produce hot spots at the nanoparticlemolecule-film junctions. After removing the free NPs, intensity of the 998 cm-1 band of 3-MPBA was measured from the GTF surface for quantitative determination of DA. To achieve a high sensitivity of detection, the reaction time between DA and DSP and incubation time of the DA adsorbed GTF in 3-MPBA-SNPs suspension were optimized (Figure 4A and 4B). The chemical adsorption time of DA on the DSP-GTF surface will influence the molecular density of DA on the film and finally the number of hot spots. The shorter

Figure 4. Optimization of (A) reaction time between DA and DSP and (B) incubation time of 3-MPBA-SNPs for SERS-based DA detection. The concentration of DA is 1 nM. Error bars indicate standard deviation of three replicates of different samples executing the entire assay. reaction time leads to an ineffective DA capture, and too long reaction time may lower the detection throughput. In our experiment, the intensity of 998 cm-1 band increased gradually with the prolonged reaction time and reached a plateau after 45 min reaction with the presence of 1 nM of DA. The saturated reaction indicated that no more DSP-DA complexes could be labelled by 3-MPBA-SNPs after 45 min. For later time points such as 50 and 55 min, the 998 cm-1 band intensity did not differ from that at 45 min. We next evaluated the optimized incubation time of DA adsorbed gold film in the SNP solution which was 40 min. Quantitative Detection of DA. Under optimal conditions, the dual-recognition-based SERS strategy was performed by the reaction of DA at a known concentration with DSP as recognition probe and then with 3-MPBA-SNP as the label probe. The immobilized 3-MPBA-SNPs on the gold substrate gave Raman emission under the excitation of 638 nm laser to produce a detectable signal at individual films for different concentrations of DA. Figure 5A-C depict the false-color mapping results of the band intensity at 998 cm-1 obtained from 0 pM, 10 pM and 1 nM of DA, respectively. The SERS signal enhanced as the concentration of DA increased, which was consistent with the SEM results (Figure S10). The calibration plot in Figure 5D shows a good linear relationship between the 998 cm-1 band intensity and the logarithmic value of DA concentration in a range of 1 pM to 1 µM. The regression equation was I = 11848.2 + 957.7 × log c with a correlation coefficient of 0.992, where I was the SERS intensity of the 998 cm-1 band in the presence of DA, and c was the concentration of DA. The detection limit at a signal-to-noise ratio of 3 was estimated to be 0.3 pM. The achieved LOD value of our SERS-based assay was comparable to the results of dual-recognition-based electrochemical luminescent methods, and was much lower than the values of dual-recognition-based colorimetric assays (See Supporting Information, Table S1). It should be noted that such a detection performance was obtained by mapping a small area (50 µm × 50 µm) on a large chip surface (3 mm × 3 mm) with a relative long mapping step (5 µm) in comparison with the 1.5 µm laser spot. With the decrease of the target concentration, the number of particle-film junctions decreased, leading to a sparse distribution of the formed hot spots on the gold film which could not be efficiently detected under the current mapping conditions. That is to say, the LOD is limited to a large extent by the experiment parameters. The sensitivity can be simply improved by altering the SERS measurement conditions including increasing the mapping area and decreasing the step interval as demonstrated by chuong and coworkers.36 However, this will inevitably cause an


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ACS Applied Materials & Interfaces tivity of the assay. Error bars indicate standard deviation of three replicates of different samples executing the entire assay.

Figure 5. False-color heat maps of the band intensity at 998 cm−1, indicating SERS hot spots measured for (A) 0 pM, (B) 10 pM and (C) 1 nM of DA solution. Scale bar: 10 µm (D) Peak intensity at 998 cm−1 as a function of DA concentration from 1 pM to 1 µM. Error bars indicate standard deviation of three replicates of different samples executing the entire assay. increase of the assay time. In future studies, we will try to find a better balance between the detection sensitivity and the assay time by lowering the chip size to sub-mm scales. Selectivity and Reproducibility of the SERS Assay. One disadvantage of the current optical assays for DA is that the signal response is easily disturbed by other species coexisting in cerebral systems. In order to evaluate whether our method is specific for DA, the influence of possible foreign substances such as ascorbic acid, uric acid, lactate, glucose, amino acids, catechol and epinephrine on the present assay was tested. As depicted in Figure 6, the intense SERS response was acquired only in the presence of DA. Whereas no obvious SERS signal was observed in the presence of a considerable excess of foreign substances, indicating that there are no significant interactions between any of other interferences and the recognition molecules (DSP and 3-MPBA) used in the assay. The high selectivity was mainly attributed to the unique molecular structure of DA with a catechol moiety and an amine group to react with boronate and DSP, respectively. This result clearly indicated that the SERS assay could effectively identify DA, thus serving as a very selective and promising optical strategy for DA sensing. Additonally, to examine the reproducibility of the method, we measured 10 samples at a DA concentration of 10 nM. As shown in Figure S11, the relative stand-1 ard deviation (RSD) of the 998 cm peak intensities was calculated to be 6.2%, indicating the good reproducibility of the SERS response.

Detection of DA in Cerebrospinal Fluid Samples. As previously reported, the expression level of DA in the cerebral extracellular fluid of patients is typically low (from pM to sub-nM),7,47 which represents a major challenge for the application of current DA bioassays. In order to evaluate the applicability and reliability of the present SERS detection, pretreated human CSF samples spiked with two different levels of DA were analyzed by the proposed procedure. Table S2 displays the results acquired from the analysis of these samples. The DA concentrations in the two samples were determined to be 42.8 and 55.3 pM, respectively, which were basically in agreement with the literature values.7,47 Additionally, the average recoveries varied in a range from 93.7% ± 6.5% to 109.3% ± 7.3% for three determinations, suggesting good accuracy and acceptable precision of the method. From these results, one may further confirm that the dual-recognition-based SERS assay does not suffer interference from the matrix in biological fluids, thus presenting potential applicability in the detection of DA for the physiological and pathological studies. CONCLUSION In summary, we have demonstrated the use of dual-molecule recognition-directed hot spot assembly for the first time as a facile sensing strategy for DA by SERS. Compared with other DA detection methods reported in the literature, this plasmonic bioassay displays several advantages. First, neither complicated nanofabrication nor expensive bio-recognition element (e. g., antibody and nucleic acid probe) was involved. Second, reliable quantification was realized by fast mapping and averaging spectral signatures of numerous SERS hot spots immobilized on a rigid gold film surface. Third, the method shows very high sensitivity and specificity for DA, making it capable of directly detecting DA in complex SCF samples. Lastly, the "signal on" sandwich assay shows nearly zero background that is beneficial to avoid the potential interference during the assay. Given this combination of advantages, we expect that the concept for a DA assay presented here will add an item to the sensing toolbox in the future studies of dopaminergic neuronal system and DA-related clinical disease diagnosis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Wavelength dependent SERS spectra of 3-MPBA-SNPs, AFM images, zeta potential analysis, SERS spectra in control experiments, optimization of immobilization time of DSP on the gold substrate, the effect of modification time and amount of 3-MPBA on the SERS response of SNPs, reproducibility analysis, table comparing performance of the method with previous DA assays.

AUTHOR INFORMATION Corresponding Author * Email: [email protected] * Email: [email protected] Figure 6. Selectivity of the dopamine assay. DA concentration is 10 nM and concentration of other substances is 100 nM. SERS response was only observed for DA implying the excellent selec-

ORCID Kun Zhang: 0000-0001-8759-7636 Baohong Liu: 0000-0002-0660-8610



Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful for the financial support from the National Science Foundation of China (21605025, 21175028 and 21375022) and the Interdisciplinary (Engineering-Medical) Research Fund of Shanghai Jiao Tong University (YG2017QN36). K.Z. acknowledges the financial support of faculty start-up grant from the Children’s Hospital of Shanghai, Shanghai Jiao Tong University.

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