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Quick Serological Detection of a Cancer Biomarker with an Agglutinated Supramolecular Glycoprobe Xiao-Peng He,† Xi-Le Hu,† Hong-Ying Jin,† Jiemin Gan,§ Huili Zhu,*,§ Jia Li,*,‡ Yi-Tao Long,*,† and He Tian† †

Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China ‡ National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shoujing Road, Shanghai 201203, PR China § Huadong Hospital Affiliated to Fudan University, 221 West Yan’an Road, Shanghai 200040, PR China S Supporting Information *

ABSTRACT: While serology represents the forefront technique for cancer diagnosis, current clinical methods for the detection of serum biomarkers have flaws in terms of the need of complicated manipulations, long analytical time, and high cost. Here, we develop a supramolecular glycoprobe for the quick serological detection of a cancer biomarker. The probe formed by agglutination between self-assembled glyco-gold nanoparticles and a lectin shows subtle optical variations upon the competitive recognition of a glycoprotein biomarker secreted by cancer cells, tumor-bearing mice, as well as clinical cancer patients, with no response to a series of controls including the serum of hepatitis patients. This research provides an insight into the development of effective tools for serological diagnosis of cancer.

events,18,19 glyco-AuNPs have been prepared to detect lectins, viruses, and bacteria.20−25 We show here the development of a supramolecular glycoprobe for the quick serological detection of a cancer biomarker. Strain-promoted click chemistry,26,27 which avoids the use of metal catalysts, combined with a thiol-gold bonding interaction, was exploited for the one-pot preparation of a glyco-AuNP. The as-prepared particles can subsequently form a supramolecular glycoprobe through specific glycoligand-lectin recognitions (Figure 1a). Subtle optical changes were observed upon the competitive interaction between the glycoprobe with a glycoprotein biomarker specifically secreted by liver cancer cells (Figure 1b). Importantly, the biomarker can be selectively captured in the serum of tumor-bearing mice and clinical liver cancer patients but not in that of normal and hepatitis controls.

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espite the rapid development of clinical sciences, cancer is still among the deadliest diseases worldwide. Whereas the treatment of midadvanced cancers with conventional chemotherapeutic regimes often elicits drug resistance and lethal side effects,1−5 the ability to diagnose cancer more effectively at an earlier stage could benefit the overall survival rate of patients.6−8 However, the latter mission has been compromised by the insufficient specificity of present cancer biomarkers as well as the drawbacks of current diagnostic techniques. Although the radiant tumor imaging and traumatic biopsy are needed for confirmation, the serological test is the forefront technique for high-throughput screening of cancer. However, the mainstream clinical techniques such as the enzyme-linked immunosorbent assay and polymer chain reaction have flaws in terms of complex preparation procedures, long detection time, and high analytical costs. As a consequence, development of simple and economic methods is in great demand for the serological diagnosis of cancer. With the advancement of modern materials science and nanotechnology, increasing functional materials for chemical biology and biomedicine have been developed. Among them, gold nanoparticle (AuNP), owing to its unique optical properties such as the easily tunable optical signals upon aggregation-disaggregation processes, has been extensively employed to produce sensing systems.9−17 Considering the importance of intercellular glycoligand− receptor recognitions, which modulate a number of biological © XXXX American Chemical Society

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EXPERIMENTAL SECTION General. All purchased chemicals and reagents are of analytical grade. Hydrogen tetrachloroaurate (III) hydrate (HAuCl4·XH2O, 99.9%-Au) was purchased from J&K Chemical. Sodium citrate was obtained from J&K Chemical. 2-(2-(2(2-Mercaptoethoxy)ethoxy)ethoxy)ethanol, Lens culinaris lectin (LcA), and bovine serum albumin (BSA) were purchased from Received: June 25, 2015 Accepted: August 3, 2015

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DOI: 10.1021/acs.analchem.5b02384 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. (a) Structure of azido mannoside 1 and cyclooctyne disulfide 2, and a scheme depicting the self-assembly of the glyco-AuNP followed by formation of the supramolecular glycoprobe via glycoligand−receptor recognitions. (b) Scheme depicting the use of the glycoprobe to detect αfetoprotein-L3 (AFP-L3) immobilized on a microplate from various biological samples.

Sigma-Aldrich. 1H NMR and 13C NMR spectra were recorded on a Bruker AM 400 MHz spectrometer with tetramethylsilane (TMS) as the internal reference. Absorption spectra were measured on a Varian Cary 500 UV−vis spectrophotometer. High-resolution mass spectra (HRMS) were recorded with a Waters Micromass LCT mass spectrometer. Transmission electron microscopy (TEM) images were obtained on a JEOL 100CX transmission electron microscope operating at an accelerating bias voltage of 100 kV. Dynamic light scattering (DLS) was carried out on a Horiba LB-550 dynamic light scattering nano-analyzer. High-performance liquid chromatography (HPLC) was measured on Shimadzu Prominence series equipment. Microplate reader (M5) was obtained from the U.S. The AFP-L3 ELISA kit was obtained from the Enzyme-linked Biotechnology Company. One-Pot, Stepwise Construction of Glyco-AuNPs. AuNPs were prepared by the citrate-mediated reduction of HAuCl4. A stirred aqueous solution of HAuCl4 (0.29 mM, 50 mL) was heated to reflux, and then a trisodium citrate solution (38.8 mM, 5 mL) was added quickly. Then, the solution was heated under reflux for an additional 10 min and then cooled to room temperature. The as-prepared AuNP solution (20 mL) was mixed with a THF solution of cyclooctyne disulfide 2 (10 μM, 4 mL) and an aqueous solution of thiol-PEG (10 μM, 4 mL), and the resulting mixture was stirred at 40 °C for 2 h. Subsequently, an aqueous solution of 1 (10 μM, 4 mL) was added, stirring at 40 °C for another 4 h. The resulting mixture was centrifuged at 8000 rpm for 17 min and the residual solid product redispersed in Tris-HCl buffer solution (20 mL). This process was repeated three times to remove excessive reactants.

The as-obtained glyco-AuNPs (dissolved in Tris-HCl buffer containing 10 nM BSA) were used directly for aggregation with lectins. Formation of Man-AuNP@LcA. To a solution of ManAuNP in Tris-HCl (0.01 M, pH 7.4) was added LcA (10 nM). The resulting mixture was stirred at room temperature for 5 min, and the resulting solution was used as is for AFP-L3 detection. AFP-L3 Detection. A 96-well microplate kit coated with an anti-AFP-L3 antibody (AFP (H-9):sc-166335, Santa Cruz Biotechnology, Inc.) was used to immobilize AFP-L3 in test samples. In a typical assay, 50 μL of test sample was immobilized on the kit according to manufacturer’s instruction, followed by addition of 100 μL of Man-AuNP@LcA. Then kit was shaken for 10 min, and the optical absorbance of each well was measured at 525 nm using a microplate reader. Standards of defined concentrations were run in each assay, allowing for the construction of a calibration curve by plotting absorbance versus concentration. The AFP-L3 glycoprotein concentrations in the sample were then calculated from this calibration curve. Cell Culture. Hep-G2 and HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY). HCT-116 cells were maintained in McCoy’s 5A Medium (Sigma-Aldrich, Shanghai, China) supplemented with 10% FBS and A549 maintained in Ham’s F-12 Nutrient Mixture (Invitrogen, Carlsbad, CA) supplemented with 10% FBS. All the cell lines were passaged every 3−4 days and cultured in a 37 °C humidified incubator under an atmosphere of 5% CO2 in air. B

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Analytical Chemistry Small Interfering RNA (siRNA) Transfection. All RNA oligonucleotides were synthesized and purified at Genepharma (Shanghai, China). One-day before the transfection, Hep-G2 cells were seeded at a density of 1.5 × 105 cells/well in a complete medium without antibiotics in 24-well plates. Synthesized siRNA against AFP were transfected into HepG2 cells using the X-tremeGENE siRNA Transfection Reagent (Invitrogen Inc., USA) according to the manufacturer’s protocol. The efficiency of RNA interfering was confirmed by real-time quantitative PCR. Sequences of the synthesized oligonucleotide were as follows: AFP sense, 5′-AAUUGCAGUCAAUGCAUCUUUCACC-3′ AFP antisense, 5′-GGUGAAAGAUGCAUUGACUGCAAUU-3′ Real-Time Quantitative PCR. Total RNA was isolated from cells using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. Complementary DNA generated using a PrimeScript RT reagent kit (TaKaRa, Dalian, China) was analyzed by quantitative PCR using SYBR Premix Ex TaqTM. Real-time PCR was performed using a 7300 real-time PCR system (Applied Biosystems, CA). GAPDH was detected as the housekeeping gene. Human primers for qPCR were as follows: GAPDH forward, 5′-ATCACTGCCACCCAGAAGAC-3′ GADPH reverse, 5′-ATGAGGTCCACCACCCTGTT-3′ AFP forward, 5′-GGAAGTCTGCTTTGCTGAAGA-3′ AFP reverse, 5′-CACACCGAATGAAAGACTCGT-3′ HepG2 Xenograft. Female athymic BALB/c nu/nu mice (4−5 weeks old, 16−18 g) were obtained from Sino-British SIPPR/BK Lab. Animal Ltd., with the certification number of 2008001638201. The animals were housed in specific pathogen-free (SPF) conditions at Key Laboratory of Brain Functional Genomics, Ministry of Education, East China Normal University, and were acclimatized for at least 3 days prior to use. In total, 5 × 106 Hep-G2 cells with a volume of 0.2 mL were subcutaneously injected into the right flank of the athymic nude mice. The cell suspension was mixed with the same volume of Matrigel and kept on ice. When the tumors reached a mean volume of around 300−400 mm3, the serum of the mice was collected.

Figure 2. (a) Man-AuNP in the presence of increasing lentil lectin (LcA, from top to bottom: 0, 10, 30, 50, 100, and 200 nM). (b) Comparison of the absorbance change (where I0 and I are the absorbance of the glyco-nanoparticle at 525 nm in the absence and presence of LcA, respectively) of Man-AuNP constructed by the onepot or the control method in the presence of increasing LcA. (c) Absorbance change of Man-AuNP in the absence and presence of LcA and Man-AuNP@LcA in the presence of increasing competing free Dmannose. (d) Dynamic light scattering of AuNP (black), Man-AuNP (red), and Man-AuNP in the presence of 30 nM (blue), 50 nM (violet), and 100 nM (green) LcA. (e) Transmission electron microscope (TEM) and dark-field microscope (DFM) images of Man-AuNP, Man-AuNP@LcA, and Man-AuNP@LcA in the presence of 10 mM free D-mannose.

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RESULTS AND DISCUSSION Construction of the Supramolecular Glycoprobe. The key building block used is a cyclooctyne tailed with a disulfide ring (2) (Scheme S1). Disulfide 2 was coated to bare AuNPs by the thiol-gold bonding interaction, forming an alkynyl shell on the surface. Then, the core−shell structure was directly subjected to a copper-free click reaction28 with mannosyl azide 1. After centrifuging, the resulting mannose-coated AuNP (Man-AuNP) was used as is for the agglutination with a lectin, owing to the fact that no particular catalyst or byproduct existed in the system. Next, we used the mannose-selective, dimeric LcA (Lens culinaris lectin)29 for agglutination of Man-AuNP. We observed a gradual bathochromic shift accompanied by peak broadening of the Man-AuNP upon the addition of increasing LcA in the UV−vis spectrum (Figure 2a). This is in agreement with the typical aggregation behavior of the AuNPs.20−25 To prove that the one-pot self-assembly does not compromise the sensitivity of the Man-AuNP, we also synthesized the triazole-connected mannosyl disulfide prior to surface functionalization (Scheme S1). After isolation, the compound was coated to AuNP and centrifuged. A comparison between the one-pot and two-step protocol (control) indicated

a consistent LcA sensitivity over a wide concentration range (Figure 2b and Figure S1), suggesting that the former is wellsuited for the simple construction of the glycoprobe. To test the reversibility of the probe formation, free D-mannose was added to the supramolecular system. We observed a concentration dependent disaggregation of the system in the UV−vis spectrum (Figure 2c and Figure S2). Dynamic light scattering (DLS) suggested that, while the particle size of the AuNP did not change substantially when coated by the mannosyl shell, the presence of increasing LcA gradually enlarged the size (Figure 2d and Figure S3), probably indicating the aggregation of Man-AuNPs. We also used spectroscopic techniques to characterize the probe formation (Figure 2e). Transmission electron microscope image showed a high-density supramolecular aggregation of the Man-AuNPs in the presence of LcA. In contrast, addition of free D-mannose disaggregated the glycoprobe (competed). A similar phenomenon was observed using the dark-field C

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Analytical Chemistry microscope (DFM) on the basis of the localized surface plasmon resonance (LSPR) scattering property of the AuNP.30 We observed a strong scattering signal produced by the coexistence of Man-AuNP and LcA, whereas the presence of free D-mannose competitively quenched the signal. Serological Detection of AFP-L3. We then explored the applicability of the supramolecular glycoprobe in analyzing a biomarker for liver cancer. α-Fetoprotein (AFP) is a serological biomarker conventionally used for the diagnosis of hepatocellular carcinoma (HCC).31 However, high-level AFP can also exist in the serum of hepatitis patients and during pregnancy; therefore, false-positive results are frequently produced by referring to the clinical standard of AFP.32 Malfunctional protein glycosylation has been recognized as a signature of many diseases and a core-fucosylated glycoform of AFP (AFP-L3, which has high binding affinity with LcA) has been approved as a biomarker for HCC.33 While current biochemical detection of AFP-L3 relies on the immunofluorescence technique, more concise and cost-effective means for the marker is needed. Considering its sensitive optical signal changes upon competitive glycoligand−lectin recognitions, we employed Man-AuNP@LcA to probe AFP-L3. We used a commercial AFP-L3 kit that has a capture antibody to immobilize the marker and Man-AuNP@LcA as a replacement of the primary and secondary antibodies (Figure 1b). We first determined the level of AFP-L3 secreted in the culture supernatant of a hepatoma cell line (Hep-G2). The marker was captured on a microplate (by the anti-AFP-L3 capture antibody of the kit) and then detected by ManAuNP@LcA using ELISA as the control. We note that our method simply involves an incubation of the supramolecular glycoprobe with the surface-immobilized AFP-L3, followed by reading of the optical density using a common microplate reader. The equilibrium was reached within an incubation time of 10 min (Figure S4). To quantify AFP-L3, a calibration curve was established by serial dilutions of AFP-L3 (Figure S5). We observed a time-dependent increase of AFP-L3 by culturing the cells for 12, 24, and 48 h (Figure 3a). In addition, by increasing the cultured cell number (400 000, 600 000, and 800 000 cells mL−1), the secreted AFP-L3 level increased synchronically (Figure 3b). These results are in good agreement with those produced by ELISA. To test the specificity and selectivity of the glycoprobe, several control cells were used. We determined that transient knockdown of the AFP expression level of Hep-G2 (si-AFP) caused a sharp decrease of AFP-L3 (Figure 3c), which was in good agreement with the relative mRNA level of AFP determined by polymerase chain reaction (PCR) (Figure 3d). We also observed that ManAuNP@LcA did not produce any signal for a control hepatocyte cell line (L-02) as well as other cancer cell lines including human lung (A549), cervix (HeLa), and colon (HCT-116). This observation accords well with the fact that these control cells do not express AFP that is the substrate for core-fucosylation to produce AFP-L3. The quantification data are listed in Table S1. The data above suggest the satisfactory sensitivity and specificity of Man-AuNP@LcA. DFM was also used to demonstrate the biomarker detection (Figure S6). The scattering of Man-AuNP@LcA could not be observed after the incubation with surface-immobilized AFPL3, probably suggesting the disaggregation of the supramolecular glycoprobe by competitive interaction with the marker. In contrast, strong scattering images were obtained

Figure 3. (a) Quantification of AFP-L3 (α-fetoprotein) in the supernatant of Hep-G2 cells (human liver cancer, 400 000 mL−1) cultured for 12, 24, and 48 h with Man-AuNP@LcA using ELISA as the control. (b) Quantification of AFP-L3 in the supernatant of increasing Hep-G2 cells (N-40 = 400 000 mL−1; N-60 = 600 000 mL−1; N-80 = 800 000 mL−1) cultured for 24 h with Man-AuNP@ LcA using ELISA as the control. (c) Quantification of AFP-L3 in the culture supernatant of different cells. (d) Quantification of the AFP mRNA level in different cells with polymerase chain reaction (***P < 0.001; n.d. = not detectable).

after incubation with the control cells, suggesting the specificity of Man-AuNP@LcA for AFP-L3 secreted by hepatoma cells. Having established the effectiveness of Man-AuNP@LcA for detection of cell-secreted AFP-L3, we interrogated its ability to probe the marker secreted by a mouse model. Female athymic BALB/c nu/nu mice bearing Hep-G2 tumor xenograft were established according to a previous report.34 The serum of five tumor-bearing mice and five control mice was treated with Man-AuNP@LcA in a high-throughput manner. We observed the existence of high-level AFP-L3 in the serum of the xenograft group but not in that of the control group (Figure 4 and Table S2). Meanwhile, DFM images showed that the plasmonic signal of the supramolecular glycoprobe could be detected after interaction with the control, but not the xenograft group, probably due to the competitive disaggregation of ManAuNP@LcA upon incubation with the latter (Figure S7).

Figure 4. Quantification of AFP-L3 in the serum of Hep-G2 xenograft mice with Man-AuNP@LcA using ELISA as control (M1−M5 indicates the number of the mouse and C1−C5 indicates the number of the control mouse) (**P < 0.05). D

DOI: 10.1021/acs.analchem.5b02384 Anal. Chem. XXXX, XXX, XXX−XXX

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These data substantiated the usefulness of the glycoprobe for AFP-L3 detection with real samples. Eventually, human serum specimens were used to demonstrate the potential clinical applicability of Man-AuNP@LcA. The specimens were divided into three groups, which included patients diagnosed with liver cancer (17 samples), those diagnosed with hepatitis (10 samples), and control (9 samples). The sera were treated with Man-AuNP@LcA in a highthroughput manner using the 96-well microplate. We observed evidently higher levels of AFP-L3 in the cancer group than in both the hepatitis and control groups (Figure 5 and Table S3);

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02384. Additional experimental section, figures, and tables (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the 973 project (Grant 2013CB733700), the National Natural Science Foundation of China (Grant 21176076), the Key Project of Shanghai Science and Technology Commission (Grant 13NM1400900), and the Fundamental Research Funds for Central Universities (Grant 222201414010). Prof. Yi Zang and Yue Zhang are warmly thanked for help in the cellular experiments. Prof. Xiongwen Zhang is warmly thanked for the animal experiments.

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Figure 5. Quantification of AFP-L3 (α-fetoprotein) in the serum of liver cancer patients, hepatitis patients, and control group with ManAuNP@LcA using ELISA as the control.

the results are in good agreement with those produced by ELISA. We note that hepatitis frequently produces a high AFP level, which is comparable to that produced by HCC, leading to false-positives.32 Our probe might have the potential to effectively prelude these false positives in a rapid manner. Further systematic clinical assays are in progress.

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CONCLUSION

In summary, we have shown the simple preparation of a supramolecular glycoprobe by the reversible agglutination between glyco-nanoparticles and a lectin. The probe has proven suitable for the quick serological detection of a glycoprotein biomarker for liver cancer. The marker can be selectively captured in the supernatant of cultured cancer cells, serum of tumor-bearing mice as well as that of clinical cancer patients. In contrast, the fact that the probe did not show evident response to a series of control sera highlights its good specificity for AFP-L3 even in complex biological fluids. Notably, the detection involves simply the coincubation of the glycoprobe with surface-immobilized biomarker on a commercial microplate within 10 min. This research provides an insight into the development of clinical tools for the quick serological diagnosis of diseases. E

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DOI: 10.1021/acs.analchem.5b02384 Anal. Chem. XXXX, XXX, XXX−XXX