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Dual Amplification Fluorescence Assay for Alpha Fetal Protein Utilizing Immuno-Hybridization Chain Reaction and Metal-Enhanced Fluorescence of Carbon Nanodots Dangdang Xu, Cui Liu, Chengyu Li, Chongyang Song, Yafeng Kang, Chu-Bo Qi, Yi Lin, Dai-Wen Pang, and Hong-Wu Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11659 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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ACS Applied Materials & Interfaces

Dual Amplification Fluorescence Assay for Alpha Fetal Protein Utilizing ImmunoHybridization Chain Reaction and MetalEnhanced Fluorescence of Carbon Nanodots Dang-Dang Xu1, Cui Liu1, Cheng-Yu Li1, Chong-Yang Song1, Ya-Feng Kang1, Chu-Bo Qi 2, Yi Lin1, DaiWen Pang1, Hong-Wu Tang1*

1

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, People’s Republic of China 2

Hubei Cancer Hospital, Wuhan, 430079, People’s Republic of China

_________________________ *Corresponding Author. E-mail: [email protected]. Phone: 0086-27-68756759. 1 ACS Paragon Plus Environment


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ABSTRACT: As an emerging fascinating fluorescent nanomaterial, carbon nanodots (CDs) have attracted much attention due to their unique properties such as small size, antiphotobleaching and biocompatibility. However, its use in biomedical analysis is limited due to its low quantum yield. Herein, we constructed a dual amplification fluorescence sensor by incorporating immuno-hybridization chain reaction (HCR) and metal-enhanced fluorescence (MEF) of CDs for the detection of alpha fetal protein (AFP). The immunoplasmonic slide and detection antibodies conjugated oligonucleotide initiator are served to capture and probe AFP molecules, respectively. Then CDs tagged hairpin nucleic acids were introduced to trigger HCR, in which the hairpin nucleic acid and oligonucleotide initiator are complementary. The interaction between CDs and the gold nano-island film greatly improves the radiative decay rate, increases the quantum yield and enhances the fluorescence emission of the CDs. Furthermore, HCR provides secondary amplification of fluorescence intensity. Therefore, the MEF-capable immuno-hybridization reactions provide obvious advantages and result in exceptional sensitivity. In addition, the sandwich immunoassay method offers high specificity. The results show wide linearity between the fluorescence intensity and AFP concentration over 5 orders of magnitude (0.0005-5 ng/mL), and the detection limit reaches as low as 94.3 fg/mL. This method offers advantages of high sensitivity and reliability, wide detection range and versatile plasmonic chips, thus it presents an alternative for the technologies in biomedical analysis and clinical applications.

KEYWORDS: Metal-enhanced fluorescence (MEF); carbon nanodots (CDs); sandwich immunoassays; hybridization chain reaction (HCR); alpha fetoprotein (AFP) 2 ACS Paragon Plus Environment

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INTRODUCTION

In most of the fluorescence-based assays, fluorophore brightness (or quantum yield) and photostability are the major concerns to achieve sensitive results. In recent years, the use of metallic surface plasmon on metal nanostructures to enhance the brightness and photostability of fluorophores in the near-field, is offering some opportunities to increase the classical photophysical properties of the known fluorophores. Many reports have shown that surface plasmons affect emission of the near-field fluorophores, and increase their quantum yield by raising the radiative decay rate of the excited fluorophores, thus more or less enhancing their fluorescence emission1-5. This is known as metal-enhanced fluorescence (MEF) and has been reported for various fluorophores1-5 and quantum dots6-9 that are in close proximity to the nanostructures of some noble metals. Hybridization chain reaction (HCR) which was firstly reported by Dirks and coworker in 200410, is a new kind of isothermal nucleic acid amplification in vitro without heterotherm or other auxiliary enzymes. The reactant mainly contains chain initiators and two hairpin nucleic acid probe (H1 and H2) in the process of HCR. H1 and H2 are stably coexist in the solution and form DNA nanowires by self-assembly only in the presence of the chain initiators. After triggering by the trigger probe, hairpin structure H1 and H2 successively opened into the copolymer with multiple repeat unit, which is the product of alternating hybrid by H1 and H2, until the H1 and H2 are depleted to realize signal amplification. HCR has been applied as an important approach for DNA amplification10-15. Compared with other amplification reaction, 3 ACS Paragon Plus Environment


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HCR is attracting considerable interests because by this approach short chains are amplified by one-step response under isothermal conditions without auxiliary enzymes and complicated equipment16, 17. As an emerging fascinating fluorescent carbon material, carbon dots (CDs) are receiving more and more attention due to their unique properties, such as antiphotobleaching, small size, green synthesis, high biocompatibility, low toxicity, abundant and cheap raw materials18-25. It has been demonstrated that CDs have wide potential applications in bioimaging26, optoelectronic devices27, photocatalysis28 and so on. Recently, several chemosensors for detecting biomolecules29 and metal ions30, 31 have been developed based on the quenching and recovering of CDs fluorescence. The applications of CDs in biosensing, bioimaging and even theranostics, greatly depend on the fluorescence brightness of this material. However, CDs has low quantum yield32, which is not conducive to biomedical applications. Furthermore, there are few reports on CDs-based metal-enhanced fluorescence chemosensors and improvement of their luminescence. As a major serum glycoprotein that is mainly produced in liver cells of embryo, AFP disappears from the blood about two weeks after birth under normal circumstances, thus AFP concentration in serum of normal adults is much lower than that of infants33. In adults, the concentration of serum AFP of about 80% patients with liver cancer increases significantly and the positive rate of AFP is about 50% in germ cell tumor34–36. The concentration of serum AFP also appears increase to some extent in other gastrointestinal tumors, such as pancreatic cancer, lung cancer and liver cirrhosis34–36. Therefore, AFP has been used as a specific tumor marker for diagnosing hepatocellular cancer. Generally, AFP level exceeding the threshold of 25 4 ACS Paragon Plus Environment

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ng/mL in serum is suspected to be associated with the occurence of liver cancer or other related diseases37-41. Therefore, it is crucial to sensitively detect the concentration of serum AFP in highly reliable predictions. So far, many methods such as enzyme linked immunosorbent assay (ELISA)42, 43, fluoroimmunoassay44, flow injection chemiluminescence45, chemiluminescence enzyme immunoassay46, etc. have been used to detect AFP in clinical aspects. However, most of these methods suffer from significant false positives, enzyme engagement, low sensitivity, complex operation, sophisticated laboratories or harmful reagents. Herein, we first constructed a dual amplification fluorescence sensor based on immunohybridization chain reaction and metal-enhanced fluorescence of carbon dots (CDs) for detection of AFP. Scheme 1 shows the basic strategy of this assay. The target AFP molecules are captured by antibodies conjugated plasmonic slide, and the detection antibodies conjugated with an oligonucleotide initiator are used to bind to AFP captured on the slide. Then CDs tagged DNA hairpins (H1 and H2) are introduced to trigger hybridization chain reaction, in which DNA hairpins (H1 and H2) and oligonucleotide initiator are complementary. Then the copolymer with multiple repeat units is generated, until H1 and H2 are depleted to attain signal amplification. The interaction between CDs and the gold film, greatly improves the radiative decay rate, increases quantum yield and enhances the fluorescence of the CDs. Furthermore, hybridization chain reaction provides secondary amplification of fluorescence intensity. In addition, dual antibody sandwich method offers high specificity. To our knowledge, this is the first attempt to develop a dual-amplification fluorescence sensor by fully utilizing the potential of CDs as a novel fluorescence nanotag.

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Scheme 1. Schematic illustration for the detection of AFP with immuno-hybridization chain reaction (HCR) and metal-enhanced fluorescent of CDs. The capture antibody-coated plasmonic slide captures AFP of interest. Thereafter, detection antibody conjugated oligonucleotide initiators are introduced and bind to AFP targets. CDs tagged DNA hairpins are introduced to trigger HCR, in which DNA hairpins and oligonucleotide initiators are complementary. EXPERIMENTAL SECTION Materials and Reagents Carbon fibers were purchased from Shanghai Tansu Manufactory. Nitric acid (65%~68%) and sodium bicarbonate (NaHCO3) were purchased from local supplies. AFP and two different monoclonal mouse anti-AFP antibodies (capture antibody (Abc) and detection antibody (Abd) ) were obtained from Biocell Biotechnology Co. Ltd (Zhengzhou, China). Chloroauric acid (HAuCl4), 3-mercaptopropionic acid, 3-aminopropyltriethoxysilane (APTES), hydroxylamine hydrochloride (NH2OH.HCl), sodium borohydride (NaBH4), N-(3-dimethylaminopropyl)-N’ethylcarbodiimidehydrochloride (EDC), ammonium hydroxide (NH4OH, 25–28 wt%), 4morpholineethanesulfonic acid (MES), tris (2-carboxyethyl)-phosphine (TCEP), Nhydroxysuccinimide (NHS), 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfoN-hydroxysuccinimide ester sodium salt (sulfo-SMCC cross-linker), Cy3-labeled sheep anti-


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mouse IgG and analog proteins used for specificity investigation were purchased from Sigma Chemical Co. (USA). Ultrapure water (18.25MΩ·cm) made by Millipore system was used in all the experiments. All chemicals used in this work were of analytical grade. All the DNA oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. (China), and their sequences are as following:

Initiators sequence (DNAi): 5’-SH-CTGATAAGCTACAGGACATCGAATAGTC-3’ H1 sequence: 5’-NH2-TTTTTTTTATCGAATACAGGACTATTCGATGTCCTG-3’ H2 sequence: 5’-NH2-TTTTGTCCTGTATTCGATCAGGACATCGAATA-3’

Apparatus The morphology of CDs was characterized by transmission electron microscopy (TEM) (JEM100CXII, JEOL, Japan). The morphology was also observed from a Zeiss Sigma scanning electron microscope (SEM). Fourier transform infrared (FT-IR) spectra were performed by a Thermo Nicolet 360 FT-IR spectrophotometer. An inverted fluorescence microscope (Ti_U, Nikon, Japan) with a CCD camera (Nikon DS-Ri1) was used to capture fluorescence images. Gel electrophoresis was conducted on an IS-2200 gel imager (Alpha Innoteth) with a CCD camera (Nikon DS-Ri1) and JY04S-3C gel imager (Beijing, China).

Synthesis of plasmonic gold films on glass slides Plasmonic gold films were synthesized according to a previous report47. First of all, the glass slides were immersed in 3 mM HAuCl4, then 110 L NH4OH was introduced to the above 7 ACS Paragon Plus Environment


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solution for 1 min under intense stirring. After washing the above glass slides with water, the glass slides with Au clusters were immersed into freshly prepared NaBH4 (1 mM) to complete the seeding step with stirring. Thereafter, the glass slides with Au seed were immersed into the same amount of HAuCl4 and NH2OH for 5 min under shaking after the again washing and incubated 15 min to complete the growth of golden island film. The glass slides were blown dry with mild nitrogen after washing. Finally, the substrate was covered with a polydimethylsiloxane (PDMS) film with array-aperture (3 mm in diameter). The morphology of the plasmonic gold films was characterized by SEM.

Synthesis of carbon nanodots (CDs) Carbon nanodots were synthesized by chemical oxidation32. 10 M boiled nitric acid solution was introduced into 0.4 g carbon fiber with continuous stirring. The reaction was refluxed for 4 h. NaHCO3 was added into the obtained mixture under stirring until the mixture was neutral, it was dialyzed for some days after filtering by 0.22 μm membrane filters. Then the above solution was ultrafiltered through Millipore centrifugal filter devices with five different molecular weight cutoff membranes in sequence (100, 50, 30, 10, and 3 kDa). Finally, the aqueous solution of CDs was obtained.

Conjugation of capture antibodies on slides The plasmonic slides were introduced to 20 mM 3-mercaptopropionic acid for 12 h in ethanol at normal temperature. 12.8 mg EDC and 8 mg NHS were introduced to activate the plasmonic substrates in MES (0.1 M, pH 5.0) under vigorous shaking for 60 min at room temperature 8 ACS Paragon Plus Environment

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after washing with ethanol. After washing, 10 μg of capture antibody was added in a plasmonic substrate overnight with gentle shaking at room temperature. Then washing again, and 1 mL 1% BSA−PBS was added to the substrates for blocking at 37 °C for 30 min with continuous shaking for further use. The piranha solution (VH2O2/VH2SO4=1:3) was added to the normal slides (with no gold films) for 30 min at room temperature, then they were flushed with plenty of deionized water and blown dry with mild nitrogen. The cleaned slides were silanized with introduction of 2% 3aminopropyl-triethoxysilane silane solution (Vmethanol/Vwater/Vacetic

acid

= 95:5:0.1) and

incubated for 30 min under continuous shaking. Afterwards, the slides were activated with 1 mM SMCC for 1 h at 25 °C with shaking. After washing, 10 μg of capture antibody reduced with DL-Dithiothreitol (DTT) was added to the activated slide overnight with gentle shaking at room temperature. Finally, 1 mL 1% BSA−PBS was added to the slides for blocking at 37 °C for 30 min with continuous shaking for further use.

Conjugation of CDs-DNA (CDs-H1 and CDs-H2 ) and CDs-Abd The CDs were modified with DNA and detection antibody of AFP to prepare CDs-DNA and CDs-Abd which are applied for sandwich immunoassay strategy and HCR. 5 μL CDs solution was added into 100 mM EDC and 1 OD DNA ( H1 or H2 ) in 1 mL PB buffer (0.01 M, pH 6.8) at 25 °C and the mixture was incubated for 1 h under continuous shaking. Afterwards, 100 mM EDC was added successfully with vigorous stirring at 25 °C for 4 h. At last, the CDs-DNA was ultrafiltered and washed with PB (0.01 M pH 7.2) several times through Millipore centrifugal filter devices. The product was stored at 4 °C for further use. 9 ACS Paragon Plus Environment


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100 mM EDC and 100 mM NHS were introduced to 5 μL CDs solution for activation in 1 mL PB (0.01 M, pH 6.8) at 25 °C and were incubated for 30 min. Then, the above mixture were separated by refrigerated centrifugation and washed with PB (0.01 M, pH 7.2) several times. Afterwards, the reactant were resuspended in 1 mL PB (0.01 M pH 7.2) and added to 50 μg anti-AFP detection antibody for 4 h under shaking at 25 °C. Finally, 1 mL 1% BSA−PBS were introduced to the CDs-Abd for blocking at 37 °C under gentle shaking for 30 min. The product was stored at 4 °C for further use.

Conjugation of oligonucleotide initiators to detection antibodies (Abd-DNAi) Oligonucleotide initiators with 3’ thiol groups in 1 × SSC (pH 7.4) were added in 3.2 mg/mL tris(2-carboxyethyl)-phosphine (TCEP) to reduce disulfide bond. The reaction last for 1 h at room temperature. Afterwards, the solution was washed and centrifuged to recover reduced initiators. The above substrate was introduced to 11.5 mg/mL detection antibody and 1 L of 5 mg/mL sulfo-SMCC cross-linker for 2 h at 25 °C. Purification was realized by incubation with a desalting column equilibrated with PBS to remove excess cross-linkers. The same concentration of desalted antibodies with cross-linkers and reduced initiators were incubated overnight at 4 °C under vigorous shaking. Initiator coupling antibodies were separated and enriched by membrane spin columns and stored at 4 °C for further use.

Investigation of the feasibility of this method Oligonucleotide initiators and DNA – H1 and H2 were respectively heated to 95 °C for 2 min, immediately annealed and kept at the room temperature. Afterwards, oligonucleotide initiators 10 ACS Paragon Plus Environment

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and DNA (H1 and H2) were incubated at different molar ratio for 1 h at 25 °C. In the meantime, Abd-DNAi and CD-DNA (CD-H1 and CD-H2) were also incubated at different molar ratios for 60 min at 25 °C. The above reactions were analyzed by agarose gel electrophoresis.

AFP detection in buffer solution Firstly, in order to verify the feasibility of the as-proposed method, AFP detection was operated in buffer solution. Various concentrations of AFP were added to the plasmonic and normal immunoslide, then the substrates were incubated for 15 min at 37 °C. After rising, 15 L AbdDNAi were introduced to the both slides for 20 min at 37 °C following thorough washing to remove excess Abd-DNAi. Thereafter, 15 L CDs-H1 and CDs-H2 were heated to 95 °C for 2 min, immediately annealed and kept at the room temperature. Furthermore, 15 L above CDsH1 and CDs-H2 solutions at a 1:1 molar ratio were incubated with the above plasmonic and normal immunoslide, respectively for 25 min at 25 °C. The surfaces were washed several times to remove excessive CDs-H1 and CDs-H2. Meanwhile, 15 L CDs-Abd were incubated with the immunoslide captured AFP to investigate the influence of HCR on the variation of fluorescence intensity. The reaction lasted for 20 min at 37 °C. Finally, the substrates were washed several times to remove excessive CDs-Abd.

Specificity investigation The specificity of the method was verified by the evaluation of 5 ng AFP and 500 ng other proteins including BSA, CEA, PSA, Thrombin, IgG are operated under the same conditions as in buffer. Afterwards, the results were compared with the data for AFP. 11 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION

The morphology of the plasmonic slide To realize the metal-enhanced fluorescence, the plasmonic slide was synthesized according to the reported procedure with slight modification47, and seed growth method was used to synthesize the tortuously plasmonic slide with abundant nanogaps. The morphology of the substrate was characterized by scanning electron microscopy (SEM), which revealed that the plasmonic slide surface was uniformly covered with tortuous, elongated, nanoscale gold islands. Importantly, the substrates possess properties of plasmon resonances (Figure 1A).

Figure 1. (A) SEM micrograph of the plasmonic slide showing the nanoscale gold island morphology. (B) TEM micrograph of the as-prepared CDs. FTIR spectra (C) and Zeta potentials (D) of the as-prepared CDs.


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Characterization of CDs High-quality hydrophilic CDs were synthesized according to chemical oxidation method32. The TEM micrograph of CDs (Figure 1B) shows that the diameter of CDs is 3.7 ± 0.8 nm. The FTIR spectrum exhibits strong absorption at 1728 and 3415 cm-1 which should be assigned to C = O and O-H stretching vibrations respectively, and the peaks at 1400 and 1242 cm-1 are ascribed to COO- symmetric stretching vibration and C-O stretching vibration, respectively (Figure 1C). Therefore, the IR spectrum clearly shows that the CDs possess rich amount of carboxyl groups. Moreover, Figure 1D shows zeta potentials of CDs (−36.8 mV), which further prove the existence of abundant carboxyl groups of CDs. Under the excitation at 300 ~ 500 nm UV irradiation or visible light, the aqueous solution of CDs clearly show excitation wavelength dependent fluorescence emission, which is consistent with the previous report32 (Figure S1). When it is excited with 360 nm UV irradiation, the as-prepared CDs exhibit a strong fluorescence intensity and slightly wide emission spectrum (peak ~515 nm). Therefore, excitation at 360 nm is employed throughout the assays when using CDs as the fluorescence tags.

Verification of the conjugation of Abc on the plasmonic slide and normal slide The experiments were designed to verify that Abc and Abd were successfully conjugated on both plasmonic and normal slide. Cy3-labeled sheep anti-mouse IgG respectively reacted with Abc-plasmonic slide and Abc-normal slide with gentle agitation for 30 min. The plasmonic slide and normal slide without conjugating Abc and conducted with identical reaction procedure were used as the controls. After rinsing, the substrates were tested under a 13 ACS Paragon Plus Environment


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fluorescence microscope. The fluorescence of Cy3 was obvious on both plasmonic slide and normal slide, but the controls (without Cy3) exhibited no fluorescence (Figure S2). These results show that Abc was successfully conjugated to both plasmonic and normal slide.

Characterization of CDs-DNA and CDs-Abd The success of conjugation reactions were monitored by garose gel electrophoresis. As can be seen from Figure S3A, the charge and relative molecular mass of CDs-DNA (H1 or H2) are higher than that of CDs, but the charge influence on its migration speed is greater than that of molecular weight. So the migration of CDs-DNA is faster than CDs in gel electrophoresis. However, for CDs-Abd and CDs, the influence of molecular weight on its migration speed is greater than that of the charge, thus the migration of CDs is faster than CDs-Abd in gel electrophoresis (Figure S3B). We found that the zeta potentials of CDs-DNA (CDs-H1: -42.1 mV, CDs-H2: -42.3 mV) and CDs-Abd (-43.1 mV) are different from bare CDs (-36.8 mV) (Figure S3C). In addition, the fluorescence spectra of the CDs show slight blue shift upon its

binding with H1, H2 and Abd (Figure S3D), which is ascribed to its surface structure change after conjugating with DNA and Abd, leading to slightly increased energy gap of the materials. Therefore, these data demonstrate that the preparation of CDs-DNA and CDs-Abd is successful.

Verification of oligonucleotide initiators conjugating to detection antibodies (Abd-DNAi) The success of conjugation reactions was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The electrophorogram shows that the position of Abd is lower than that of Abd-DNAi due to the larger relative molecular mass of Abd-DNAi, indicating 14 ACS Paragon Plus Environment

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the successful coupling of Abd-DNAi (Figure S3E). Figure S3E also demonstrates that the average number of oligonucleotide initiators per Abd is approximately 1 ~ 2.

Verification of the feasibility of this method The HCR took place successfully with oligonucleotide initiators and DNA (H1 and H2), and the reaction was barely affected by initiators coupled Abd and DNA (H1 and H2) coupled CDs (Figure S4A). We chose 1:10:10 molar ratio of Abd-DNAi/CD-H1/CD-H2 to conduct the subsequent experiments. Furthermore, the as-proposed detection system was investigated under different conditions (Abd without conjugating oligonucleotide initiators reacted with CD-H1 and CD-H2; Abd-DNAi reacted with CD and without conjugating H1 and H2; AbdDNAi reacted with CD-H1 and CD-H2) to optimize the fluorescence emission from Abcplasmonic slide. As shown in Figure S4B, the substances without participating in the reaction could be removed from the system and hardly affects the fluorescence intensity of the assay. On the whole, the as-proposed detection system is feasible.

AFP detection in buffer solution The detection of AFP by immuno-HCR-mediated strategy was first conducted on the plasmonic slide and normal slide in buffer. The detection was evaluated by adding different contents of AFP under the optimal reaction conditions (Figure S5). AFP could be enriched on the substrate with the sandwich structure formed by Abc and Abd. As shown in Figure 2, the fluorescence intensities of the system increase with the increase of AFP concentration. The fluorescence emission on the plasmonic slide shows the enhancement of up to ~7-fold over the 15 ACS Paragon Plus Environment


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normal slide owing to the effect of metal enhancing fluorescence at the same concentrations of AFP. Importantly, an exceptional wide linearity between the fluorescence intensity and AFP concentration (from 0.0005 to 5 ng/mL) on the plasmonic slide was obtained (Figure 2D). The corresponding linear equation is F=50.5374 logC+197.8480 (C is AFP concentration and F is the fluorescence intensity of the system), and the coefficient of correlationt is 0.992. The limit of detection of this approach reaches 94.3 fg/mL. This result is comparable to the reported approaches for AFP assay (Table 1)48-55. Since the detection limit of this approach is much lower than the threshold value for AFP in serum, the as-proposed immunoassay possess great potential for clinically diagnosing AFP related diseases at early stage. The exceptionally high sensitivity of this approach is ascribed to the intensified fluorescence of CDs through MEF and further signal amplification by HCR. To our knowledge, the reports on CDs-based metalenhanced fluorescence sensors are few. Although some report investigated the influence factors for fluorescence enhancement and improved quantum yield of CDs by different metal nanomaterials, the applications of CDs based MEF are limited and mainly focus on optoelectronic devices56-59. In this sense, we not only constructed a fluorescence sensor based on metal-enhanced fluorescence of CDs for the detection of AFP, but also provided a secondary amplification of fluorescence by HCR. This


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240 HCR on plasmonic slide HCR on normal slide

200

Intensity

160 120 80 40 0 0.005

0.0005

0.5

0.05

5

AFP (ng/ml)

250

250

HCR on plasmonic slide HCR on normal slide

200

200

150

150

Intensity

Intensity

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100 50

F=50.5374 logC+197.8480 R2=0.9916

100 50 0

0 0.0005

0.005

0.5

0.05

5

-4

-3

AFP (ng/ml)

0 -1 -2 Log([AFP] (ng/ml))

1

Figure 2. (A) Fluorescence diagram of the system upon the addition of increasing amount of AFP of HCR-plasmonic slide and HCR-normal slide. (B) The quantification of the fluorescence intensity in A. (C) The histogram of contrastive fluorescence intensity between HCR on plasmonic slide and normal slide. (D) Calibration curve for AFP detection by the asproposed detection system (RSD < 5%). method offers achievement of high sensitivity and reliability, wide detection range and versatile plasmonic chips and paves a new avenue in biomedical analysis and clinical diagnosis

Table 1. Comparison of this approach and some other known methods for AFP assay

Method Homogeneous immunoassay based

Detection limit

Linear range

Reference

714 fM

/

48

0.5-10 µg/mL

49

single gold nanoparticle counter assay

(51.9 pg/mL)

Oligonucleotide-linked

0.74 ng/mL

immunosorbent assay (OLISA) 17 ACS Paragon Plus Environment


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Magnetic bead-based microfluidic

12.5 ng/mL

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12.5-800 ng/mL

50

SELEX chip Sandwich-type electrochemiluminescence immunosensor based magnetic

0.2 pg/mL

0.0005-5 ng/mL

51

capture probes and quantum dots Sandwich immunoassay and surface

4 pg/mL

0.002-20 ng/mL

52

/

0−350 ng/mL

53

0.317µg/dL

0.317-35 µg/dL

54

plasmon field-enhanced fluorescence Carbon dot-linked immunosorbent assay Ratiometric fluorescent immunoassay based on carbon dot-doped silica nanoparticles Photoelectrochemistry immunoassay

0.2 pg/mL

0.5-100 ng/mL

55

based on graphitic carbon nitride This method

94.3 fg/mL

0.0005-5 ng/mL

/

In the meantime, the same process for the assay of AFP using CDs tagged Abd on the plasmonic slide was performed and used for a comparative study in order to verify the influence of HCR. Notably, the assay of AFP without HCR on the plasmonic slide shows much weaker signal in the range of 0.0005-5 ng/mL (Figure 3). It shows that the assay of AFP with HCR on the plasmonic slide provides a photoluminescence enhancement of up to ~5.5-fold compared to that without HCR on the plasmonic slide. Furthermore, the assay of AFP without HCR on the normal slide was also performed with CDs-Abd. As shown in Figure 4, the comparison shows that the plasmonic substrate provides ~3.2-fold fluorescence enhancement of CDs over the normal glass substrate when HCR is not conducted for the both cases. More interestingly, when


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HCR is applied using CDs as the fluorescence tag, the same plasmonic slide shows ~17.2-fold signal amplification comparing with normal slide where no HCR is conducted (Figure 5 and Figure S6). These results clearly illustrate that the combination of HCR and MEF is advantageous in the design of the biosensors.

250

HCR on plasmonic slide Plasmonic slide

Intensity

200 150 100 50 0 0.0005

0.005

0.05

0.5

5

AFP (ng/ml)

Figure 3. (A) Fluorescence photograph of the reaction chambers for the as-proposed immunoassay upon the addition of increasing amount of AFP of HCR-plasmonic slide and plasmonic slide. (B) The histogram of contrastive fluorescence intensity between HCRplasmonic slide and plasmonic slide. 80

Plasmonic slide Normal slide

70 60

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50 40 30 20 10 0 0.0005

0.005

0.05

0.5

5

AFP (ng/ml)

Figure 4. (A) Fluorescence photograph of the reaction chambers for the as-proposed immunoassay upon the addition of increasing amount of AFP on plasmonic slide and normal slide. (B) The histogram of contrastive fluorescence intensity between plasmonic slide and normal slide.



150

100

Intensity

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Hybridization chain reaction on plasmonic slide Plasmonic slide Hybridization chain reaction on normal slide Normal slide

50

0

Figure 5. The histogram of contrastive fluorescence intensity in HCR on plasmonic slide, plasmonic slide, HCR on normal slide and normal slide when the sensors were applied for 0.05 ng/mL AFP. Specificity evaluation In order to evaluate the specificity of the method for AFP assay, we investigated the possible interference of some analog proteins. The target molecules are specifically captured onto the substrate due to the antigen-antibody recognition, the coexisting species should not contribute much to the signal response. However, a variety of biologically relevant species including BSA, PSA, Thrombin, CEA and IgG, were investigated to consider their influences on the assay (Figure 6). In the experiment, the concentration of all the inspected coexisting analogs is 500 ng/mL and the concentration of AFP is only 5 ng/mL for comparison. As can be seen from Figure 6, no obvious fluorescence is observed when the analogs are 100-fold higher than AFP, because their fluorescence intensities are almost the same as the blank. Therefore, these analogs exhibit no obvious interference on AFP detection. In conclusion, the as-proposed method can be potentially applied in highly specific detection of AFP in complex samples.


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Fluorescence Intensity

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240 220 200 180 160 140 120 100 80 60 40 20 0

AFP

BSA CEA

PSA Thrombin IgG

Blank

Figure 6. Comparison of the fluorescence intensities of the assay system (buffer solution) in the presence of AFP, BSA, PSA, Thrombin, CEA and IgG, respectively.

Analysis of AFP in serum samples The reliability of this method in clinical applications was assessed by the assay of real samples. We measured AFP contents in whole serum of 11 cancer patients by this approach. Considering the AFP levels from the actual samples of cancer patients may exceed the upper limit of the linear response range, sample 1-5 and 6-11 were diluted 103-fold and 106-fold with standard serum, respectively. Table 2 shows the results of the AFP levels detected using this method, and as a comparison, the data obtained by traditional chemiluminescence immunoassay (CLIA) is also shown. As shown in Table 2, the results by the two approaches are highly consistent. In addition, the AFP level of a healthy donor detected using this method is also shown in Table 2, and it is significantly lower than that of all the samples from the cancer patients. Interestingly, sample 1 and 2 which have close AFP levels and slightly lower than the threshold value, exhibit pathological difference – patient 1 and 2 are diagnosed as rectal carcinoma and hepatocellular



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carcinoma, respectively. This result suggests that certain cases of hepatocellular carcinoma may exhibit low levels of AFP, thus combinational detection of different biomarkers for carcinomas is vital importance because the detection of a single biomarker in serum may not provide sufficient sensitivity or specificity for diagnosis. However, in the cases of high levels of AFP (sample 3-11 as shown in Table 2), the nine patients are pathologically diagnosed as hepatocellular carcinoma. It is worth noting that the variation between the nine cases is tremendous since the AFP level normally depends on the tumor size and cancer stage. Table 2.

AFP levels (ng/mL) in human sera obtained by this method and traditional CLIA method.

Sample No. a

CLIA b 21.6

This method c (mean ±SD) 22.8 ±1.3

Pathological diagnosis d RC

1 2

21.8

22.9 ±1.2

HCC

3

93.1

90.0 ±4.5

HCC

4

94.9

104.1 ±5.1

HCC

5

144.2

151.9 ±7.0

HCC

6

714.4

763.7 ±38.2

HCC

7

1860.2

1818.9 ±90.2

HCC

8

5105.1

4963.7 ±247.1

HCC

9

7080.3

7448.5 ±372.3

HCC

10

18381.6

18162.9 ±907.5

HCC

11

41703.6

40594.3 ±2012.7

HCC

Healthy donor

/

0.2 ±0.02

/

Threshold value

25

a

Samples 1-11 are whole serum from cancer patients with abnormal contents of AFP where 15 and 6-11 were diluted 103-fold and 106-fold respectively by standard serum prior to the assay. 22 ACS Paragon Plus Environment

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b

The values were measured by chemiluminescence immunoassay (CLIA) method using standard AFP kit. c Each sample was measured thrice (RSD< 5%). d HCC: hepatocellular carcinoma; RC: rectal carcinoma. CONCLUSIONS In summary, we successfully constructed a dual amplification fluorescence sensor based on the combination of immuno-hybridization chain reaction and metal-enhanced fluorescence of CDs for detection of alpha fetal protein. When HCR is applied using CDs as the fluorescence tag, the plasmonic slide shows ~17.2-fold total signal amplification comparing with normal slide where no HCR is conducted. Owing to the significant fluorescence enhancement, there is wide linearity over 5 orders of magnitude between the fluorescence intensity and AFP concentration (0.0005-5 ng/mL), and the detection limit for AFP reaches as low as 94.3 fg/mL in buffer system. More importantly, the platform provides highly sensitive and specific results for the assay of real samples, thus validating its potentiality in clinical analysis that serves as a new and significant alternative in clinical diagnosis based on biomarker detection. Moreover, the flexible construction of the sensor also provides the possibility to develop other sensing systems through specific recognition pairs based on immuno-hybridization chain reaction and metal-enhanced fluorescence for other targets.

ASSOCIATED CONTENT Supporting Information Excitation dependent fluorescence spectra of the CDs; Fluorescence images of different substrates after reacted with Cy3-labeled sheep anti-mouse IgG; Characterization of CDs-DNA



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and CDs-Abd and verification of oligonucleotide initiators conjugating to detection antibodies by garose gel electrophoresis, zeta potentials, fluorescence spectrum and SDS-PAGE; Verification of the feasibility of this method by agarose gel electrophoresis image and fluorescence imaging; The comparison of fluorescence intensities in the presence of different contents of AFP under optimal reaction conditions (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: 0086-27-68756759. Fax: 0086-27-68754067. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81572086, 81772256 and 21535005) and Wuhan Municipal Basic Research Project (2016060101010052).

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