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Plasmonic and Photothermal Immunoassay via Enzyme-Triggered Crystal Growth on Gold Nanostars Yahua Liu, Min Pan, Wenxiao Wang, Qunying Jiang, Fuan Wang, Dai-Wen Pang, and Xiaoqing Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04517 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 4, 2019

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Analytical Chemistry

Plasmonic and Photothermal Immunoassay via EnzymeTriggered Crystal Growth on Gold Nanostars Yahua Liu, Min Pan, Wenxiao Wang, Qunying Jiang, Fuan Wang, Dai-Wen Pang and Xiaoqing Liu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China ABSTRACT: Immunoassay is commonly used for the detection of disease biomarkers, but advanced instruments and professional operating are often needed with current techniques. The facile readout strategy for immunoassay is mainly limited to the gold nanoparticles-based colorimetric detection. Here, we show that photothermal nanoparticles can be applied for biosensing and immunoassay with temperature as readout. We develop a plasmonic and photothermal immunoassay that allows straightforward readout by color and temperature based on crystal growth without advanced equipment. It is demonstrated that alkaline phosphatase-triggered silver deposition on the surface of gold nanostars causes large blue shift in the localized surface plasmon resonance of the nanosensor, accompanied with photothermal conversion efficiency changes. This approach also allows dualreadout of immunoassays with high sensitivity and great accuracy for the detection of prostate-specific antigen in complex samples. Our strategy provides a promising way for point-of-care testing and may broaden the applicability of programmable nanomaterials for diagnostics.

Highly sensitive detection of disease biomarkers with simplicity is an important challenge for early diagnosis, treatment and effective management of cancer.1 Among reported methods, enzyme-linked immunosorbent assay (ELISA) has been commonly used for the detection of trace biomarkers.2-4 ELISA assay combines the antigen-antibody recognition with biocatalytic property of an enzyme such as alkaline phosphatase (ALP), which is widely used for its excellent catalytic activity.5-7 A major advantage of ELISA is that a single enzyme molecule can catalytically transform many molecules of substrate into products, producing a measurable signal for sensitive detection of analytes.8-10 However, advanced instruments and professional operating are often needed by current techniques such as surface-enhanced Raman scattering,11 fluorescence,12,13 electrochemistry,14 and electrochemiluminescence15 in ELISA assay. To the best of our knowledge, traditional strategy without advanced equipment for immunoassay is mainly by colorimetric assay based on gold nanoparticles via naked eye,7,16,17 but the screening through color change suffers from low sensitivity. Therefore, development of simple and sensitive immunosensors that allow straightforward readout is particularly urgent for point-of-care testing and clinical diagnosis.16-19 Recently, gold nanostars (AuNS) with tunable localized surface plasmon resonance (LSPR) peak show great potential for biosensing and biomedical applications.20-23 For example, AuNS have been used for bioanalysis by glucose oxidasemediated crystal growth, because LSPR can be governed by morphology, size, particle component, and refractive index of local dielectric environment.24,25 Notably, with plasmon bands in the near-infrared optical window, AuNS can efficiently absorb light and convert it to heat, which can be transferred to surrounding environment and result in temperature increase.26

Meanwhile, mechanism studies have shown that photothermal conversion efficiency of metal nanocrystals is controlled by factors such as plasmon resonance wavelength, shell coating and assembly, through the change in the plasmon resonance energy of the nanocrystals.25,27 Compared with organic photothermal agents, the inorganic nanomaterials possess good photothermal stability and chemostability.28 It is reported that the near-infrared absorption cross section of Au nanostructures are several orders of magnitude greater than the conventional organic dyes, and the spiky gold nanostructures can significantly enhance light-to-heat conversion.29,30 Furthermore, the temperature signal which is induced by photothermal effect, can be easily detected with high sensitivity by a thermometer without using advanced analytical instruments.31,32 On the basis of these facts, we develop a plasmonic and photothermal nanosensor for dual readout immunoassay by color and temperature via enzymetriggered crystal growth on AuNS. Combination of the two analytical methods is expected to result in more reliable and precise results. We aim to provide a highly sensitive and convenient approach that may potentially facilitate point-ofcare detection for immunoassay. In this work, prostate-specific antigen (PSA) is chosen as a model analyte in ELISA assay, since PSA is an important biomarker for the early diagnosis of prostate cancer, and total PSA assay is crucial for clinical diagnosis.33,34 We first investigated LSPR shifts and photothermal conversion of the nanosensor by ALP-triggered crystal growth on AuNS with a silver coating. Due to a large blue-shift in the LSPR bands, noticeable color and temperature changes of the AuNS solution were observed in response to ALP addition, enabling convenient readout without advanced equipment. Without using any signal amplification, as low as 0.5 pM of ALP can be detected. This approach is then expanded to ALP-linked

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immunosorbent assay for cancer biomarker detection with straightforward readout of diagnostics by observation of color and temperature. Successful quantification of PSA in complex samples was achieved with a detection limit of 0.95 ng/mL. The nanosensor also demonstrated satisfying performance for monitoring PSA variation in real clinical samples. This plasmonic and photothermal detection via enzyme-triggered crystal growth provides a sensitive method for enzyme assay and immunoassay, making dual readout by color and temperature possible with great simplicity and accuracy. EXPERIMENTAL SECTION Chemicals and Reagents. Hydrogen tetrachloroauratetrihydrate (HAuCl4·3H2O), sodium citrate, silver nitrate (AgNO3), polyvinylpyrrolidone (PVP, MW=10000), L-glutathione in the reduced form (GSH), bovine serum albumin (BSA), L-ascorbic acid (AA), ascorbic acid phosphate (AAP), alkaline phosphatase (ALP) from bovine intestinal mucosa, and thrombin (ThB) were obtained from Sigma-Aldrich. ALP-streptavidin conjugate (3 mg/mL, streptavidin-ALP) was purchased from Thermo. Biotin-antiPSA-antibody (ab182031, biotin-recognized antibody) was purchased from Abcam. Total prostate-specific antigen (PSA), capture anti-PSA-antibody (capture antibody), carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) were purchased from Biocell Company (Zhengzhou, China). All other chemical reagents were of analytical grade. Carboxyl-modified 96-well plate was obtained from Corning. The coating buffer consisted of 0.05 M carbonate/bicarbonate buffer (pH 9.6). The phosphate buffer solution (1×PBS) consisted of 0.01 M phosphate buffered saline, 0.137 M NaCl and 0.003 M KCl (pH 7.4). Blocking buffer solution consisted of a 1×PBS solution with 1% (w/v) bovine serum albumin (pH 7.4) added. The washing buffer (PBST) consisted of a 1×PBS solution (pH 7.4) and 0.05% (v/v) Tween 20. All solutions were prepared using ultrapure water (Millipore). Characterization. Transmission-electron microscopy (TEM) images were obtained with Hitachi HT-7700 electron microscope. Ultraviolet-visible (UV-Vis) absorption spectra were recorded with an UV-2660 UV-Vis spectrophotometer (Shimadzu, Tokyo). Dynamic light scattering (DLS) measurements were performed on Malvern Zetasizer Nano ZS 90. Spectrophotometric measurements were performed using a Multiskan GO (Thermo). Temperature changes of the AuNS solution upon 808 nm excitation was monitored by a thermocouple microprobe. All measurements were performed at room temperature. Preparation of AuNS. Gold nanoparticles (AuNPs) with an average diameter of 17 nm were prepared using the citrate reduction method.35 Briefly, a solution of sodium citrate (4 mL, 1%) was added to a rapidly stirred boiling aqueous solution of HAuCl4 (50 mL, 0.5 mM). After 30 min of boiling, the red mixture was allowed to cool to room temperature to get the gold seed. The as-prepared AuNPs were stored at 4 °C for further use. AuNS was prepared through a modified method of seedmediated growth.36 The as-formed seeds were functionalized with polyvinylpyrrolidone (PVP) by mixing the seed with PVP under stirring for 24 h at room temperature. Then the mixture was washed three times with ethanol, and dispersed in 5 mL ethanol. To produce small asymmetric gold nanostars, 82 μL of aqueous 50 mM HAuCl4 solution was mixed with 15 mL of

10 mM PVP solution in DMF, followed by rapid addition of the PVP-capped AuNPs solution (43 μL). After 15 min, the color of the solution changed from pink to colorless and finally to blue, indicating the formation of AuNS. The AuNS was washed three times with ethanol to remove excess PVP, and re-dispersed in ultrapure water containing 0.05% Tween. Plasmonic and Photothermal Detection of ALP activity. Analysis of ALP was performed by incubating ALP solution with 80 μL of Tris-HCl buffer (100 mM, pH 9.5) containing 16 μL AuNS, 0.7 mM AgNO3 and 0.5 mM AAP at 37 °C for 30 min. Absorbance spectra was measured from 450 to 850 nm. For photothermal measurement, the solutions were irradiated vertically by an 808 nm near-infrared (NIR) laser at a power density of 1 W/cm2 for 5 min. The temperature changes were recorded by a thermocouple microprobe with water as a control solution. Immunoassay of PSA. Carboxyl-modified 96-well plate was incubated with capture antibody of PSA (20 μg/mL) at 4 °C overnight. The plate was washed with PBST and blocked with blocking buffer (1% BSA in PBS) for 1 h at 37 °C. Subsequently, the plate was washed three times with PBS, in which 100 μL PSA was added for 1 h incubation at 37 °C. The well was washed three times with PBST, and 100 μL of biotinrecognized antibody (1 μg/mL) was added and incubated for 1 h at 37 °C. Next, the plate was washed three times, and 100 μL of streptavidin-ALP (0.5 μg/mL) was added and incubated for 1 h at 37 °C. Finally, the well was washed, and 80 μL of 100 mM Tris-HCl (pH 9.5) containing 16 μL AuNS, 0.7 mM AgNO3 and 0.5 mM AAP was added into the plate for 30 min incubation at 37 °C. The solution was employed for colorimetric and photothermal sensing. ELISA for Real Clinical Samples. Serum samples from patients that are identified to be PSA positive or negative were obtained from Hubei Cancer Hospital. The samples were centrifuged, aliquoted, and stored at -20 °C for further use. The procedure for the real sample detection was similar to that of the standard sandwich assay, expect that the PSA was replaced by real clinical samples from different patients. All serum samples were diluted 20 times before adding to the plates. RESULTS AND DISCUSSION Characterization of AuNPs and AuNS Polyvinylpyrrolidone-modified gold nanoparticles (PVPAuNPs) were prepared as the seed for the growth of gold nanostars (AuNS). Transmission-electron microscopy (TEM) images showed that the size of AuNPs kept nearly the same after PVP modification (Figure S1). However, surface plasmon resonance peak of the AuNPs shifted from 524 nm to 530 nm, and the hydrodynamic size obtained from dynamic light scattering (DLS) was enlarged from 24.2 to 33.1 nm (Figure S1). Morphology and size of the as-prepared AuNS was characterized by TEM and DLS. Figure S2A showed that AuNS comprised a central core from which multiple sharp tips protruded, and had a size distribution of 83.6 ± 4.9 nm. DLS analysis indicated that AuNS had a hydrodynamic diameter of 85.4 nm and a negative zeta potential of -20.2 mV. The AuNS displayed broad plasmon band with two absorbance peaks at 560 nm and 770 nm (Figure S2B), which were attributed to plasmon modes localized at the core and the tips, respectively. Principle and Feasibility of the Plasmonic and Photothermal Immunoassay


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Analytical Chemistry Principle of the proposed immunoassay was shown in Scheme 1. When target antigen is present, the antigen, capture antibody (Ab1) and biotin-recognized antibody (Ab2) will form a sandwiched immunocomplex. Streptavidin-alkaline phosphatase (streptavidin-ALP) can be conjugated to Ab2 through biotin-avidin interaction, and used as the labeling enzyme for enzyme-linked immunosorbent assay (ELISA). Following the ALP-catalyzed dephosphorylation of ascorbic acid phosphate (AAP) and production of ascorbic acid (AA), Ag+ adsorbed on the surface of AuNS will be reduced to Ag0. The ALP-triggered silver deposition onto AuNS thus results in crystal growth and formation of silver-coated AuNS (Au/Ag NS). It is well known that the plasmon wavelength of metal nanostructures is sensitive to the dielectric properties of the surrounding medium. The silver coating is supposed to lead to blue shifts in the plasmon resonance peak and color change of the AuNS solution, due to hybridization of the dielectric constants of gold and silver. Moreover, photothermal conversion of noble metal nanostructures can be controlled by shell coating through the change in the plasmon resonance energy. If plasmon resonance is shifted away from illumination wavelength and the light absorption changes, photothermal conversion efficiency of AuNS and surrounding environmental temperature will change correspondingly. Therefore, based on ALP-triggered crystal growth on AuNS, the nanosensor allows dual readout of immunoassay by color and temperature.

was also confirmed by TEM comparing the morphology and size of AuNS and Au/Ag NS. As shown in Figure 1C and 1D, the sharp protrusions of AuNS disappeared, and the size of the nanoparticles grown from 83.6 nm to 88.4 nm after crystal growth. Similarly, photothermal transduction efficiency of the AuNS subjected to different components was studied (Figure 1E). The solution without AuNS showed negligible temperature changes, indicating no photothermal heating upon illumination. All the AuNS without silver deposition showed about 30 C increase with prolonged irradiation time and then reached equilibrium. It can be seen that these systems exhibited similar noticeable temperature changes, since the LSPR peak was similar to the illumination laser wavelength of 808 nm. On the contrary, silver-coated AuNS with blue-shift of LSPR displayed negligible changes that were close to the background temperature when irradiated at 808 nm. This result was attributed to the fact that the plasmon resonance was shifted from 770 to 540 nm, which was far away from the illumination laser wavelength. Therefore, the light absorption was decreased, photothermal conversion efficiency of the Au/Ag NS was greatly inhibited, and no notable increase in temperature was observed.

Figure 1. ALP-triggered Ag crystal growth on AuNS. (A) UV-Vis spectra of AuNS samples in the absence and presence of reagents. (B) Photographs of the AuNS solution in the presence of different reagents. (C) TEM image of pristine AuNS. (D) TEM image of AuNS after crystal growth. (E) Temperature curves of the representative AuNS samples and pure water.

Scheme 1. Plasmonic and photothermal immunoassay based on enzyme-triggered crystal growth. On the basis of these considerations, feasibility of the plasmonic and photothermal assay is investigated. First of all, different components of potential reactants were incubated to study influence of crystal growth on LSPR, color, and photothermal transduction efficiency of AuNS. As shown in Figure 1A and 1B, neither color nor localized surface plasmon resonance (LSPR) peak of AuNS was changed in the absence of streptavidin-ALP, AAP, or Ag+. Only in the presence of AA or in the presence of both ALP and AAP, there was a blueshift of LSPR more than 200 nm, with a color change from blue to orange. These results imply that it is the ALP catalyzed generation of AA that leads to silver deposition onto AuNS. The successful deposition of silver on the surface of AuNS

Plasmonic and Photothermal Assay of ALP For sensitive plasmonic and photothermal ELISA, we first studied ALP-triggered crystal growth on AuNS in the presence of AAP and silver ions. The plasmonic and photothermal properties of the AuNS were investigated in response to ALP activity. Several experimental parameters were optimized to achieve a good response of ALP activity and ALP-linked immunoassay. The reaction of the ALP-guided crystal growth on AuNS was carried out in a Tris-HCl solution (0.1 M, pH 9.5) at 37 °C. Other factors such as Ag+ concentration, AAP concentration, and incubation time for the enzymatic reaction were also studied (Figure S3). The LSPR peak shift (Δλ) of AuNS increased gradually as the concentration of Ag+ increased from 0 to 1.5 mM, and the Δλ reached a platform at a concentration of 0.7 mM. It is supposed that higher concentration of Ag+ could accelerate the reduction reaction of Ag+, and more deposited silver could also be obtained. What’s more, with the increasing concentration of AAP, the Δλ also



increased and reached the maximum shift at 0.5 mM AAP. The enzymatic reaction time could also influence the enzymatic hydrolysis reaction. At the optimal Ag+ and AAP concentration, the Δλ of AuNS was gradually increased as reaction time prolonged and reached a nearly constant value after 30 min, suggesting that the incubation time was suitable for probing the targets. Furthermore, with the increasing concentration of ALP, the LSPR peak shifted to a greater extent, which demonstrated more silver deposition on the AuNS at higher ALP concentration. As a consequence, a detection solution containing 0.7 mM Ag+ and 0.5 mM AAP was employed to incubate with ALP and AuNS for 30 min in the subsequence study.

corresponding linear regression equation was Δλ = 240.24c 0.71 (R2 = 0.998) (Figure S4), where Δλ was the LSPR blueshift and c was the ALP concentration. Without using any amplification method, this plasmonic nanosensor allows quantitative detection of ALP with a low detection limit of 9.54 pM, which is better than previous reported methods.5,7 As shown in inset of Figure 2B, the color of the solution changed from blue to purple and finally turned to orange with increasing ALP concentration, realizing convenient and direct detection of ALP with naked eye. To demonstrate photothermal effect of the nanosensor for ALP detection, the system with different ALP concentrations were irradiated with 808 nm laser for 5 min. As the time of irradiation increased, the temperature of the solution was gradually increased and reached a constant value after 5 min (Figure 2C). Figure 2D revealed that the photothermal effect of the AuNS solutions was dependent on ALP concentration. Temperature increase of AuNS was greatly inhibited with increasing ALP concentration from 0 to 50 pM, and then reached a constant value in a concentration range from 50 pM to 2 nM. A linear relationship was obtained between the temperature increase and the concentration of ALP over the range from 0.5 to 20 pM. Without combining amplification technique, highly sensitive detection of ALP can be achieved with a detection limit of 0.5 pM, which is nearly 20-fold enhancement comparing to the plasmonic assay. Furthermore, the temperature increment is hardly affected by ambient temperature (Figure S5), which demonstrates that the temperature signal is reliable and robust. From these results it can be seen that this nanosensor integrates both colorimetric and temperature dual-signals into one assay, and allows not only preliminarily screening via naked-eye observation of color but also accurate diagnosis by temperature signal with significantly enhanced sensitivity.

Figure 2. Plasmonic and photothermal assay of ALP. (A) Absorption spectra of the AuNS solution subjected to different concentrations of ALP: 0, 0.001, 0.01, 0.05, 0.1, 0.2, 0.5, 1, and 2 nM (from right to left) in the presence of 0.7 mM Ag+ and 0.5 mM AAP. Incubation time: 30 min. (B) Blue shift of the LSPR absorbance band (Δλ) as a function of ALP concentration as depicted in (A). Inset: corresponding photographs of the AuNS samples in the presence of different concentrations of ALP: 0, 0.01, 0.05, 0.1, and 1 nM. (C) Temperature curves of the AuNS samples in the presence of different concentrations of ALP: 0, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 1, and 2 nM (from top to bottom) upon irradiation at 808 nm. (D) Temperature increase (ΔT) as a function of ALP concentration. Inset: linear relationship between the temperature increase (ΔT) of AuNS and ALP concentration. Under the optimal experimental conditions, absorption spectra of the system with different concentrations of ALP were recorded. As the concentrations of ALP were increased from 0 to 2 nM, the LSPR peaks of AuNS were gradually blue-shifted about 220 nm (Figure 2A). The result is consistent with the gradual hydrolysis of AAP by ALP and the deposition of silver on the surface of AuNS. Figure 2B shows dependence of the LSPR peak shift on the concentrations of ALP. The Δλ of AuNS increased continuously with increasing ALP concentration from 0 to 1 nM, and then leveled off to a constant value in a concentration range from 1 to 2 nM. The

Figure 3. Dual readout of immunoassay by color and temperature. (A) Absorption spectra of the AuNS solution subjected to different concentrations of PSA: 0, 5, 10, 20, 50, 100, 150, 200, and 350 ng/mL (from right to left) in the presence of 0.7 mM Ag+ and 0.5 mM AAP. (B) Blue shift of the LSPR absorbance band (Δλ) as a function of PSA concentration. Inset: photographs of the AuNS samples on


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Analytical Chemistry microwells in the presence of different concentrations of PSA. (C) Temperature curves of the AuNS dispersions at different concentrations of PSA: 0, 5, 20, 50, 70, 100, 150, 200, and 500 ng/mL (from top to bottom) under irradiation with 808-nm laser. (D) Temperature increase (ΔT) as a function of PSA concentration. Dual Readout of Immunoassay by Color and Temperature The sensitive detection of ALP using the plasmonic and photothermal nanosensor allows development of immunoassay via ALP-triggered crystal growth on AuNS. Here prostatespecific antigen (PSA) was used as a model analyte for the plasmonic and photothermal ELISA. As shown in Figure S6, with higher concentration of capture antibody, biotinrecognized antibody and streptavidin-ALP, the enzymatic hydrolysis reaction accelerated, and a larger LSPR peak shift of AuNS was obtained. Accordingly, a detection solution containing 20 μg/mL capture antibody, 1 μg/mL biotinrecognized antibody and 0.5 μg/mL streptavidin-ALP was adopted for the following immunoassay. Under the optimized conditions, different concentrations of PSA were detected via dual readout of immunoassay by color and temperature. As shown in Figure 3A, with increased concentration of PSA, the LSPR peak of AuNS gradually blue-shifted from 771 to 550 nm with the solution color changing from blue to orangered (Figure 3, inset). Figure 3B shows dependence of Δλ on the concentration of PSA. The Δλ increased gradually as the concentration of PSA increased from 0 to 350 ng/mL, and then leveled off to a constant value in a concentration range from 350 ng/mL to 1 μg/mL. As shown in Figure S7A, the Δλ of AuNS increased linearly with the concentration of PSA from 10 to 200 ng/mL (Δλ = 0.95c - 7.75, R2 = 0.990). A detection limit was calculated to be 1.53 ng/mL of PSA, which was lower than a cut-off point of 4.0 ng/mL for clinical test of prostate cancer biomarker PSA.

Figure 4. Selectivity of the sensor for the detection of PSA. The analyte concentration is 0.5 μg/mL for PSA, 1 μg/mL for other targets. Δλ corresponds to the blue shift of LSPR absorption band of the AuNS in the presence of different analytes. Inset: photographs of the AuNS samples on microwells after adding different analytes. Then we studied photothermal ELISA for PSA detection. It is known that light-to-heat conversion profiles is related to the

absorption spectra of the nanoparticles,27 and plasmon resonance spectra of AuNS is strongly dependent on size and morphology, especially spike length and number of spikes.37 Therefore, the PSA-dependent ALP-triggered crystal growth on AuNS by silver deposition results in blue-shift of LSPR and low photothermal conversion efficiency, and thus inhibits temperature increase of the solution. As shown in Figure 3C, the temperature of AuNS solutions were gradually increased as the time of the laser irradiation increased from 0 to 300 s. The AuNS solutions displayed lower photothermal conversation efficiency upon 808 nm irradiation with increasing PSA concentration, as the LSPR band of Au/Ag NS shifted more greatly from the irradiation wavelength, and the absorbance at 808 nm decreased more strongly. As the concentrations of PSA were increased from 0 to 500 ng/mL, the temperature increase was less efficient (Figure 3D). These results are consistent with the gradual deposition of silver on AuNS and blue-shift of LSPR peak. The photothermal ELISA allows readout of immunoassay by temperature. A quantitative relationship between ΔT and PSA concentration in the range of 0 - 150 ng/mL was obtained, with a detectable concentration of 1 ng/mL (Figure S7B). Overall, besides colorimetric readout of immunoassay by naked eye, the temperature changes can be easily readout by a thermometer with high sensitivity, which is suitable for POC detection of PSA in resource-limited regions where expensive apparatus are not accessible. In addition, the sensitivity of the present plasmonic and photothermal nanosensor for PSA detection is comparable and even higher than some other analytical methods (Table S1). As a result, this plasmonic and photothermal nanosensor enables versatile and sensitive immunoassay, and allows dual readout with simplicity and high reliability.

Figure 5. Plasmonic and photothermal immunoassay of serum PSA. (A) Absorption spectra of the AuNS solution in the presence of 0.7 mM Ag+ and 0.5 mM AAP subjected to different concentrations of PSA. (B) Blue shift of the LSPR absorbance band (Δλ) as a function of PSA concentration. (C) Temperature curves of the AuNS dispersions in serum with different PSA concentrations upon irradiation at 808 nm. (D) Temperature increase (ΔT) as a function of PSA concentration.



PSA concentration: 0, 1, 5, 10, 20, 50, 70, 100, 150, 200, 350, and 500 ng/mL. Furthermore, we evaluated selectivity of the immunoassay for PSA detection by utilizing other common biological species including bovine serum albumin (BSA), alphafetoprotein (AFP), glutathione (GSH), carcinoembryonic antigen (CEA), and thrombin (ThB). As shown in Figure 4, the presence of PSA in the standard assay solution leads to 200 nm blue-shift of LSPR peak and noticeable color change of the AuNS solution, but the introduction of other common biological species results in negligible LSPR shift and color change.

methods, and provides more reliable and precise results, especially for the detection of complex samples. These results strongly demonstrate that the proposed method could be applied to clinical diagnosis with high accuracy. CONCLUSION In summary, we proposed a plasmonic and photothermal immunoassay based on crystal growth on AuNS. The nanosensor allows detection of alkaline phosphatase with a low detection limit, and enables convenient readout of assay via color and temperature. This method provides remarkable sensitivity and selectivity for dual-readout immunoassay without advanced instruments. The immunosensor can be applied for detection of PSA in real clinical sample. It is envisioned this work may inspire bioanalysis and immunoassay using different plasmonic nanoparticles or functional materials with good photothermal efficiency, and facilitate developing point-of-care diagnostics.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

Figure 6. Immunoassay of PSA in real clinical samples readout by color (A) and temperature (B). P1 to P3 indicate different prostate cancer patients that are clinically diagnosed to be positive, while N1 to N5 indicate non-prostate cancer patients that are clinically diagnosed to be negative.

Characterization of gold nanoparticles and gold nanostars, optimization experimental conditions, UV-Vis spectra and photothermal effect (PDF)

The immunosensor was further applied to the detection of PSA in complex samples. As shown in Figure 5A, the LSPR peak gradually blue-shifted with the increased PSA concentration, and the Δλ reached a platform at the concentration of 200 ng/mL (Figure 5B). The Δλ had a liner range with the increased PSA at the concentration ranging from 20 to 150 ng/mL, and the detection limit of PSA using this method can reach a value of 0.95 ng/mL (Figure S8A). With the irradiation of the laser, the temperature of the solution was gradually increased with time, and ΔT gradually lowered as the concentration of PSA increased from 0 to 500 ng/mL (Figure 5C,D). The corresponding linear regression equation is ΔT = -0.34c + 29.00 (R2 = 0.999), where ΔT is the temperature increase and c is the PSA concentration (Figure S8B). An obvious temperature decrease of 1 °C was observed even at 1.0 ng/mL. The result suggested that the plasmonic and photothermal immunoassay was applicable for detection of complex biological samples. Furthermore, we applied the approach for the detection of PSA in real clinical samples by analyzing serum from eight patients. The PSA-positive samples were collected from clinical patients whose PSA levels were higher than the cut-off value, whereas PSAnegative samples were below the cut-off value. The positive samples showed color changes as well as shift in absorbance, while the negative controls displayed no changes at all (Figures 6A and S9). This difference enabled preliminary screening of PSA in complex biological samples via nakedeye observation of color. Figure 6B exhibited that the temperature of positive samples was distinctly lower than that of the negative samples. Furthermore, there was dramatic difference even between each positive sample, indicating that the photothermal assay had a higher sensitivity than colorimetric method. It can be seen that dual readout methodology combines advantages of the two analytical

Corresponding Author

AUTHOR INFORMATION * Xiaoqing Liu, E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes Any additional relevant notes should be placed here.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (grant nos. 21503151, 81602610), the National Natural Science Foundation of Jiangsu Province, China (grant nos. BK20161248, BK20160381), the Fundamental Research Funds for the Central Universities (grant no. 2042018kf1006), and 1000 Young Talent Program of China (F. W. and X. L.)

REFERENCES (1) Liu, X.; Dai, Q.; Austin, L.; Coutts, J.; Knowles, G.; Zou, J.; Chen, H.; Huo, Q. A One-Step Homogeneous Immunoassay for Cancer Biomarker Detection Using Gold Nanoparticle Probes Coupled with Dynamic Light Scattering. J. Am. Chem. Soc. 2008, 130, 2780-2782. (2) de la Rica, R.; Stevens, M. M. Plasmonic ELISA for the Detection of Analytes at Ultralow Concentrations with the Naked Eye. Nat. Protoc. 2013, 8, 1759-1764. (3) Xianyu, Y.; Wu, J.; Chen, Y.; Zheng, W.; Xie, M.; Jiang, X. Controllable Assembly of Enzymes for Multiplexed Lab-on-a-Chip Bioassays with a Tunable Detection Range. Angew. Chem. Int. Ed. 2018, 57, 7503-7507. (4) Lai, W.; Tang, D.; Que, X.; Zhuang, J.; Fu, L.; Chen, G. Enzyme-Catalyzed Silver Deposition on Irregular-Shaped Gold Nanoparticles for Electrochemical Immunoassay of AlphaFetoprotein. Anal. Chim. Acta 2012, 755, 62-68.


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