DOI: 10.1002/chem.201405884
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& Analytical Methods
Rolling-Circle Amplification Detection of Thrombin using SurfaceEnhanced Raman Spectroscopy with Core-Shell Nanoparticle Probe Xuemei Li,*[a] Linlin Wang,[a, b] and Chunxiang Li[b]
thrombin, the aptamer sequence was released; this allowed the remaining single-stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0 10 12 to 1.0 10 8 m and a detection limit of 4.2 10 13 m, and showed good performance in real serum samples.
Abstract: An ultrasensitive surface-enhanced Raman spectroscopy (SERS) sensor based on rolling-circle amplification (RCA)-increased “hot-spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO2 core-shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer-by-layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of
Introduction
nanowires and nanochips have been widely developed for the detection of proteins,[16] nucleic acids,[17] pathogens[18] and small bioactive molecules.[19] In order to improve Raman signals, signal amplification methods based on rolling circle amplification (RCA),[20, 21] and DNA recycling[22, 23] have been introduced in SERS bioassays. Among these strategies, gold nanoparticles (AuNPs) or silver nanoparticles (AgNPs) were used as supporting substrates, which have relatively weak enhancing ability and the Raman reporters are vulnerable to detachment from the surface of metallic nanoparticles upon direct exposure to the environment, leading to poor reproducibility of SERS sensing. Therefore, a protective shell has been used to surround the metallic SERS substrates and the Raman reporter, preventing them from leaching-out of the Raman reporters and improving their water solubility and stability as well as the reproducibility of the SERS signals.[24, 25] Very recently, gold nanostar@Raman label@SiO2 core-shell nanoparticles have been used as SERS reporters for the detection of adenosine triphosphate (ATP).[26] However, there have been few reports on the combination of signal amplification strategies with coreshell nanoparticles for SERS sensing. Thrombin plays a crucial role in many physiological and pathological processes, such as blood coagulation, thrombosis, inflammation, angiogenesis, tumor growth and metastasis. Thrombin can be used as a therapeutic target and a biomarker for diagnosis of some diseases, such as pulmonary metastases and diseases associated with coagulation abnormalities.[27] Numerous assays have been developed for the detection of thrombin by using nucleic acid aptamers.[28] In the present work, gold nanoparticle@Raman label@SiO2 nanoparticles are produced by sandwiching the Raman reporter between a gold nanoparticle core and a thin silica shell by
Due to their relatively low level in the early stage of disease, ultrasensitive assays of disease-marker proteins are of tremendous importance in clinical settings for disease diagnosis, molecular diagnostics and biomedical development.[1, 2] A number of techniques, including fluorescence,[3, 4] colorimetry,[5, 6] electrochemistry,[7, 8] chemiluminescence,[9] surface plasmon resonance (SPR)[10, 11] and surface-enhanced Raman scattering (SERS),[12, 13] have attracted substantial research efforts toward the detection of nucleic acids and proteins. Especially, there has been great interest in the development of signal amplification strategies combined with these detection tools to improve detection sensitivity.[14] Recently, there has been great interest in the development of SERS-based analytical techniques for chemical and biomedical applications, since it can provide molecular-level identification in samples, nondestructive and noninvasive detection, and enhancement that is several orders magnitude greater than previously reported methods.[15] Various SERS plasmonic platforms ranging from nanoparticles to nanopost arrays, [a] Prof. Dr. X. Li, L. Wang Shandong Provincial Key Laboratory of Detection Technology of Tumor Markers, School of Chemistry and Chemical Engineering Linyi University, Linyi 276005 (P. R. China) E-mail: [email protected] [b] L. Wang, Dr. C. Li State Key Laboratory Base of Eco-chemical Engineering College of Chemistry and Molecular Engineering Qingdao University of Science and Technology Qingdao 266042 (P. R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201405884. Chem. Eur. J. 2015, 21, 1 – 7
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Full Paper a layer-by-layer method. RCA is an isothermal, enzymatic process mediated by some DNA polymerases in which long single-stranded DNA (ssDNA) molecules are synthesized on a short circular ssDNA template with a single DNA primer. With the core-shell nanoparticles as SERS tags, a new SERS-based strategy that is focused on increasing the number of “hotspots” in the signaling system by RCA was developed for detection of thrombin. Taking advantage of the high enhancement factor and stability of the core-shell nanoparticle probe and the signal-amplification effects of RCA, the detection limit of the system for thrombin was found to be as low as 0.42 pm.
Characterization of the core-shell SERS nanoparticles Figure 1 A and B show the TEM images of the AuNPs before and after coating of SiO2 layer, respectively. The average diameters of AuNPs were estimated to be 25 nm. Growth of the silica shell was achieved by Stçber’s method and the thickness of the silica shell was 3.8 nm according to our measurements.
Results and Discussion Principle of the sensing strategy Figure 1. TEM images of: A) the bare AuNPs, and B) AuNP@RBITC@SiO2 nanoparticles. Inset: the magnification of one sphere.
The configuration and operation principle for the detection of thrombin by the RCA–SERS strategy with core-shell tags is depicted in Scheme 1. The double-stranded hybrids of S1 and S2 were immobilized on the MB through amide-bond formation between the COOH group on the MB and the NH2 on S1. In the presence of thrombin, the aptamer sequence S2 bound to thrombin and folded to a complex structure, which was released into solution. After the circular template was formed by incubating the padlock probe and T4 ligase with the remained S1, which acted as primers, the RCA reaction was initiated by adding Phi29 DNA polymerase and deoxynucleotides (dNTPs).[7] The RCA process produced long ssDNA in situ for the attachment of a large number of S4–AuNP@RBITC@SiO2 probes. The modified MBs were then measured with a Raman spectrometer. The intensity of the Raman signal reflects the amount of thrombin in this detection system.
Figure 2 A reveals the UV–visible absorption spectra of the bare AuNPs, AuNP@RBITC@SiO2, DNA, and DNA conjugated AuNP@RBITC@SiO2. The bare AuNPs exhibited the characteristic absorbance at about 520 nm while a wide absorbance peak from 520 to 620 nm was seen for AuNP@RBITC@SiO2. The redshift wide peak may be ascribed to rhodamine B isothiocyanate (RBITC) and the higher refractive index of SiO2 with respect to that of water. After modification with S4, an obvious peak at 260 nm, which corresponded to that of DNA, was observed. The SERS tag nanoparticles were further characterized with fluorescent spectra. As shown in Figure 2 B, free RBITC in solution exhibited an obvious peak at 580 nm (line a), while the fluorescence intensity of AuNP@RBITC@SiO2 decreased sharply, due to the quenching effect of AuNP and encapsulation effect of SiO2 shell. SERS characterization
Scheme 1. Schematic illustration of the detection of thrombin by RCA–SERS strategy with core-shell tags. The structure of AuNP@RBITC@SiO2 is shown in the square frame.
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It has been well established in the literature that both fluorescent and nonfluorescent dyes can be used as resonant Raman reporters, because fluorescence emission is efficiently quenched by gold particles, not interfering with Raman measurement.[29] Silica-encapsulated RBITC–AuNP tags prepared by layer-by-layer method were used as SERS reporters as the protective shells can prevent desorption of Raman reporters and adsorption
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Full Paper Signal amplification of RCA To prove the signal amplification of the RCA strategy, the control experiment without T4 ligase was carried out as shown in Figure 4. In the absence of target DNA (Figure 4 a) or T4 ligase (Figure 4 b), the RCA reaction did not take place. There were very small peaks in the spectra, which might be due to the nonspecific adsorption of the SERS tags on the surface of the MBs. When thrombin was present it interacted with its aptamer, leading to the dissociation of the duplex DNA. The primer S1 was free to hybridize with the padlock DNA. After rollingcircle amplification was carried out, each primer would have created a long ssDNA that hybridized with a number of S4– AuNP@RBITC@SiO2. The longer the ssDNA, the more probe tags were attached to it. In the RCA reaction, the length of the ssDNA product is related to the concentration of polymerase, dNTPs, reaction temperature and time. In order to control the length of RCA product consistently in all assays, the RCA reaction was carried out for 1 h in the same reaction conditions. Thus, each protein molecule recognized by S4–AuNP@RBITC@SiO2 contains a large number of hot-spot groups.[32] The differences in the SERS spectra are the result of the different amounts of hot-spots among the arrays. The intensity of the Raman signal reflects the amount of thrombin in this detection system and the strongest Raman band at 1642 cm 1 was used for the quantitative evaluation of thrombin.
Figure 2. A) UV spectra of: DNA (a), RBITC (b), S4-AuNP@RBITC@SiO2 (c), AuNP@RBITC@SiO2 (d) and AuNPs (e). B) Fluorescence-emission spectra of: RBITC isothiocyanate in solution (a), AuNP@RBITC@SiO2 core-shell nanoparticles (b), and blank (c).
of external species. As shown in Figure 3, the laser excitation of the samples provided discrete vibrational peaks at 1349, 1506 and 1645 cm 1, which were attributed to the ring C C stretching vibrations of rhodamine.[30] The peaks at 1200 and 1273 cm 1 were ascribed to the in-plane benzene v9 mode and the in-plane C H or C C H bending mode, respectively.[31] The intensity of the Raman signal reflects the amount of thrombin in this detection system and the strongest Raman band at 1642 cm 1 was used for the quantitative evaluation of thrombin. Figure 4. SERS spectra obtained from RCA detection of thrombin: in the absence of the thrombin (a), in the absence of T4 ligase (b), RCA detection of 10 nm thrombin (c).
Characterization of the core-shell SERS nanoparticles: sensitive detection of thrombin Under the optimum conditions, the sensitivity of the present method was investigated upon addition of different concentrations of thrombin as shown in Figure 5. Improved SERS signal was observed with the increase of thrombin. With the measurement of the normalized Raman intensity of the 1645 cm 1 peak, the change of normalized Raman intensity was quantitatively analyzed with the concentration of thrombin. As shown in Figure 5 b, the normalized Raman intensity was a good linear fit to the logarithm of thrombin concentration in the range from 1.0 10 12 to 1.0 10 8 m. The regression equation was DI = 0.243 log C + 3.23276 (DI is the Raman intensity sub-
Figure 3. SERS spectra obtained from free AuNP@RBITC@SiO2 core-shell nanoparticles. The as-prepared nanoparticles were 1000 times diluted (a), 50 times diluted (b) and not diluted (c). Inset: molecular structure of RBITC. Chem. Eur. J. 2015, 21, 1 – 7
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Full Paper tracted the blank, C is the concentration of thrombin), and the regression coefficient of the linear curve was R2 = 0.983. A detection limit of 4.2 10 13 m of thrombin was estimated using 3s, which is more sensitive than previously reported SERS detection of thrombin with 4-aminobenzenethiol (4-ABT)-encoded gold nanoparticle hot-spots.[33]
weights and isoelectric points, including human serum albumin (HSA), bovine serum albumin (BSA), myoglobin, lysozyme, and trypsin. As shown in Figure 6, Even at a concentration as high as 10 nm, the responses from the interferences are very low and close to the background. This demonstrates that the RCA–SERS based biosensor is highly specific and has a good selectivity for the detection of thrombin from other proteins.
Figure 6. The selectivity of the proposed protein assay system toward 1.0 nm thrombin. Five non-target proteins at high concentration (10 nm) were used to assess specificity.
Application of the strategy in biological sample To investigate the applicability of the probing methods in a real biological environment, human blood serum (HBS) was selected. Serum samples spiked with different concentrations of thrombin were analyzed with our method. As presented in Table 1, a series of five parallel measurements of samples yielded a relative standard deviation less than 7.3 %, recoveries were obtained with 94–108 %. These results indicate that the proposed assay components do not cross-react with the complex matrix of serum.
Figure 5. A) SERS spectral responses with different concentration of thrombin: 0 (a), 1.0 pm (b), 5.0 pm (c), 10 pm (d), 50 pm (e), 100 pm (f), 500 pm (g), 1.0 nm (h) and 10 nm (i). B) Corresponding peak intensities at 1642 cm 1 for RCA–SERS detection of thrombin with varying concentrations. The error bars are standard deviations across triplicate assays.
Table 1. Determination of thrombin in serum samples.
Notably, the sensitivity of the current method had increased by one or two orders of magnitude as compared to our previously reported method,[34, 35] and was also comparable with or even exceeded the reported detection limit by the RCA-based electrochemical method[36] and by the RCA-based chemiluminescent method.[37] The high sensitivity of the present method could be attributed to the high effect of RCA as compared with the assay without RCA (details are shown in Scheme S1 and Figure S2 in the Supporting Information). Moreover, the highly sensitive response of the assay for DNA detection displayed satisfactory reproducibility. The standard deviation of eight duplicated tests at 10 pm was found to be less than 7.6 %.
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Detection [pm]
RSD [%] n=5
Recovery [%] n=5
1 2 3 4 5
5.0 20 100 500 1000
4.7 21.6 95.2 530.7 981.2
6.6 7.3 5.9 5.2 6.7
94 108 95 106 98
In summary, we have developed a sensitive SERS sensor for detection of thrombin, in which AuNP@RBITC@SiO2 was employed as the nanotag and RCA as enhancement strategy to further increase hot-spots. The SERS sensor exhibited good specificity and high sensitivity toward thrombin with a limit of detection of 0.42 pm. Encapsulation of a large number of RBITC molecules inside the sandwich-structured nanoparticles contributed to the high sensitivity. Furthermore, RCA can
Control experiments were also performed to examine the selectivity of these probing methods. Some proteins were chosen as possible interference, based on their molecular &
Spiked [pm]
Conclusion
Selectivity of the present strategy
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Full Paper solution under rapid stirring, and the mixture was allowed to equilibrate for 20 min. Then, 3 mL of PAA solution (4 g L 1), which had been adjusted to pH 7.0 with 0.1 m NaOH, was added dropwise with vigorous stirring and stirred for 3 h. The solution of polymerencapsulated nanospheres was centrifuged twice to remove excess polymer molecules and redispersed into water. A coupling agent, 3-aminopropyltrimethoxysilane, was added to this solution and was equilibrated for 20 min. Then the silica shell was grown up by the hydrolysis and condensation of TEOS using ammonia as a catalyst in alcohol solution. 2-Propanol (17 mL) and 200 mL ammonia (25 %) were successively added under gentle stirring, followed by addition of 8 mL of TEOS in four portions over a time interval of 1 h. Afterward, the mixture was allowed to react for 12 h, and the mixture was centrifuged at 4500 rpm for 15 min. Finally, the precipitated dye-coded Raman tags were redispersed in water.
greatly enhance the Raman intensity of microarrays by creating many more hot-spot groups for each biomolecular recognition event.
Experimental Section Apparatus SERS analysis was performed on a Renisaw Invia Raman spectrometer (RamLab-010) at an excitation laser of 633 nm. A microscope equipped with a 50 objective was used to focus the incident excitation laser. The laser power on the sample was 5 mW, and the accumulation time was 10 s, the Raman spectra were calibrated with the WiRE Raman software version 2.0 from Renisaw Ltd. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was performed with a DYCZ-28C electrophoresis power supply equipped with WD-9413A gel documentation and analysis systems from Beijing Liuyi Instrument Factory (Beijing, China). UV/Vis absorption spectra were obtained with a Cary 50 Series spectrophotometer (Varian, Australia). The sizes of the AuNPs were verified by transmission electron microscopy (TEM) using a Jeol JEM-2100EX microscope (Japan).
DNA functionalization of AuNP@RBITC@SiO2 TEPSA (200 mL, 200 mm) was mixed with 400 mL of AuNP@RBITC@SiO2 aqueous solution and then incubated, overnight. COOH-terminated AuNP@RBITC@SiO2 was obtained after centrifugation and redispersed in 200 mL of PBS solution (0.1 m, pH 7.4) for later use. The functionalization of DNA was carried out by carbodiimide chemistry. First, 100 mL of solution containing 50 mm NHS and 200 mm EDC was added to the AuNP@RBITC@SiO2 solution to activate the COOH group. After incubation for 2 h, 50 mL of 1.0 10 8 m aminomodified DNA (S4) was added. After standing, overnight, the solution was centrifuged and washed using PBS, and was dispersed in PBS solution for later use.
Chemicals All oligonucleotides used in the present study were purchased either from Takara Biotechnology Co., Ltd. (Dalian, China); the sequences are provided in Supporting Information Table S1. Carboxyl-modified magnetic beads (MBs; ~ 1.0 mm, 10 mg mL 1) were obtained from BaseLine Chrom Tech Research Centre (Tianjin, China). Tetraethyl orthosilicate (TEOS), Na2SiO3·9 H2O and poly(acrylic acid) were purchased from Aladdin Industrial Inc. (Shanghai, China). Rhodamine B isothiocyanate (RBITC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and (3-mercaptopropyl)tri-methoxysilane were purchased from Sigma–Aldrich. Tri(2-carboxyethyl)phosphine hydrochloride (TCEP, 98 %) was purchased from Alfa Aesar (MA, USA). 3-Triethoxysilylpropyl succinic anhydride (TEPSA), human serum albumin (HSA) and bovine serum albumin (BSA) were purchased from Sigma–Aldrich. T4 DNA ligase, Phi29 DNA polymerase and deoxyribonucleoside 5’-triphosphates (dNTPs) were purchased from Thermo Scientific. Other chemicals employed were all of analytical grade and double distilled water was used throughout the experiments.
Preparation of MBs modified with dsDNA The binding of the amino-modified primer with carboxyl-coated MBs was carried out as follows. Briefly, carboxyl-modified MBs (40 mL) were transferred into a 1.5 mL Eppendorf tube and were washed three times with PBS buffer (pH 7.4). After magnetic separation, 0.1 m of EDC (150 mL) and 0.1 m of imidazol-HCl buffer were added to the MBs for 40 min to activate the carboxylate groups on the MBs. Then, the MBs were washed and resuspended in a 100 mL mixture of 10 mm amino-modified primer S1 and 20 mm S2, followed by incubation for 12 h at 37 8C with gentle shaking. Finally, the formed MB–dsDNA conjugates were separated and washed with 200 mL of 0.01 m PBS buffer three times, resuspended in 200 mL of PBS buffer, and stored at 4 8C for later use.
Preparation of AuNPs
Thrombin recognition and RCA
Gold nanoparticles were prepared by citrate reduction of HAuCl4 according to the literature.[38] 1 % (w/v) HAuCl4 solution was added to 50 mL of ultrapure water and heated to boiling for 20 min with stirring, and then an appropriate volume of 1 % (w/v) sodium citrate was added. The mixture was kept boiling for another 20 min until the color of solution remained unchanged; then the mixture was kept stirring for a further 15 min. After cooling to room temperature, the AuNPs solution was placed in the refrigerator. The final gold nanoparticles prepared by this method had an average diameter of approximately 25 nm as determined by TEM.
The MB–dsDNA biocomplex was incubated with 100 mL of PBS containing various concentrations of thrombin at room temperature for 40 min. Then the S2 released from the MB–dsDNA biocomplex was separated with a magnetic field. Padlock probe (10 mL of a 10 nm solution) was introduced to the mixture, and incubated at 37 8C for 30 min. Then 10 units of T4 DNA ligase was added and incubated at 22 8C for 1 h. Subsequently, the RCA reaction was carried out for 1 h with the addition of 10 mm dNTPs (5 mL), 10 U mL 1 Phi29 DNA polymerase (1 mL), and washed three times. The RCA products in solution were then analyzed using a 1 % agarose gel and imaged using PAGE; the results indicated that RCA was successfully performed (Figure S1 in the Supporting Information).
Preparation of silica-encapsulated AuNP SERS tags (AuNP@RBITC@SiO2)
Measurement of Raman spectra
Silica-encapsulated AuNP SERS tags were synthesized by a layerby-layer method according to the literature.[39] Briefly, 6 mL of RBITC Raman reporter (1.0 10 4 m) solution was added to 3 mL of AuNP Chem. Eur. J. 2015, 21, 1 – 7
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Afterward 50 mL of S4-modified AuNP@RBITC@SiO2 and the resulting MBs were incubated at 37 8C for 1 h. Finally, the mixture was
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Full Paper separated and washed with washing buffer to remove excess oligonucleotide–AuNP@RBITC@SiO2. SERS spectra were measured with a Renisaw Raman spectrometer with a 633 nm laser. The laser power was 5 mW, and the resolution of SERS spectra was over 1 cm 1. The collection time for each spectrum was 10 s.
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Cell culture and thrombin extraction HeLa and K562 cell lines were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 100 IU mL 1 penicillin/streptomycin. The cell density was determined by using a hemocytometer prior to any experiments. Approximately one million cells dispersed in RPMI 1640 cell medium were centrifuged at 3000 rpm for 5 min and rinsed with 5 mL of dye-free medium three times and were then redispersed in medium (1 mL). Finally, the cells were disrupted by sonication for 20 min at 0 8C and the lysate was centrifuged at 18 000 rpm for 20 min at 4 8C to remove the cell debris.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21275083) and the Scientific and Technical Development Project of Linyi (201312023). Keywords: analytical methods · nanoparticles · spectroscopy · rolling-circle amplification · thrombin
Raman
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Tagging along: An ultrasensitive surface-enhanced Raman spectroscopy (SERS) sensor based on rolling-circle amplification (RCA)-increased “hot-spot” is developed for the detection of thrombin, with core-shell nanoparticles (see figure) as Raman reporter.
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X. Li,* L. Wang, C. Li && – && Rolling-Circle Amplification Detection of Thrombin using Surface-Enhanced Raman Spectroscopy with Core-Shell Nanoparticle Probe
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& Analytical Methods
Rolling-Circle Amplification Detection of Thrombin using SurfaceEnhanced Raman Spectroscopy with Core-Shell Nanoparticle Probe Xuemei Li,*[a] Linlin Wang,[a, b] and Chunxiang Li[b]
thrombin, the aptamer sequence was released; this allowed the remaining single-stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0 10 12 to 1.0 10 8 m and a detection limit of 4.2 10 13 m, and showed good performance in real serum samples.
Abstract: An ultrasensitive surface-enhanced Raman spectroscopy (SERS) sensor based on rolling-circle amplification (RCA)-increased “hot-spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO2 core-shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer-by-layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of
Introduction
nanowires and nanochips have been widely developed for the detection of proteins,[16] nucleic acids,[17] pathogens[18] and small bioactive molecules.[19] In order to improve Raman signals, signal amplification methods based on rolling circle amplification (RCA),[20, 21] and DNA recycling[22, 23] have been introduced in SERS bioassays. Among these strategies, gold nanoparticles (AuNPs) or silver nanoparticles (AgNPs) were used as supporting substrates, which have relatively weak enhancing ability and the Raman reporters are vulnerable to detachment from the surface of metallic nanoparticles upon direct exposure to the environment, leading to poor reproducibility of SERS sensing. Therefore, a protective shell has been used to surround the metallic SERS substrates and the Raman reporter, preventing them from leaching-out of the Raman reporters and improving their water solubility and stability as well as the reproducibility of the SERS signals.[24, 25] Very recently, gold nanostar@Raman label@SiO2 core-shell nanoparticles have been used as SERS reporters for the detection of adenosine triphosphate (ATP).[26] However, there have been few reports on the combination of signal amplification strategies with coreshell nanoparticles for SERS sensing. Thrombin plays a crucial role in many physiological and pathological processes, such as blood coagulation, thrombosis, inflammation, angiogenesis, tumor growth and metastasis. Thrombin can be used as a therapeutic target and a biomarker for diagnosis of some diseases, such as pulmonary metastases and diseases associated with coagulation abnormalities.[27] Numerous assays have been developed for the detection of thrombin by using nucleic acid aptamers.[28] In the present work, gold nanoparticle@Raman label@SiO2 nanoparticles are produced by sandwiching the Raman reporter between a gold nanoparticle core and a thin silica shell by
Due to their relatively low level in the early stage of disease, ultrasensitive assays of disease-marker proteins are of tremendous importance in clinical settings for disease diagnosis, molecular diagnostics and biomedical development.[1, 2] A number of techniques, including fluorescence,[3, 4] colorimetry,[5, 6] electrochemistry,[7, 8] chemiluminescence,[9] surface plasmon resonance (SPR)[10, 11] and surface-enhanced Raman scattering (SERS),[12, 13] have attracted substantial research efforts toward the detection of nucleic acids and proteins. Especially, there has been great interest in the development of signal amplification strategies combined with these detection tools to improve detection sensitivity.[14] Recently, there has been great interest in the development of SERS-based analytical techniques for chemical and biomedical applications, since it can provide molecular-level identification in samples, nondestructive and noninvasive detection, and enhancement that is several orders magnitude greater than previously reported methods.[15] Various SERS plasmonic platforms ranging from nanoparticles to nanopost arrays, [a] Prof. Dr. X. Li, L. Wang Shandong Provincial Key Laboratory of Detection Technology of Tumor Markers, School of Chemistry and Chemical Engineering Linyi University, Linyi 276005 (P. R. China) E-mail: [email protected] [b] L. Wang, Dr. C. Li State Key Laboratory Base of Eco-chemical Engineering College of Chemistry and Molecular Engineering Qingdao University of Science and Technology Qingdao 266042 (P. R. China) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201405884. Chem. Eur. J. 2015, 21, 1 – 7
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Full Paper a layer-by-layer method. RCA is an isothermal, enzymatic process mediated by some DNA polymerases in which long single-stranded DNA (ssDNA) molecules are synthesized on a short circular ssDNA template with a single DNA primer. With the core-shell nanoparticles as SERS tags, a new SERS-based strategy that is focused on increasing the number of “hotspots” in the signaling system by RCA was developed for detection of thrombin. Taking advantage of the high enhancement factor and stability of the core-shell nanoparticle probe and the signal-amplification effects of RCA, the detection limit of the system for thrombin was found to be as low as 0.42 pm.
Characterization of the core-shell SERS nanoparticles Figure 1 A and B show the TEM images of the AuNPs before and after coating of SiO2 layer, respectively. The average diameters of AuNPs were estimated to be 25 nm. Growth of the silica shell was achieved by Stçber’s method and the thickness of the silica shell was 3.8 nm according to our measurements.
Results and Discussion Principle of the sensing strategy Figure 1. TEM images of: A) the bare AuNPs, and B) AuNP@RBITC@SiO2 nanoparticles. Inset: the magnification of one sphere.
The configuration and operation principle for the detection of thrombin by the RCA–SERS strategy with core-shell tags is depicted in Scheme 1. The double-stranded hybrids of S1 and S2 were immobilized on the MB through amide-bond formation between the COOH group on the MB and the NH2 on S1. In the presence of thrombin, the aptamer sequence S2 bound to thrombin and folded to a complex structure, which was released into solution. After the circular template was formed by incubating the padlock probe and T4 ligase with the remained S1, which acted as primers, the RCA reaction was initiated by adding Phi29 DNA polymerase and deoxynucleotides (dNTPs).[7] The RCA process produced long ssDNA in situ for the attachment of a large number of S4–AuNP@RBITC@SiO2 probes. The modified MBs were then measured with a Raman spectrometer. The intensity of the Raman signal reflects the amount of thrombin in this detection system.
Figure 2 A reveals the UV–visible absorption spectra of the bare AuNPs, AuNP@RBITC@SiO2, DNA, and DNA conjugated AuNP@RBITC@SiO2. The bare AuNPs exhibited the characteristic absorbance at about 520 nm while a wide absorbance peak from 520 to 620 nm was seen for AuNP@RBITC@SiO2. The redshift wide peak may be ascribed to rhodamine B isothiocyanate (RBITC) and the higher refractive index of SiO2 with respect to that of water. After modification with S4, an obvious peak at 260 nm, which corresponded to that of DNA, was observed. The SERS tag nanoparticles were further characterized with fluorescent spectra. As shown in Figure 2 B, free RBITC in solution exhibited an obvious peak at 580 nm (line a), while the fluorescence intensity of AuNP@RBITC@SiO2 decreased sharply, due to the quenching effect of AuNP and encapsulation effect of SiO2 shell. SERS characterization
Scheme 1. Schematic illustration of the detection of thrombin by RCA–SERS strategy with core-shell tags. The structure of AuNP@RBITC@SiO2 is shown in the square frame.
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It has been well established in the literature that both fluorescent and nonfluorescent dyes can be used as resonant Raman reporters, because fluorescence emission is efficiently quenched by gold particles, not interfering with Raman measurement.[29] Silica-encapsulated RBITC–AuNP tags prepared by layer-by-layer method were used as SERS reporters as the protective shells can prevent desorption of Raman reporters and adsorption
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Full Paper Signal amplification of RCA To prove the signal amplification of the RCA strategy, the control experiment without T4 ligase was carried out as shown in Figure 4. In the absence of target DNA (Figure 4 a) or T4 ligase (Figure 4 b), the RCA reaction did not take place. There were very small peaks in the spectra, which might be due to the nonspecific adsorption of the SERS tags on the surface of the MBs. When thrombin was present it interacted with its aptamer, leading to the dissociation of the duplex DNA. The primer S1 was free to hybridize with the padlock DNA. After rollingcircle amplification was carried out, each primer would have created a long ssDNA that hybridized with a number of S4– AuNP@RBITC@SiO2. The longer the ssDNA, the more probe tags were attached to it. In the RCA reaction, the length of the ssDNA product is related to the concentration of polymerase, dNTPs, reaction temperature and time. In order to control the length of RCA product consistently in all assays, the RCA reaction was carried out for 1 h in the same reaction conditions. Thus, each protein molecule recognized by S4–AuNP@RBITC@SiO2 contains a large number of hot-spot groups.[32] The differences in the SERS spectra are the result of the different amounts of hot-spots among the arrays. The intensity of the Raman signal reflects the amount of thrombin in this detection system and the strongest Raman band at 1642 cm 1 was used for the quantitative evaluation of thrombin.
Figure 2. A) UV spectra of: DNA (a), RBITC (b), S4-AuNP@RBITC@SiO2 (c), AuNP@RBITC@SiO2 (d) and AuNPs (e). B) Fluorescence-emission spectra of: RBITC isothiocyanate in solution (a), AuNP@RBITC@SiO2 core-shell nanoparticles (b), and blank (c).
of external species. As shown in Figure 3, the laser excitation of the samples provided discrete vibrational peaks at 1349, 1506 and 1645 cm 1, which were attributed to the ring C C stretching vibrations of rhodamine.[30] The peaks at 1200 and 1273 cm 1 were ascribed to the in-plane benzene v9 mode and the in-plane C H or C C H bending mode, respectively.[31] The intensity of the Raman signal reflects the amount of thrombin in this detection system and the strongest Raman band at 1642 cm 1 was used for the quantitative evaluation of thrombin. Figure 4. SERS spectra obtained from RCA detection of thrombin: in the absence of the thrombin (a), in the absence of T4 ligase (b), RCA detection of 10 nm thrombin (c).
Characterization of the core-shell SERS nanoparticles: sensitive detection of thrombin Under the optimum conditions, the sensitivity of the present method was investigated upon addition of different concentrations of thrombin as shown in Figure 5. Improved SERS signal was observed with the increase of thrombin. With the measurement of the normalized Raman intensity of the 1645 cm 1 peak, the change of normalized Raman intensity was quantitatively analyzed with the concentration of thrombin. As shown in Figure 5 b, the normalized Raman intensity was a good linear fit to the logarithm of thrombin concentration in the range from 1.0 10 12 to 1.0 10 8 m. The regression equation was DI = 0.243 log C + 3.23276 (DI is the Raman intensity sub-
Figure 3. SERS spectra obtained from free AuNP@RBITC@SiO2 core-shell nanoparticles. The as-prepared nanoparticles were 1000 times diluted (a), 50 times diluted (b) and not diluted (c). Inset: molecular structure of RBITC. Chem. Eur. J. 2015, 21, 1 – 7
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Full Paper tracted the blank, C is the concentration of thrombin), and the regression coefficient of the linear curve was R2 = 0.983. A detection limit of 4.2 10 13 m of thrombin was estimated using 3s, which is more sensitive than previously reported SERS detection of thrombin with 4-aminobenzenethiol (4-ABT)-encoded gold nanoparticle hot-spots.[33]
weights and isoelectric points, including human serum albumin (HSA), bovine serum albumin (BSA), myoglobin, lysozyme, and trypsin. As shown in Figure 6, Even at a concentration as high as 10 nm, the responses from the interferences are very low and close to the background. This demonstrates that the RCA–SERS based biosensor is highly specific and has a good selectivity for the detection of thrombin from other proteins.
Figure 6. The selectivity of the proposed protein assay system toward 1.0 nm thrombin. Five non-target proteins at high concentration (10 nm) were used to assess specificity.
Application of the strategy in biological sample To investigate the applicability of the probing methods in a real biological environment, human blood serum (HBS) was selected. Serum samples spiked with different concentrations of thrombin were analyzed with our method. As presented in Table 1, a series of five parallel measurements of samples yielded a relative standard deviation less than 7.3 %, recoveries were obtained with 94–108 %. These results indicate that the proposed assay components do not cross-react with the complex matrix of serum.
Figure 5. A) SERS spectral responses with different concentration of thrombin: 0 (a), 1.0 pm (b), 5.0 pm (c), 10 pm (d), 50 pm (e), 100 pm (f), 500 pm (g), 1.0 nm (h) and 10 nm (i). B) Corresponding peak intensities at 1642 cm 1 for RCA–SERS detection of thrombin with varying concentrations. The error bars are standard deviations across triplicate assays.
Table 1. Determination of thrombin in serum samples.
Notably, the sensitivity of the current method had increased by one or two orders of magnitude as compared to our previously reported method,[34, 35] and was also comparable with or even exceeded the reported detection limit by the RCA-based electrochemical method[36] and by the RCA-based chemiluminescent method.[37] The high sensitivity of the present method could be attributed to the high effect of RCA as compared with the assay without RCA (details are shown in Scheme S1 and Figure S2 in the Supporting Information). Moreover, the highly sensitive response of the assay for DNA detection displayed satisfactory reproducibility. The standard deviation of eight duplicated tests at 10 pm was found to be less than 7.6 %.
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Detection [pm]
RSD [%] n=5
Recovery [%] n=5
1 2 3 4 5
5.0 20 100 500 1000
4.7 21.6 95.2 530.7 981.2
6.6 7.3 5.9 5.2 6.7
94 108 95 106 98
In summary, we have developed a sensitive SERS sensor for detection of thrombin, in which AuNP@RBITC@SiO2 was employed as the nanotag and RCA as enhancement strategy to further increase hot-spots. The SERS sensor exhibited good specificity and high sensitivity toward thrombin with a limit of detection of 0.42 pm. Encapsulation of a large number of RBITC molecules inside the sandwich-structured nanoparticles contributed to the high sensitivity. Furthermore, RCA can
Control experiments were also performed to examine the selectivity of these probing methods. Some proteins were chosen as possible interference, based on their molecular &
Spiked [pm]
Conclusion
Selectivity of the present strategy
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Full Paper solution under rapid stirring, and the mixture was allowed to equilibrate for 20 min. Then, 3 mL of PAA solution (4 g L 1), which had been adjusted to pH 7.0 with 0.1 m NaOH, was added dropwise with vigorous stirring and stirred for 3 h. The solution of polymerencapsulated nanospheres was centrifuged twice to remove excess polymer molecules and redispersed into water. A coupling agent, 3-aminopropyltrimethoxysilane, was added to this solution and was equilibrated for 20 min. Then the silica shell was grown up by the hydrolysis and condensation of TEOS using ammonia as a catalyst in alcohol solution. 2-Propanol (17 mL) and 200 mL ammonia (25 %) were successively added under gentle stirring, followed by addition of 8 mL of TEOS in four portions over a time interval of 1 h. Afterward, the mixture was allowed to react for 12 h, and the mixture was centrifuged at 4500 rpm for 15 min. Finally, the precipitated dye-coded Raman tags were redispersed in water.
greatly enhance the Raman intensity of microarrays by creating many more hot-spot groups for each biomolecular recognition event.
Experimental Section Apparatus SERS analysis was performed on a Renisaw Invia Raman spectrometer (RamLab-010) at an excitation laser of 633 nm. A microscope equipped with a 50 objective was used to focus the incident excitation laser. The laser power on the sample was 5 mW, and the accumulation time was 10 s, the Raman spectra were calibrated with the WiRE Raman software version 2.0 from Renisaw Ltd. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was performed with a DYCZ-28C electrophoresis power supply equipped with WD-9413A gel documentation and analysis systems from Beijing Liuyi Instrument Factory (Beijing, China). UV/Vis absorption spectra were obtained with a Cary 50 Series spectrophotometer (Varian, Australia). The sizes of the AuNPs were verified by transmission electron microscopy (TEM) using a Jeol JEM-2100EX microscope (Japan).
DNA functionalization of AuNP@RBITC@SiO2 TEPSA (200 mL, 200 mm) was mixed with 400 mL of AuNP@RBITC@SiO2 aqueous solution and then incubated, overnight. COOH-terminated AuNP@RBITC@SiO2 was obtained after centrifugation and redispersed in 200 mL of PBS solution (0.1 m, pH 7.4) for later use. The functionalization of DNA was carried out by carbodiimide chemistry. First, 100 mL of solution containing 50 mm NHS and 200 mm EDC was added to the AuNP@RBITC@SiO2 solution to activate the COOH group. After incubation for 2 h, 50 mL of 1.0 10 8 m aminomodified DNA (S4) was added. After standing, overnight, the solution was centrifuged and washed using PBS, and was dispersed in PBS solution for later use.
Chemicals All oligonucleotides used in the present study were purchased either from Takara Biotechnology Co., Ltd. (Dalian, China); the sequences are provided in Supporting Information Table S1. Carboxyl-modified magnetic beads (MBs; ~ 1.0 mm, 10 mg mL 1) were obtained from BaseLine Chrom Tech Research Centre (Tianjin, China). Tetraethyl orthosilicate (TEOS), Na2SiO3·9 H2O and poly(acrylic acid) were purchased from Aladdin Industrial Inc. (Shanghai, China). Rhodamine B isothiocyanate (RBITC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and (3-mercaptopropyl)tri-methoxysilane were purchased from Sigma–Aldrich. Tri(2-carboxyethyl)phosphine hydrochloride (TCEP, 98 %) was purchased from Alfa Aesar (MA, USA). 3-Triethoxysilylpropyl succinic anhydride (TEPSA), human serum albumin (HSA) and bovine serum albumin (BSA) were purchased from Sigma–Aldrich. T4 DNA ligase, Phi29 DNA polymerase and deoxyribonucleoside 5’-triphosphates (dNTPs) were purchased from Thermo Scientific. Other chemicals employed were all of analytical grade and double distilled water was used throughout the experiments.
Preparation of MBs modified with dsDNA The binding of the amino-modified primer with carboxyl-coated MBs was carried out as follows. Briefly, carboxyl-modified MBs (40 mL) were transferred into a 1.5 mL Eppendorf tube and were washed three times with PBS buffer (pH 7.4). After magnetic separation, 0.1 m of EDC (150 mL) and 0.1 m of imidazol-HCl buffer were added to the MBs for 40 min to activate the carboxylate groups on the MBs. Then, the MBs were washed and resuspended in a 100 mL mixture of 10 mm amino-modified primer S1 and 20 mm S2, followed by incubation for 12 h at 37 8C with gentle shaking. Finally, the formed MB–dsDNA conjugates were separated and washed with 200 mL of 0.01 m PBS buffer three times, resuspended in 200 mL of PBS buffer, and stored at 4 8C for later use.
Preparation of AuNPs
Thrombin recognition and RCA
Gold nanoparticles were prepared by citrate reduction of HAuCl4 according to the literature.[38] 1 % (w/v) HAuCl4 solution was added to 50 mL of ultrapure water and heated to boiling for 20 min with stirring, and then an appropriate volume of 1 % (w/v) sodium citrate was added. The mixture was kept boiling for another 20 min until the color of solution remained unchanged; then the mixture was kept stirring for a further 15 min. After cooling to room temperature, the AuNPs solution was placed in the refrigerator. The final gold nanoparticles prepared by this method had an average diameter of approximately 25 nm as determined by TEM.
The MB–dsDNA biocomplex was incubated with 100 mL of PBS containing various concentrations of thrombin at room temperature for 40 min. Then the S2 released from the MB–dsDNA biocomplex was separated with a magnetic field. Padlock probe (10 mL of a 10 nm solution) was introduced to the mixture, and incubated at 37 8C for 30 min. Then 10 units of T4 DNA ligase was added and incubated at 22 8C for 1 h. Subsequently, the RCA reaction was carried out for 1 h with the addition of 10 mm dNTPs (5 mL), 10 U mL 1 Phi29 DNA polymerase (1 mL), and washed three times. The RCA products in solution were then analyzed using a 1 % agarose gel and imaged using PAGE; the results indicated that RCA was successfully performed (Figure S1 in the Supporting Information).
Preparation of silica-encapsulated AuNP SERS tags (AuNP@RBITC@SiO2)
Measurement of Raman spectra
Silica-encapsulated AuNP SERS tags were synthesized by a layerby-layer method according to the literature.[39] Briefly, 6 mL of RBITC Raman reporter (1.0 10 4 m) solution was added to 3 mL of AuNP Chem. Eur. J. 2015, 21, 1 – 7
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Afterward 50 mL of S4-modified AuNP@RBITC@SiO2 and the resulting MBs were incubated at 37 8C for 1 h. Finally, the mixture was
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Cell culture and thrombin extraction HeLa and K562 cell lines were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 100 IU mL 1 penicillin/streptomycin. The cell density was determined by using a hemocytometer prior to any experiments. Approximately one million cells dispersed in RPMI 1640 cell medium were centrifuged at 3000 rpm for 5 min and rinsed with 5 mL of dye-free medium three times and were then redispersed in medium (1 mL). Finally, the cells were disrupted by sonication for 20 min at 0 8C and the lysate was centrifuged at 18 000 rpm for 20 min at 4 8C to remove the cell debris.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21275083) and the Scientific and Technical Development Project of Linyi (201312023). Keywords: analytical methods · nanoparticles · spectroscopy · rolling-circle amplification · thrombin
Raman
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Tagging along: An ultrasensitive surface-enhanced Raman spectroscopy (SERS) sensor based on rolling-circle amplification (RCA)-increased “hot-spot” is developed for the detection of thrombin, with core-shell nanoparticles (see figure) as Raman reporter.
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X. Li,* L. Wang, C. Li && – && Rolling-Circle Amplification Detection of Thrombin using Surface-Enhanced Raman Spectroscopy with Core-Shell Nanoparticle Probe
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