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Peptide-Conjugated Gold Nanoprobe: Intrinsic Nanozyme-Linked Immunsorbant Assay of Integrin Expression Level on Cell Membrane Xueyun Gao, Liang Gao, Meiqing Liu, Guifu Ma, Yaling Wang, Lina Zhao, Qing Yuan, Fuping Gao, Ru Liu, Jiao Zhai, Zhifang Chai, and Yu-liang Zhao ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b04261 • Publication Date (Web): 05 Oct 2015 Downloaded from http://pubs.acs.org on October 5, 2015

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Peptide-Conjugated

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Gold

Nanoprobe:

Intrinsic

Nanozyme-Linked Immunsorbant Assay of Integrin Expression Level on Cell Membrane Liang Gao,§Meiqing Liu,§ Guifu Ma, Yaling Wang, Lina Zhao, Qing Yuan, Fuping Gao, Ru Liu, Jiao Zhai, Zhifang Chai, Yuliang Zhao and Xueyun Gao* CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China.

§

L. Gao and M. Liu contributed

equally to this work. *To whom correspondence should be addressed. E-mail: [email protected]. ABSTRACT: Precisely quantifying the membrane proteins expression level on cell surfaces is of vital importance for early cancer diagnosis and efficient treatment. We demonstrate that gold nanoparticle bioconjugated by a rationally-designed peptide as nanoprobe possesses selective labeling and accurate quantification capacity of integrin GPIIb/IIIa on human erythroleukemia cell line. On one hand, through selective recognition and marking of integrin, two-photon photoluminescence of the nanoprobe is exploited for direct observation of protein spatial distribution on cell membrane. More importantly, utilizing intrinsic enzyme-like catalysis property of the nanoprobe, the expression level of integrin on human erythroleukemia cells can be quantitatively counted in an amplified and reliable colorimetric assay without cell lysis and protein extraction process. In addition, the analysis of the correlation between the gold nanoparticle and the membrane protein via relevant inductively coupled plasma mass spectrometry measurement verifies the reliability of the new analytical method. It is anticipated that this facile and efficient strategy holds a great promise for a rapid, precise and reliable quantification of 1 Environment ACS Paragon Plus

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interested functional membrane proteins on cell surface. KEYWORDS: nanozyme· enzyme-linked immunosorbent assay· gold nanoparticles· membrane protein quantification· two-photon imaging Variations of spatiality and number of membrane proteins are closely related to cancer proliferation, angiogenesis and metastasis.1 Thus, accurate membrane proteomic quantification is very critical for early diagnosis and effective therapy for tumor owing to the aberrantly expressed biomarkers between normal and cancer cells.2,3 So far, a series of techniques have been established to analyze cell membrane proteins expression level, such as flow cytometry,4 enzyme-linked immunosorbent assay (ELISA)5 and mass spectrometry-based proteomic methodologies.6 Unfortunately, these methods require cell lysis and protein extraction approaches by which target protein wastage is inevitable. Moreover, the direct analyzing traces of cancer-related proteins in crude or complex biological samples is limited against the high background of other abundant proteins.2 To tackle this issue, a powerful nonradioactive element/isotope analysis tool,7 inductively coupled plasma mass spectrometry (ICP-MS) has recently been developed as an ultrasensitive technique to quantification of cell membrane biomarker. The platform is started from devising rare earth or noble metal probe with different metallic chemical forms (nanoparticle, cluster or molecular), in which antibody, aptamer and peptide serve as guiding moieties to specifically recognize target protein overexpressed on the corresponding cells.8-10 Furthermore, the expression of target protein is expediently counted by the metallic element via ICP-MS on the basis of accurate formula of the probe and the particular recognition ratio of probe to the protein are defined. However, mass cytometry analysis is time-consuming and relies on complex instrumentations and expertise. It remains a worthy task to set out a rapid, reliable, sensitive and accurate membrane protein quantification method. As a pioneering research, Yan and co-workers corroborated Fe3O4 nanoparticles with amazing intrinsic enzyme mimetic property.11 Since then, past decade witnessed nanomaterial-based artificial enzymes (nanozymes), alternative of natural enzymes have been widely applied in numerous fields,

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including biosensor, cancer diagnostics and therapy, neuroprotection and pollutant removal.12 Especially, several types of artificial enzymes were utilized for catalytic oxidation of peroxidase substrates in the presence of hydrogen peroxide (H2O2) for bioanalysis.13 That is, conjugating antibody to the nanozyme, rather than a traditional enzyme like horse radish peroxidase (HRP), enables the application of nanozyme technology for immunoassays. These modified ELISA mostly employs the particular antigen-antibody recognition and superior catalytic power of nanozyme to realize target protein content signal amplification. Until now at least three immunoassay formats concerning nanozyme-based microplate ELISA were established. The first type belongs to antigen-down style11,14,15 and the second one is widely accepted as conventional sandwich immunoassay14-16. Besides, a mutation of sandwich assay where nanozyme is ferromagnetic, capable of capturing and separating antigen was also developed.11,14 Regrettably, all aforementioned strategies require membrane protein extraction approaches from cell rather than analyzing the protein on cell membrane directly. Similar to the principle of classic microplate-based ELISA, peroxidase nanozyme has aroused much interest on quantifying tumor cells by aiding its efficient targeting capacity to special antigen or receptor on objective cells.17-20 Since the biomarker (antigen or receptor) is normally overexpressed on cancer cells compared to normal ones, considerable attention has focused on exploring the approach to differentiate cancer cell types and reflect relevant metabolic activity. Yet owing to the lack of standard curve, the method is restricted to the evaluation of cells number, rather than the membrane protein expression level. Herein, our original motivation is to give a systematic investigation on exploring a cancer cell immunoassay to analyze membrane protein expression based on profiling calibration plot by peroxidase mimetic microplate-based ELISA. Integrin GPIIb/IIIa, as the model protein in the proof-of-concept research, is a member of integrin family of cell membrane receptors whose expression level is closely related to platelet aggregation and cancer pathogenesis, including metastasis in prostate cancer and human erythroleukemia (HEL) cells.21-23 In this report, as shown in Scheme 1a, we aim to devise peptide-decorated gold nanoparticles


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(Peptide-AuNPs) through chemical reduction and accompanying ligand exchange. With the aid of bioconjugation, Peptide-AuNPs as a nanoprobe can selectively recognize integrin on HEL cells membrane. Furthermore, Peptide-AuNPs act as robust enzyme-mimetic catalyst that the number of integrin on HEL cells can be counted in an amplified and reliable manner by a cancer cell immunoassay method. The prerequisite for quantification is setting up standard curve through microplate-based ELISA concerning oxidation of 3, 3', 5, 5'-Tetramethylbenzidine (TMB) in the presence of H2O2 catalyzed by Peptide-AuNPs as a function of integrin concentration. Simultaneously, thanks to gold nanoparticles’ unique nonlinear optical property, two-photon photoluminescence will be observed for directly visualizing spatial position of the protein on cell membrane. Additionally, gold nanoparticle-based ICP-MS analysis will be undertaken to confirm the feasibility of this protocol. This simple and highly efficient strategy holds a great promise for universal detection and quantification of other functional proteins on cell membrane directly. RESULTS AND DISCUSSION Constructing the nanoprobe involves preparation of gold nanoparticles followed by a ligand exchange reaction. At first, 2-aminoethanethiol modified gold nanoparticles (AuNPs) were synthesized according to the previous report24 with a minor modification. Transmission electronic microscopy (TEM) image in Figure 1a reveals the typical spherical morphology of AuNPs as-prepared. Then AuNPs dispersion was mixed with a rationally-designed peptide (H2N-CCYKKKKQAGDV-COOH) targeting to integrin GPIIb/IIIa. Notably, the sequence of the peptide contains three functional components. The first domain is CC, comprised of –SH group with the ability to capture AuNPs. Domain two is YKKK serving as the sequence to avoid steric hindrance. The third domain is KQAGDV, derived from a subunit sequence of fibrinogen, proved to be specifically bound to integrin GPIIb/IIIa.25 After purification and concentration, a well-dispersed Peptide-AuNPs solution was obtained. Through statistical analysis of representative 50 Peptide-AuNPs by TEM characterization, the mean size is about 3.79±0.56 nm (Figure 1b). The above size of nanoprobe not only ensures it possesses intriguing catalytic capacity,26 but also facilitates size


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match between a single Peptide-AuNP and integrin ligand-binding domain.25 Moreover, dynamic light scattering (DLS) measurement (Figure 1c) shows average hydrated diameter of nanoparticles increases from 4.5 to 5.0 nm after peptide decoration. The reason can be ascribed to the longer chain of peptide compared to that of 2-aminoethanethiol. The as-prepared nanoprobe is substantially stable in double-distilled water for a long storage period at 4 oC, which could be accounted for strong electrostatic repulsion of positively-charged surface (+33.0 mV). The limited size and good mono-dispersity of the nanoprobe is speculated as the prerequisite for further efficient recognition and accurate quantification analysis. To preliminarily elucidate the successful bioconjugation of peptide on the surface of AuNPs, ultraviolet-visible (UV-VIS), Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) were performed. UV-VIS spectra of AuNPs and Peptide-AuNPs in Figure 2a depict obvious peak at around 520 nm, sign of gold nanoparticles’ characteristic local surface plasmon resonance. Similar pattern implies ligands exchange doesn’t induce obvious nanoparticles aggregation. Besides, as shown in Figure 2b, unlike FTIR curve of AuNPs which presents the typical absorption peaks of surface 2-aminoethanethiol, profile of Peptide-AuNPs shows two new characteristic absorption peaks centered around 1647 and 1539 cm-1, assigned to the bands of amide I (C=O stretching vibration) and amide II (C–N stretching and N–H bending vibration) of the peptides, respectively.27,28 XPS analysis was conducted to further demonstrate the ligands exchange on the surface of AuNPs. The high resolution XPS pattern of deconvoluted C1s spectrum of Peptide-AuNPs consists of three different chemically shifted components, indicative of the existence of sp3 carbons (C–C or C–S, 285.0 eV), primary amine carbons (C–N, 286.3 eV) and amide carbons (N–C=O, 288.2 eV) (see Figure 2d). 29,30 As a comparison, amide carbons signal can hardly be detected on AuNPs (Figure 2c). Moreover, to gain insights into the binding force between AuNPs and peptide, Au and S content before and after bioconjugation was roughly measured by XPS. The relevant results in Figure S1 indicate that the integrated peak area of Au and S decreases about 35% after ligands exchange, respectively. We deduce that peptide partly 5 Environment ACS Paragon Plus

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displacing the capping agent through forming Au–S bond and the larger steric hindrance of peptide lead to the decline of Au and S content. Determining the accurate composition of the nanoprobe is essential to quantify the target protein precisely via ICP-MS methods.8,9 The exact number of Au atoms in each nanoprobe was analyzed by ICP-MS. According to the size and lattice parameters of gold nanoparticle,31 the concentration of Peptide-AuNPs can be clarified to fulfill the following quantitative analysis requirements (See Part I, SI). Next bicinchoninic acid (BCA) assay was manipulated to quantitatively evaluate the peptides assembled on a single nanoparticle. After calculation the peptide concentration before and after modification, it can be assessed that mean 11 peptides were bioconjugated on a single AuNP (See Part I, SI). The current study focuses on setting out analytical methodology for cell membrane protein quantification in virtue of catalytic property of the nanoprobe. In the presence of H2O2, Peptide-AuNPs catalyze the oxidation of TMB to ox-TMB (charge-transfer complex) with the maximum absorbance at 652 nm monitored by absorption spectroscopy,32 showing the solution color alterations from colorless to deep blue (see Figure S2a). As a comparison, H2O2 alone does not produce the significant color change. The typical absorbance curves for the catalytic reaction are present in Figure 3a. The maximum absorbance at 652 nm rises in a time and catalyst concentration-dependent manner. In particular, the color development of the system in the initial oxidation period (within 1 h) at a series of catalyst concentration behaves a linear increase, respectively (see Figure S2b). The result suggests that the optimized analytical time can be selected within one hour to assure the accuracy of the analysis. Moreover, as shown in Figure 3b and 3c, similar to other nanomaterial-based peroxidase mimics,11,16 the catalytic activity of Peptide-AuNPs is relied on pH and temperature variation. In this case, the optimal pH and temperature were chosen as pH 4.0 and 37 oC. In addition, to understand the mechanism of peroxidase mimetic reaction, generation of ●OH radical from H2O2 catalyzed by Peptide-AuNPs was


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confirmed by the electron spin resonance (ESR) technique. As shown in Figure 3d (blue curve), mixing Peptide-AuNPs, H2O2 and trapping agent 5, 5-dimethyl-1-pyrroline n-oxide (DMPO) aqueous suspension leads to formation of a large amount of DMPO-OH spin adducts, which can be easily distinguished by a typical four-line spectrum with intensity ratio 1:2:2:1 and equivalent hyperfine splitting constants (aN=14.9 G, aH=14.9 G). It might be ascribed to O–O bond fragmentation to form ●

OH radical through partial electron transfer from Peptide-AuNPs to H2O2.33,34 The presence of ●OH as

oxidant to oxidize TMB is expected to accelerate the reaction. In contrast, under the similar conditions, in the absence of Peptide-AuNPs, DMPO-OH formation does not occur (Figure 3d, red curve), indicating that Peptide-AuNPs as enzyme-like catalyst trigger the above reaction. The steady state kinetics investigated by initial rate method was adopted to confirm the kinetic parameters for better assessing the nanozyme activity. In a certain range of H2O2 and TMB concentrations, typical Michaelis-Menten curves were obtained for the oxidation reaction, if accepting pH 4.0 and 37 oC as the standard conditions (Figure 4a, 4c, S3a and S3c). Apparent Michaelis-Menten constant (Km) and catalytic constant (Kcat) were further estimated using Lineweaver−Burk plot (Figure 4b, 4d, S3b and S3d).11 As listed in Table 1, the apparent Km value of Peptide-AuNPs with H2O2 as the substrate is much higher than that of HRP, which is consistent with the higher concentration of H2O2 for Peptide-AuNPs to achieve maximum activity. Additionally, the apparent Km value of Peptide-AuNPs with TMB as the substrate is almost the same as HRP, disclosing that Peptide-AuNPs has a similar affinity for TMB compared to HRP. Notwithstanding apparent Kcat values indicate the relatively less catalytic activity of Peptide-AuNPs than that of HRP35, considering our nanoprobe possesses reliable physicochemical stability, proper catalytic activity as well as potential optical/mass property, it could offer a good opportunity to achieve integrative protein detection strategy. It should be noted that compared with AuNPs, Peptide-AuNPs behave a little bit less catalytic activity toward peroxidase substrates which is reflected by higher apparent Km and lower Kcat. The result is in accordance with previous independent studies that surface coatings tune the nanozyme’s activities via shielding 7 Environment ACS Paragon Plus

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nanozyme core from the substrate which in turn decreases their activities.36,37 The decrease of interfaced active gold atoms ascribed to peptide capping is a contributing factor to the decline of nanozyme activity in our case (Figure S1a). Taken together, through aforementioned studies, a series of preferred catalytic parameters were selected except otherwise statement. That is, experiments were carried out at 37 °C under pH 4.0 environment, accepting 200 mM H2O2 as well as 800 µM TMB as substrates. Accurate cancer cell immunoassay requires rational cell labeling efficiency. We thereupon chose HEL cells as model cell line and the incubation time of nanoprobe with cells was first optimized through ICP-MS analysis by digesting a large population of cells to determine the abundance of Au. It should be emphasized that the dosage of nanoprobe was fixed at 120 nM. ICP-MS result in Figure S4 shows the average mass of Au on a single cell rises rapidly as the incubation time varies from 5 to 20 min and remains stable for longer incubation condition. The test implies that integrin is saturated completely when 120 nM nanoprobe was incubated with cells for 30 min. Above condition is selected in the following labeling experiments except otherwise stated. Equally importantly, the nanoprobe’s specificity and distribution state on plasma membrane warrants vivid verification before quantifying integrin. It is widely known that Au nanoparticles are non-fluorescent under one-photon excitation due to their low emission quantum yields. Comparatively, aggregated Au nanoparticles display enhanced two-photon photoluminescence (TPPL) due to plasmon coupling which is potential for bioimaging application.38 In this case, TPPL microscopy was employed to provide evidence that Peptide-AuNPs can selectively recognize integrin on HEL cells’ cytomembrane. As shown in Figure 5a, after coculture nanoprobe of optimized dosage with paraformaldehyde fixed HEL cells, apparent red photoluminescence on cells plasma membrane was observed under femtosecond laser irradiation at 800 nm. The result is in accordance with the previous study that aggregated Au nanospheres behaves rather broad TPPL spectrum ranged from 570 nm to 700 nm.38,39 The nonlinear optical property of coupled Peptide-AuNPs allows us to deduce inhomogeneous spatial distribution of the integrin. In striking contrast, vacant cells do not emit noticeable photoluminescence


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(Figure 5b) when TPPL image was collected at the same setting as above (Figure 5b). The accurate targeting of integrin by nanoprobe is further delineated by a peptide blocking study.40 After being blocked by 2 mM free peptide, the red photoluminescence on cell membrane has almost vanished (Figure 5c). Another test was conducted by coculturing nanoprobe and free peptide with HEL cells simultaneously. As expected, the nanoprobe marks plasma membrane with dimmer photoluminescence (Figure 5d), suggesting nanoprobe and free peptide compete for the same binding site on integrin. In addition, the overlapped study was used to document the exact molecular targeting of the nanoprobe. As dipicted in Figure 5e, green fluorescence accounts for FITC-labelled GPIIb/IIIa through antibody bioconjugation while red photoluminescence originates from the emission of nanoprobe under two-photon irradiation. The evident overlapping between the images above discloses that tag and antibody target to the same protein. To implement the concept of utilizing intrinsic catalysis property of Peptide-AuNPs to quantify integrin expression, the key point is setting up calibration plot and rendering nanozyme realization target protein signal amplification. Standard curve concerning oxidation of TMB through heterogeneous catalysis by Peptide-AuNPs is plotted as a function of integrin concentration. As illustrated in the profile of Figure 6a, the assay employs a sandwich enzyme immunoassay technique14-16. In practice, polyclonal antibody specific for human integrin GPIIb/IIIa was pre-coated onto the surface of microplate. Then gradient amount of integrin was sandwiched by the immobilized antibody and nanoprobe. After that, all unbound material was washed away before substrate TMB and H2O2 were supplemented to initiate the catalytic reaction. As shown in Figure 6b, the standard curve is plotted relating the absorbance at 652 nm to the corresponding delivered integrin concentration. As expected, the concentration of integrin displays an excellent linear relationship to the level of ox-TMB conversion catalyzed by the immobilized Peptide-AuNPs (R2=0.99). The result confirms that gradient amount of integrin captures corresponding gradient amount of Peptide-AuNPs, triggering catalyst-driven proportional chromogenic reaction. More importantly, noticing 11 fold increase of the amount of


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integrin is accompanied by an 11 fold enhancement of the absorbance, it is safe to draw conclusion that no obvious aggregation or coupling of the nanozyme on the surface of microplate was observed in this case. Cancer cell immunoassay was immediately undertaken to inquire into quantitative determination of integrin expression on HEL cells, as illustrated in Figure 7a. Practically, Peptide-AuNPs with optimized dosage was preliminarily incubated with various number of cells (0.5, 1.0, 1.5, 2.0, 2.5×104 cells per well) in 96-well plate to ensure integrin was saturated occupied. After the unbound nanoprobe was discarded and cells were thoroughly rinsed, the color evolution was launched via oxidation of substrate catalyzed by Peptide-AuNPs on cells membrane. The unambiguous result present in Figure 7b (blue curve) discloses that the reaction system presents gradational absorption enhancement upon increasing the cell number from 0.5×104 to 2.5×104 (R2=0.99). Further assessment of the elemental Au content against cell number exihibits a linear increasing fashion (R2=0.99, Figure 7b, red curve). It is reasonable to postulate that gradient number of cancer cells with gradient amount of integrin successfully seize corresponding gradient amount of Peptide-AuNPs. Thus, catalytic production of ox-TMB elevates proportionally. Prior to evaluating integrin express level on cells by comparing the optical density obtained from cancer cell immunoassay with that of the standard curve, it is imperative to consider multiple factors influencing the accuracy of the modality originated from the systematic distinction of experimental group and standard curve group. On one hand, catalytic reaction conditions including the volume and concentration of the delivered catalyst and substrates, catalytic time and temperature were deliberately kept consistent with each other. On the other hand, a single well’s diameter of microplate or 96-well plate is about 6.5 mm, whereas the thickness of HEL cell is approxiamtly 3-5 µm. It is presumed that nanozyme distributes on the cell membrane in a quasi-two-dimensional mode, similar to that in tranditional ELISA. After preliminarily excluding the aforementioned two feasibilities of derivation, remarkable discrimination on nanoprobe’s disperity between microplated-based ELISA and cancer cell


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immunoassay can not be neglected. Through attentively analyzing the results of standard curve and two-photon photoluminescence images, it can be inferred that Peptide-AuNPs are distributed in a more crowded manner on cytomembrane than on the surface of plate. It is highly plausible that the decline of surface area and active sites of Peptide-AuNPs inevitably result in the compromise of nanozyme’s catalytic efficacy.41 It is therefore vital to introduce a coefficient to calibrate the relevant discrimination. As shown in Figure 6b, standard curve reveals that integrin concentration rising from 31. 25 to 375 ng is accompanied by absorbance intensity changes from 0.025 to 0.30. That is, both independent and dependent variable behave an overall increase of 11 fold. In the case of cancer cell immunoassay, a linearly positive association between absorbance and the cells number is observed (Figure 7b, blue curve). In detail, when cells number varies from 0.5×104 to 2.5×104, the absorbance intensity changes from 0.16 to 0.58. It means a 4 fold elevation of cells number is correlated with a 2.6 fold enhancement of absorbance. Considering a 4 fold cells number enhancement is accompanied by a 4 fold increment of Au content (Figure 7b, red curve), it is claimed that the coupled nanozyme on the cytomembrane retains 72% of catalytic activity compared to that of the isolated Peptide-AuNPs arranged on the surface of microplate (See detailed δ definition in Part II, SI). Taken together, after tentatively introducing 0.72 as the coefficent, integrin express level on HEL cell is assessed by comparing the optical density derived from cancer cell immunoassay to that of the standard curve obtained from microplate-based ELISA. The concrete calculation procedure is present in Part II, SI, examplied by determination integrin number on 0.5×104 HEL cells. The ultimate integrin expression on a single HEL cell is calculated as 6.4×106. In order to verify the reliability of the analytical method above, the average number of integrin per cell was estimated by ICP-MS in parallel. The abundance of Au on the known number of cells can be precisely exchanged to the integrin number according to the nanoprobe formula and recognition ratio (Figure 7b, red curve). The concrete calculation process is present in Part III, SI. On the basis that one nanoprobe comprises 1.68×103 Au atoms, the amount of nanoprobe per cell is calculated as about 5.0×106. For recognition ratio, it was advocated that one peptide targets to one binding pocket of


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integrin.25 Additionally, nanoprobe’s limited size (5.0 nm) resembles the diameter of head domain of integrin (approximate 7-8 nm), it can be inferred that one integrin could bind to one nanoprobe. Therefore, the calculated result above also exactly represents the average integrin number on a single cell. Definitely, integrin expression on a single HEL cell determined by the catalytic chromogenic modality is close to that evaluated by ICP-MS analysis. A noteworthy point is that protein expression derived from ICP-MS method (5.0×106 per cell) is a little bit lower than that obtained from catalytic chromogenic approach (6.4×106 per cell). The reason might arises in part from metallic atom/ion signal inevidable attenuation in the transport flow or an incomplete atomization/ionization of the nanoparticles for mass analysis.42 To further prove the reliability of the proposed system, we quantified a series of integrin level by utilizing variable amount of peptide to partially block the aimed protein. As shown in Table S1, the protein level derived from catalytic chromogenic approach and ICP-MS method is very close with each other in the same manner. In a word, all these results delineate that enzyme-like catalysis method together with the nanoprobe is reliable for providing accurate quantitative information of aimed protein. CONCLUSIONS In summary, in virtue of rationally-designed peptides as multifunctional ligands, we construct Peptide-AuNPs as novel nanoprobe to spatially visualize and quantitatively determine integrin GPIIb/IIIa on cell membrane. The nanoprobe integrates capacities of selective recognition and intrinsic enzyme-like catalysis. Under the assistance of NIR two-photon technique, integrin is visualized directly which ensures the spatial specialty and avoids autofluorescence interference from impurity. Additionally, after a series of optimal catalytic parameters were selected, intrinsic enzyme-like catalysis by nanoprobe amplifies colorimetric signal to quantify integrin on cell membrane directly, rapidly and accurately. Meanwhile, utilizing sensitive metallic mass signal of the bioconjugate, integrin expression can be facilely calculated according to the accurate formula of nanoprobe. Note that the protein expression counted by two means above are consistent with each other, it is presumed that catalytic chromogenic


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method is reliable for providing quantitative information of marked membrane protein. The simple and highly efficient strategy is anticipated to be extended for precisely monitoring other biomarkers of interest and assist in accessing the progression of the relevant diseases. METHODS SECTION Chemicals and Reagents. The peptide (H2N-CCYKKKKQAGDV-COOH) was synthesized by a solid phase method (China Peptides Co. Ltd, Purity: 95%). Hydrogen tetrachloroaurate (III) (HAuCl4.3H2O), cystamine dihydrochloride, bovine serum albumin (BSA), 5, 5-dimethyl-1-pyrroline n-oxide (DMPO), 3, 3', 5, 5'-tetramethylbenzidine (TMB), paraformaldehyde (POM) were purchased from Sigma. Sodium borohydride (NaBH4), hydrogen peroxide (H2O2), nitric acid (HNO3) and hydrochloric acid (HCl) were obtained from Beijing Chemical Reagent Co., China. HEL cell line was from Cancer Institute and Hospital, Chinese Academy of Medical Sciences. Cell culture medium RPMI 1640 and fetal bovine serum (FBS) were purchased from Hyclone. Integrin GPIIb/IIIa antibody and goat anti-mouse IgG-FITC were obtained from Santa Cruz Biotechnology. Human Platelet Membrane Glycoprotein GPIIb/IIIa (integrin GPIIb/IIIa) ELISA Kit was purchased from Beijing JieHuiBoGao Biological Technology Co. Ltd. All other materials were commercially available and used as received unless otherwise mentioned. The water with a resistivity of 18.2 MΩ⋅cm, used throughout the experiment, was purified with a Milli-Q system from Millipore Co., USA. Preparation of 2-Aminoethanethiol-Modified Gold Nanoparticles (AuNPs). In a typical experiment, 400 µL of 213 mM cystamine dihydrochloride was added into 40 mL of 1.42 mM HAuCl4 at ambient temperature. After stirring for 20 min, 10 µL of 1250 mM freshly prepared NaBH4 aqueous solution was added into the mixture. After reduction reaction for 30 min under darkness, the red colloidal dispersion was obtained. Furthermore, as-prepared AuNPs colloidal dispersion was concentrated by superfilter tube (MWCO: 100 KDa) to cut off free ions. Preparation of Peptide-Modified Gold Nanoparticles (Peptide-AuNPs). 2 mL of 1.25 mg mL-1 13 Environment ACS Paragon Plus

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peptide aqueous solution was added to 2 mL of AuNPs solution. After the ligand-exchange reaction for 12 h, Peptide-AuNPs was purified and concentrated using superfilter tube (MWCO: 100 KDa) to cut off free peptide. The filtration containing unbound peptide was collected to determine the amount of peptide assembled on the surface of gold nanoparticles by BCA method. Then, Peptide-AuNPs was re-dispersed in ddH2O with a concentration of 240 nM and stored at 4 oC for future use. Characterization of Peptide-AuNPs. For TEM observation, the sample was dropped on carbon-copper grid. TEM images were collected by using a JEM 2100F microscope (JEOL, Japan) working at 200 kV. The average size analysis was evaluated with image software (ImageJ 1.43) by manually measuring at least 50 Peptide-AuNPs’ diameter. The mean diameter and size distribution of AuNPs and Peptide-AuNPs were further documented by a phase analysis light scattering technique (Zetasizer Nano, Malvern). FTIR spectra of AuNPs and Peptide-AuNPs were measured on a Bruker Tensor 27 spectrophotometer using KBr pressed disks. XPS was performed on the Thermo Scientific ESCALab 250Xi using 200 W monochromated Al Kα radiation. The 500 µm X-ray spot was used for XPS analysis. The base pressure in the analysis chamber was about 3×10-10 mbar. Typically the hydrocarbon C1s line at 284.8 eV from adventitious carbon is used for energy referencing. Survey scans were run in 0-1350 eV range, while detailed scans were recorded for the C1s, Au4f and S2p regions. A UV-1800 spectrophotometer (SHIMADZU, Japan) was used to record the UV-VIS spectra of various samples. ICP-MS Analysis of Peptide-AuNPs Concentration. The concentration of Peptide-AuNPs was determined by ICP-MS analysis (Thermo Elemental X7, USA). Firstly, the prepared and purified Peptide-AuNPs was pre-digested by H2O2 and HNO3 at a volume ratio of 1:3 over night. Then the sample was further digested by aqua regia at mild boiling temperature. The solution was evaporated to 0.1-0.2 mL and diluted by 2% HNO3 and 1% HCl to a final volume. Calibration plot for standard Au was obtained by injecting a series of standard aqueous Au solutions (0.1, 0.5, 1, 5, 10, 50, 100 ng mL-1


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containing 2% HNO3 and 1% HCl) into the ICP-MS system. Then completely digested Peptide-AuNPs solution was followed to measure Au content. 20 ng mL-1 of Bismuth was regarded as an internal standard. Each experiment was conducted in triplicate. Quantification was performed by internal seven-point calibration, by which the concentration of Peptide-AuNP was determined by calculation. Electron Spin Resonance Test. The ESR spectra were measured by using a Bruker model ESP 300 spectrometer operating at room temperature. H2O2 solution was mixed with DMPO in buffer at pH 4.0 and the reaction was initiated by addition of Peptide-AuNPs. Immediately, 30 µL aliquot of control or experimental sample was put in glass capillary tube and sealed. The capillary tubes were then inserted into the ESR cavity, and the spectra were recorded. Other settings were as follows: microwave power, 10.12 mW; frequency, 9.8 GHz; time constant, 40.96 ms; scan width, 100 G. Concentration-Dependent Peroxidase-Like Activity of Peptide-AuNPs. The oxidation of TMB by H2O2 catalyzed by Peptide-AuNPs produced a blue color with major absorbance peak at 652 nm. The concentration-dependent peroxidase-like activity of Peptide-AuNPs assays were operated at 37 oC with various concentrations of artificial enzyme (0, 4.80, 9.60, 14.4 and 19.2 nM) in the presence of 200 mM H2O2 and 800 µM TMB at pH 4.0. The reaction was carried out in a 96-well plate, and the absorbance of each sample at 652 nm was recorded at different time points in a time course mode. Peptide-AuNPs Mediated Oxidation of TMB at Various pHs and Temperatures. 200 µL of reaction buffer at pH ranging from 3.0 to 9.0 containing 19.2 nM Peptide-AuNPs, 200 mM H2O2 together with 800 µM of TMB was kept at 37 oC water baths for 30 min. Then the absorbance of each sample at 652 nm was recorded by UV-VIS spectrophotometer. To examine the influence of incubation temperature on the peroxidase-like activity, 200 µL of reaction buffer at pH 4.0 containing 19.2 nM Peptide-AuNPs, 200 mM H2O2 as well as 800 µM of TMB was kept at different water bath from 25 oC to 60 oC for 30 min. Then the absorbance of each sample at 652 nm was recorded by UV-VIS spectrophotometer. The absorbance of similar reaction system without nanozyme was set as control.


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Steady-State Kinetic Studies. Steady-state kinetic assays were carried out at 37 oC in a 96-well plate. When varying substrate is H2O2, chemicals were added into 200 µL of buffer solution containing 19.2 nM Peptide-AuNPs or AuNPs, 800 µM TMB and a series of concentration of H2O2 at pH 4.0. When varying substrate is TMB, chemicals were added into 200 µL of buffer solution containing 19.2 nM Peptide-AuNPs or AuNPs, 200 mM H2O2 together with a series concentration of TMB at pH 4.0. Kinetic measurements were conducted by monitoring the absorbance change at 652 nm on a microplate reader (SpectraMAX M2, Sunnyvale, California). The Michaelis−Menten constant and the maximal reaction velocity were calculated using the Lineweaver−Burk plot. The kinetic parameters were calculated using the Michaelis−Menten model based on the equation V = Vmax [S]/(Km + [S]), where [S] is the concentration of the substrate, V is the initial velocity, Vmax is the maximal velocity and Km is the Michaelis constant.11 Optimization of Time Parameters on Labeling HEL cells via ICP-MS. HEL cells were cultured in 1640 medium and 10% FBS supplemented with 1% penicillin and streptomycin at 37 °C in 5% CO2, 70% humidity environment, reseeded every three days to maintain subconfluence. When cells reached about 90% confluence, they were split from the bottom and suspended by a standard trypsin-based technique. Then, 3.7% POM PBS solution was introduced to fix cells at ambient temperature for 30 min to disrupt the endocytosis process of the cells. After centrifuging and discarding the remaining POM, the fixed cells were washed thoroughly. To investigate time parameter on labeling efficiency, fixed 2.5 ×104 HEL cells were incubated with 120 nM nanoprobe from 5 min to 45 min at room temperature. The cells were washed to remove free nanoprobe and counted by flow cytometry before pre-digested by mixed acid (VH2O2: VHNO3 at a volume ratio of 1:3) over night. After that the samples were digested by aqua regia and diluted by mixed acid (2% HNO3 and 1% HCl) to a final volume. The procedure to obtain Au standard calibration plot and the Au content in each sample were similar as described above. Each experiment was repeated three times. Specificity Investigation of Nanoprobe to Integrin GPIIb/IIIa on HEL Cell Membrane.


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Peptide-AuNPs Targets to Integrin on HEL Cell Membrane: HEL cells were first split into a glass bottomed culture dish using a standard trypsin-based technique and incubated in the culture media to allow 70% confluence overnight. After PBS was added to wash cells three times, 3.7% POM PBS solution was introduced to fix cells. Remaining POM was taken away before 3% BSA PBS solution was introduced into culture dish for 1 h to block the nonspecific recognition sites. After the remaining BSA solution was discarded, 120 nM Peptide-AuNPs suspension was cocultured with cells for 30 min at ambient temperature and then cells were washed for three times. At last, the cells were immediately observed with a 1.35 numerical aperture oil immersion 60×objective equipped by a two-photon laser scanning microscope (Olympus, FV1000MPE). TPPL of nanoprobe marked cells was collected mainly in red channel when excited by 800 nm laser with a repetition rate of 80 MHz and pulse width of 100 fs. The cells without any treatment were set as control. Blocking Protein Binding Sites Study: HEL cells were first fixed, then 3% BSA in PBS was introduced to block the nonspecific recognition sites. After that, cells were incubated with 2 mM peptide for 1 h to block the specific recognition sites. Next, 120 nM Peptide-AuNPs was cocultured with cells for 30 min at room temperature before observed by two-photon laser scanning microscope. The TPPL images were collected at the same setting as that of the experimental group. Competitive Recognition Study: HEL cells were first fixed before 3% BSA in PBS was introduced to block the nonspecific recognition sites. After that, cells were incubated with 2 mM peptide and 120 nM Peptide-AuNPs for 30 min at room temperature simultaneously before imaged by two-photon laser scanning microscope. The TPPL images were collected at the same setting as above. Immune Fluorescence Measurement: After immobilized and blocked the nonspecific recognition sites, HEL cells were incubated with antibody of integrin GPIIb/IIIa at 1:100 dilution ratio for 1 h and washed for three times with PBS. Notably the antibody is a mouse monoclonal antibody raised against peripheral blood mononuclear cells from human origin. Subsequently, goat anti-mouse IgG-FITC with dilution ratio at 1:200 was introduced and cocultured with cells for 30 min. After cells were washed for 17 Environment ACS Paragon Plus

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three times, 120 nM Peptide-AuNPs was added and incubated with cells for 30 min. Then excess nanoparticles were washed away and cells were imaged. TPPL collected in the red channel derived from Peptide-AuNPs was obtained by a femtosecond pulse laser, whereas one-photon fluorescence image was captured by a 488 nm laser. Microplate-Based ELISA to Set up Standard Curve. For the purpose of plotting standard curve to quantify integrin, we traced oxidation of TMB in the presence of H2O2 catalyzed by Peptide-AuNPs as a function of integrin concentration using a converted commercial ELISA Kit. The procedure to establish the standard curve of optical density versus integrin concentration was fulfilled by following the manufacturer’s protocol with minor modification. In practice, a polyclonal antibody specific for integrin GPIIb/IIIa was pre-coated on the surface of each well of microplate. Then 50 µL of 0, 31.25, 62.5, 125, 250 and 375 ng mL-1 standard integrin solution was mixed with 50 µL of 240 nM Peptide-AuNPs to form homogenous mixtures, respectively. 100 µL of the mixture above was added into each well of the microplate and incubated for 30 min, respectively. After that, the wells were decanted and washed for five times. Then 100 µL of mixed solution of 800 µM TMB and 200 mM H2O2 was added to the wells and the catalytic reaction proceeded at 37 oC. Thirty minutes later, optical density at 652 nm of each well was recorded by a microplate reader. Calculate the mean value of the triplicate readings for each sample. Cancer Cell Immunoassay to Determine Integrin Expression on HEL Cells. Different numbers of HEL cells (0, 1750, 3500, 5250, 7000, 8750 cells) were split into 96-well plate using a standard trypsin-based technique. The cells were cultured for at least 24 h to allow cells number reached 0, 0.5×104, 1.0×104, 1.5×104, 2.0×104, 2.5×104 cells well-1, respectively. Then cells were washed with PBS before 3.7% POM was introduced to fix them. After excess POM was suck away, cells were thoroughly rinsed with PBS. Next, 3% BSA PBS solution was added to block the nonspecific recognition sites for 1 h before 100 µL of 120 nM Peptide-AuNPs was introduced to mark HEL cells. Thirty minutes later,


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cells were rinsed to discard unbound nanoparticles. After that, 100 µL of mixed solution containing 800 µM TMB and 200 mM H2O2 was added to initiate enzyme-mimetic catalytic reaction. Thirty minutes later, optical density of the catalytic system at 652 nm was recorded with a microplate reader, respectively. Data are shown as mean± the standard error from three independent experiments. The concentration of integrin in each well was then determined by comparing the optical density of the samples to that of the standard curve. Quantitative Analysis of Integrin Expression on HEL cells via ICP-MS. Under the aforementioned optimal labbeling conditions, integrin on HEL cells was bound by nanoprobe in a saturated manner. Then cells were pre-digested by mixed acid (VH2O2: VHNO3=1:3), then digested by aqua regia and finally diluted by mixed acid (2% HNO3 and 1% HCl) to a final volume. The procedures to obtain Au standard calibration plot and the Au content in each sample were followed the standard operating process. Each experiment was repeated three times. Quantitative Analysis of Partially Blocked Integrin Level on HEL cells via Cell Immunoassay and ICP-MS Approaches. 2.5×104 fixed HEL cells were first blocked the nonspecific recognition sites, then incubated with 0, 2, 20, 200 µM peptide for 1 h to partially block the specific recognition sites, respectively. Then integrin level was quantified through catalytic chromogenic approach and ICP-MS method by following the standard operating process mentioned above, respectively. Each experiment was repeated three times. Conflict of Interest: The authors declare no competing financial interest. Acknowledgment. We acknowledge the financial support of this research by the National Key Basic Research Program of China (2013CB932703) and the Natural Science Foundation of China (21390410, 31271072, 31200751, 31500815, 11404333, 81472851 and 31300827). Supporting Information Available. The Supporting Information file contains ICP-MS measurement to determine the concentration of Peptide-AuNPs; quantitative calculation of integrin expression through catalytic chromogenic method and ICP-MS analysis. The material is available free of charge via the


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Scheme 1. (a) Synthesis of peptide-conjugated gold nanoparticles by chemical reduction and ligand exchange; (b) Nanoprobe integrates the signal generation and amplification for detection of integrin GPIIb/IIIa. (i) Selective recognition mediated coupling of gold nanoparticles inducing two-photon photoluminescence to display GPIIb/IIIa on the surface of HEL cell; (ii) Intrinsic enzyme-like catalysis amplifying signal to sensitively and accurately quantify GPIIb/IIIa expression.


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Figure 1. TEM images of (a) AuNPs and (b) Peptide-AuNPs. (c) Size distributions of AuNPs and Peptide-AuNPs evaluated from DLS results.

Figure 2. UV-VIS absorption (a) and FTIR (b) spectra of AuNPs and Peptide-AuNPs. C1s patterns of (c) AuNPs and (d) Peptide-AuNPs analyzed by XPS.


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Figure 3. (a) Time-dependent absorbance change at 652 nm of mixture containing 800 µM TMB and 200 mM H2O2 in the presence of Peptide-AuNPs with different concentrations (0-19.2 nM). (b) pH-dependent peroxidase-like activity of Peptide-AuNPs. Experiments were carried out using 19.2 nM Peptide-AuNPs as nanozyme in a reaction system containing 200 mM H2O2 together with 800 µM TMB at 37 oC. (c) Temperature-dependent peroxidase-like activity of Peptide-AuNPs. Experiments were performed at pH 4.0 containing 19.2 nM Peptide-AuNPs, 200 mM H2O2 as well as 800 µM TMB at different temperatures. The maximum point in each curve was set as 100% enzymatic activity. (d) ESR spectra of ●OH spin adduct (DMPO-OH) generated during the catalytic reaction (blue curve). Control experiments in which without H2O2 or Peptide-AuNPs (black curve); without nanozyme (red curve) were provided.


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Figure 4. Steady-state kinetic analyses of Peptide-AuNPs by (a) varying the concentrations of H2O2 (0-1000 mM) with fixed 800 µM TMB and (c) changing the concentrations of TMB (0-2 mM) with fixed 200 mM H2O2. Experiments were carried out at pH 4.0 using 19.2 nM Peptide-AuNPs as catalyst at 37 °C. Fitted curve (b) and (d) are relevant double reciprocal plots of peroxidase activity of Peptide-AuNPs using Michaelis–Menten and Lineweaver–Burk models.


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Table 1. Comparison of the apparent Michaelis–Menten constant (Km) and catalytic constant (Kcat) of Peptide-AuNPs, AuNPs and HRP Catalyst

Substance

Km [mM]

Kcat [s-1]

Peptide-AuNPs

H2O2

929

14.6

AuNPs

H2O2

558

22.2

HRP35

H2O2

0.935

4.05×103

Peptide-AuNPs

TMB

0.277

3.57

AuNPs

TMB

0.0360

4.44

HRP35

TMB

0.301

5.18×103


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Figure 5. Specificity of nanoprobe for integrin GPIIb/IIIa. Two-photon photoluminescence images of HEL cells exposed to (a) 120 nM Peptide-AuNPs only; (b) disperse medium as control; (c) 2 mM peptide for 1 h, followed by 120 nM Peptide-AuNPs; (d) 2 mM peptide and 120 nM Peptide-AuNPs simultaneously. Note that all above images were collected at the same setting. Images of HEL cells exposed to (e) integrin antibody and goat anti-mouse IgG-FITC (green one-photon fluorescence), followed by 120 nM Peptide-AuNPs (red two-photon photoluminescence).


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ACS Nano

Figure 6. (a) Schematic illustration of Peptide-AuNPs based microplate-ELISA. (b) The optical density at 652 nm as a function of concentration of integrin GPIIb/IIIa. This curve is further employed as the standard reference. Each point is the average result of triplicate analysis. The concentration of delivered Peptide-AuNPs was 120 nM. The substrates comprise of 800 µM TMB and 200 mM H2O2. The catalytic time was 30 min and catalytic temprature was set at 37 °C.

Figure 7. (a) Schematic illustration of Peptide-AuNPs mediated cancer cell immunoassay. (b) Linear regression associates HEL cells number with Au concentration (red curve) and catalytic colorimetric variation (blue curve). The concentration of delivered Peptide-AuNPs was 120 nM. The substrates comprise of 800 µM TMB and 200 mM H2O2. The catalytic time was 30 min and catalytic temprature was set at 37 °C. Each point is the average result of triplicate analysis.


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SYNOPSIS TOC

Liang Gao, Meiqing Liu, Guifu Ma, Yaling Wang, Lina Zhao, Qing Yuan, Fuping Gao, Ru Liu, Jiao Zhai, Zhifang Chai, Yuliang Zhao and Xueyun Gao *

Peptide-Conjugated Gold Nanoprobe: Intrinsic Nanozyme-Linked Immunsorbant Assay of Cell Integrin Expression

Multifunctional small peptide is designed and developed to construct gold nanoprobe integrating signal generation and amplification for detection of cell membrane protein integrin GPIIb/IIIa. Selective recognition mediated partial aggregation of gold nanoparticles inducing two-photon photoluminescence can be utilized to display integrin on the surface of human erythroleukemia cells. Intrinsic enzyme-like catalysis amplifying colorimetric signal is explored to accurately quantify integrin expression on cell membrane.


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Peptide-Conjugated Gold Nanoprobe: Intrinsic ... - ACS Publications

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