Article Cite This: ACS Appl. Nano Mater. 2019, 2, 4637−4645
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Fluorescent Mn:ZnCdS@ZnS and CdTe Quantum Dots Probes on SiO2 Microspheres for Versatile Detection of Carcinoembryonic Antigen and Monitoring T4 Polynucleotide Kinase Activity Guifen Jie,* Chunli Li, Yu Zhao, Qian Kuang, and Shuyan Niu
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Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE; Shandong Key Laboratory of Biochemical Analysis; Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong; College of Chemistry and Molecular Engineering. Qingdao University of Science and Technology, Qingdao 266042, PR China S Supporting Information *
ABSTRACT: Versatile fluorescent quantum dots (QDs) probes on SiO2 microspheres were prepared and used for detection of carcinoembryonic antigen (CEA) and monitoring T4 polynucleotide kinase (PNK) activity by integrating with amplification strategy. The highly fluorescent Mn:ZnCdS@ZnS and CdTe QDs showed different emission peaks. The amino-modified SiO2 microspheres with good morphology and no fluorescence interference were designed to link DNA and fabricate multiple branched QDs probes. First, CEA-induced target recycle amplification technique was combined with the Mn:ZnCdS@ZnS QDs labeled P2 signal probe for sensitive detection of CEA. Then, the formed DNA S1−DNA3 double strands further polymerized and proceeded exonuclease-cleavage reaction in the presence of T4 PNK, the released DNA fragments were used to trigger assembly of multibranched CdTe QDs-DNA signal probe (P1) by hybridization chain reaction (HCR), and achieved a greatly amplified fluorescence detection of PNK. The detection limits for CEA and PNK were 0.1 pg/mL and 0.001 U/mL, which was comparable to the reported methods. It is for the first time that the multiple branched QDs probes was coupled with HCR and cycling amplification strategy for versatile fluorescence detection of CEA and PNK activity. It shows great potential for early clinical diagnosis of cancer and the nucleotide kinase-target drug discovery. KEYWORDS: fluorescence strategy, multiple branched quantum dots, target recycle amplification, SiO2 microspheres, T4 polynucleotide kinase
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egy,12 and chemiluminescence13 techniques have been applied to the assay of T4 PNK. Among them, the homogeneous fluorescence methods have attracted more interest owing to their flexibility, speediness, high sensitivity, and well selectivity. Fluorescent quantum dots (QDs) have enhanced brightness, photostability, and wavelength tenability. QDs were considered to be the ideal fluorescent markers in many fields, such as biosensing, biological imaging, immunoassay, and drug delivery because of unique photophysical properties of QDs. Fluorescence biosensing methods show many advantages for the
INTRODUCTION Cancers have serious threats to human health, and can cause high mortality.1 Carcinoembryonic antigen (CEA) is a glycoprotein produced in human tumor cells, it is a common tumor marker for early clinical diagnosis of cancer.2 T4 polynucleotide kinase plays an important role in DNA recombination, replication, repair during strand damage, and interruption.3,4 Therefore, DNA repair by T4 PNK may inhibite CEA generation in tumor cells, developing sensitive methods for assays of CEA and T4 PNK is of great importance for the clinical diagnosis. So far, many analytical methods have been reported for CEA detection, such as fluorescence,5 electrochemical method,6 enzyme-linked immunoassay,7 ECL,8 photoelectric assays.9 In addition, fluorescence,10 electrochemistry,11 colorimetric strat© 2019 American Chemical Society
Received: May 29, 2019 Accepted: June 18, 2019 Published: June 28, 2019 4637
DOI: 10.1021/acsanm.9b01003 ACS Appl. Nano Mater. 2019, 2, 4637−4645
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Zn(NO3)2 (0.1 M) was added to the solution, and further reacted to obtain the resulting core−shell QDs.33 Fabrication of the Fluorescence Biosensor. DNA was prepared using TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0) buffer, the concentration of DNA involved in this experiment is 1.0 × 10−5 M. Assembly of Fluorescent Quantum Dots Probe. According to the literature.34 30 μL of 1.0 × 10−5 M long single stranded DNA (P9) was mixed with 1.0 × 10−5 M single stranded DNA P6, P7, and P8 at 95 °C for 5 min and annealed to perform hybridization reaction, forming the forked structure probe (P1). Then 100 μL of carboxyl modified CdTe QDs (80 μL of QDs, 15 μL of 0.1 M EDC, and 5 μL of 0.05 M NHS, activated at room temperature for 1 h) were added and incubated for over 6 h. This mixture was centrifuged and the precipitate was resuspended in 50 μL of deionized water, forming the fluorescent signal probe (P1-CdTe QDs) with mutual-crossed four DNA strands and stored at 4 °C for use. In a similar way, 50 μL of DNA P2 (10−5 M) was added to 50 μL of Mn:ZnCdS@ZnS core−shell quantum dots (30 μL of QDs, 15 μL of 0.1 M EDC, 5 μL of 0.05 M NHS, activated at room temperature for 1 h) and reacted at 37 °C for over 6 h. The mixture was centrifuged at 10000 rpm for 20 min, and the precipitate was then resuspended in deionized water, and stored in 4 °C for use. Different concentrations of CEA and T4 PNK were prepared in advance: 0.1 pg/mL, 1.0 pg/mL, 0.01 ng/mL, 0.1 ng/mL, 1.0 ng/mL, 10 ng/mL; T4 PNK: 0.001 U/mL, 0.005 U/mL, 0.1 U/mL, 1 U/mL, 1 U/mL, 5 U/mL. Assembly of Substrate Strands S1 and S2 on SiO 2 Microsphere. 50 μL of 1.0 × 10−5 M DNA S1 was activated with 25 μL of 0.1 M EDC and 25 μL of 0.025 M NHS at room temperature for 1 h, and then linked to 50 μL of amino-modified SiO2.35 The resulting solution was centrifuged to remove excess sequence, and the precipitate was resuspended in 50 μL of deionized water. Similarly, the carboxyl-modified DNA S2 was assembled on the SiO2 microsphere and stored at 4 °C for use. Fabrication of the Biosensing System for Detecting two Kinds of Targets. According to the literature,35 50 μL of 1.0 × 10−5 M DNA 2 was added to the solution with SiO2-DNA S1, the hybridization reaction was performed at 37 °C for 90 min. The solution was centrifuged to remove the uncombined DNA2. Then, CEA was added to the solution to bind with DNA S1 (aptamer), and the released DNA2 was collected. The sediment was resuspended in 50 μL of 1.0 × 10−5 M DNA3, the hybridization reaction was incubated at 37 °C for 2 h. Then 1 μL of phi29 polymerase (10000 U/mL), 10 μL of 10× phi29 buffer and 5 μL of dNTPs (1 mM) were added into the solution to perform polymerization reaction at 37 °C for 2 h, and the released target CEA was recycled, thus plenty of DNA2 was obtained, and the polymerized DNA S1-DNA3 duplex structure was formed at the same time. The resulting solution was centrifuged, the DNA2 in supernate was collected, and the precipitate with DNA S1-DNA3 duplex structure was resuspended in 30 μL of deionized water. Then, 1 μL of T4-PNK (10 000 U/mL), 5 μL of 10 × PNK Buffer, 1 μL of λ Exo (10 000 U/mL), 5 μL of 10 × NEB Buffer and 5 μL of 2 mM ATP were mixed with the above solution (DNA S1-DNA3), and the final volume was 50 μL. After that, phosphorylation reaction and cleavage reaction were conducted at 37 °C for 2 h, and the solution was centrifuged to remove the supernatant. Subsequently, 20 μL of 10−5 M DNA HP1 and 25 μL of 10−5 M DNA HP2 were mixed with the above solution, annealing at 95 °C slowly for 10 min to perform HCR reaction. The uncombined DNA HP1 and DNA HP2 were separated by centrifugation. 50 μL of P1-CdTe QDs were added and the solution was incubated at 37 °C for 2 h. After centrifugation at 3000 rpm, the sediment was resuspended in deionized water, then fluorescence detection was conducted. Meanwhile, the collected DNA2 was mixed with SiO2-DNA S2, and 50 μL of DNA P2−Mn:ZnCdS@ZnS QDs probe was also added to the mixture. The solution was incubated at 37 °C for 2 h, centrifugated at 5000 rpm, and washed, the sediment was then resuspended in 50 μL of deionized water for fluorescence detection.
challenging requirements in diagnosis, such as multiplexing capability, high sensitivity, and homogeneous assay.14,15 Although great advances have been made toward assay of DNA phosphorylation, it is still in high demand to improve analytical performances to satisfy the development needs of biological research, clinic diagnostics, and drug discovery. Moreover, the target biomarkers for some major diseases (for example, cancers) show very low content in the early stage, it is necessary to develop amplification techniques to achieve ultrasensitive assays of targets.16,17 So far, signal amplification has been aroused increasing interest, and led to the development of many advanced nanomaterials such as luminescent quantum dots,18 nanosheets,19 and heterostructured nanocomposites.20 DNA nanostructures of various sizes, shapes, and geometries via self-assembly,21,22 have inspired the design of multiple nucleic acid biosensors, including homogeneous fluorescence detection.23 Different QDs-based nanoprobes have been employed in the bionanotechnology field.24,25 In addition, many DNA amplification strategies have been developed, such as polymerase chain reactions,26 hybridization chain reaction amplification,27−29 and exonuclease-assisted amplification,30 nicking enzyme-assisted target recycling.31 In contrast, enzyme-assisted target recycling appears effective and competent. Thus, a combination of QD-based nanoprobes, fluorescent methods with enzyme-assisted target recycling amplification is an ideal candidate for the detection of CEA and T4 PNK activity. Herein, we reported a new strategy for versatile fluorescence detection of CEA and T4 PNK activity by using multiple branched QDs probes coupled with target recycle amplification technique. The amino-modified SiO2 microspheres were used as carrier to fabricate multiple branched QDs probes, which greatly enhanced the fluorescence signals of QDs for targets assays. Moreover, the double amplification strategy by cycling and HCR further amplified fluorescence signal and much improved the detection sensitivity. The detection limits toward CEA and PNK were 0.1 pg/mL and 0.001 U/mL, which was comparable with the reported methods, and showed great potential for early clinical diagnosis of cancer and serious human disorders.
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EXPERIMENTAL SECTION
Synthesis of Amino Functionalized SiO2 Microsphere. The amino functionalized SiO2 microsphere was prepared according to the reported methods with a slight modification.32 30 mL of ethanol, 50 mL of ultrapure water and 10 mL of NH3·H2O were added into the 250 mL three-neck round flask under magnetic stirring. Then the mixed solution of tetraethyl orthosilicate (5 mL) with ethanol (20 mL) was injected into the reaction flask drop by drop. The mixture kept stirring for 6 h, then 5 mL of 3-aminopropyltriethoxysilane (APTES) was added to the solution, and further stirred over 12 h at room temperature. Finally, the SiO2 microsphere was centrifuged and washed with ethanol repeatedly, and then resuspended in 50 mL of ultrapure water. Preparation of Mn:ZnCdS@ZnS Core−shell Quantum Dots. The Mn:ZnCdS@ZnS QDs were prepared according to the literature.33 3.5 mL of Zn(NO3)2 (0.1 M), 3.5 mL of Cd(NO3)2 (0.1 M), 0.2 mL of Mn(Ac)2 (0.05 M), 122 μL of 3-mercaptopropionic acid (MPA) and some ultrapure water were mixed homogeneously in 100 mL 3-neck bottom flask, and the final volume was 45 mL. The solution was adjusted to pH 11 using NaOH (1.0 M), then 4.5 mL of Na2S (0.1 M) was quickly added to the flask and stirred for 10 min. The mixture was refluxed at 100 °C for 1.0 h, then 3.8 mL of 4638
DOI: 10.1021/acsanm.9b01003 ACS Appl. Nano Mater. 2019, 2, 4637−4645
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Figure 1. TEM images of (A) Mn:ZnCdS@ZnS and (C) CdTe QDs; FL emission spectra of (B) Mn:ZnCdS@ZnS and (D) CdTe QDs.
Scheme 1. Schematic Representation Strategy for Fluorescence Detection of T4 PNK Activity Based on the CdTe QDs Signal Probe P1 (A) and CEA Detection Based on the Mn:ZnCdS@ZnS QDs QDs Probe P2 (B) by Enzyme-Assisted Target Recycling and HCR Amplification Technology
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DOI: 10.1021/acsanm.9b01003 ACS Appl. Nano Mater. 2019, 2, 4637−4645
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ACS Applied Nano Materials Confocal microscopy imaging was performed with 10 μL of sample solution on a microscope slide. The fluorescence of QDs on the surface of SiO2 microspheres was observed by HCX PL APO 40/0.85 objective with 80−420 nm excitation light source.
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RESULTS AND DISCUSSION Characterization of the QDs. Transmission electron microscopic (TEM) image of Mn:ZnCdS@ZnS QDs was showed in Figure 1(A). It can be seen that the size of Mn:ZnCdS@ZnS QDs is about 4−4.5 nm, and the QDs are equally distributed. Figure 1 (B) displayed the fluorescence (FL) emission of Mn:ZnCdS@ZnS QDs, the FL peak is at 570 nm with excited wavelength at 410 nm, and the fluorescence signal is very high. Figure 1C showed the TEM image of CdTe quantum dots. The average size of CdTe QDs was approximately 4.0 nm. Figure 1D presented the FL emission of CdTe QDs, the FL peak at 626 nm is very high, demonstrating that the prepared CdTe QDs possess well optical property. Moreover, the FL quantum yields of the CdTe and Mn:ZnCdS@ZnS QDs were estimated to be 61.8% and 41.9 by the literature,35,36 which is higher than the reported QDs.37 Principle for Detection of CEA and T4-PNK Activity Based on Two QDs Signal Probes by Enzyme-Assisted Target Recycling and HCR Amplification Technology. In this work, the highly fluorescent Mn:ZnCdS@ZnS QDs were used to label DNA P2, and formed a novel trifurcate structure on the surface of SiO2,38 which was further combined with enzyme-aided target recycle amplification technique for CEA detection. At the same time, CdTe QDs with excellent fluorescence property was labeled fork probe P1, and combined with HCR amplification reaction for assay of T4 PNK. Therefore, the fluorescence biosensing method was used for sensitive detection of both CEA and T4 PNK. The principle was shown in Scheme 1. In part A, the carboxylmodified DNA S1 containing aptamer for CEA was assembled on the surface of amino-modified SiO2 microsphere, then DNA 2 hybridized with S1 and formed a duplex structure. When CEA was present, the specific binding of CEA to aptamer led to quantitative release of DNA 2. Then DNA 3 was introduced to hybridize with part of S1, and further polymerized to release target CEA. As a result, plenty of DNA2 was released due to the recycling of CEA, and the double strands of DNA S1/DNA3 were formed. In the presence of T4-PNK and ATP, the hydroxyl group at the 5′-termini of DNA3 was performed phosphorylation reaction, which was immediately cleaved by λexo, and the single stranded DNA S1 was released. Then HCR amplification reaction was performed in the presence of hairpin DNA4 and DNA5, followed by hybridization with CdTe QDs labeled fork probe (P1) for fluorescence detection of T4-PNK. In Part B, DNA S2 was first assembled on the surface of SiO2 microspheres, then it can form a stable trifurcation structure by linking with DNA2 and Mn: ZnCdS@ZnS labeled fluorescent DNA probe (P2), which can be used for fluorescence detection of CEA concentration. Characterization of SiO2 Microspheres Assembled with Quantum Dots Probe. The SiO2 microspheres were used as a carrier for separation in the sensing system. Figure 2(A) and (B) showed the transmission electron microscopy (TEM) and field emission scanning electron microscopy (SEM) images of SiO2 microspheres, respectively. It can be seen that the average size of SiO 2 microspheres is
Figure 2. (A) TEM image of SiO2 microspheres. (B) Field-emission scanning electron microscopy (FE-SEM) image of the SiO 2 microspheres. (C) TEM image of the Mn:ZnCdS@ZnS QDs signal probe (P2) on the SiO2 microspheres. (D) TEM image of the CdTe QDs signal probe (P1) on the SiO2 microspheres.
approximately 80 nm, the surface of the microspheres is smooth. Figure 2 (C) showed the TEM image of SiO2−P2-Mn: ZnCdS@ZnS QDs. When CEA was present, abundant DNA2 was produced by cycling amplification, which was captured by DNA S2 on the SiO2 microspheres, and then combined with QDs signal probe to form trigeminal structure. It was observed that small particles were attached to the surface of the microsphere and the surface was rough, proving that the QDs signal probe was successfully linked to the microspheres. Figure 2 (D) showed the TEM image of the SiO2−P1-CdTe QDs. When T4 PNK was present, the fork structure signal probe P1 was bounded to the surface of SiO2 microspheres after HCR reaction, it can be obviously seen that a layer of material is covered on the surface. This indicates that the P1 signal probe was assembled onto the surface of the microspheres. The TEM results verified that the QDs probes were successfully combined with the SiO2 microspheres for fluorescence sensing analysis. Feasibility Study for Fluorescence Detection on CEA Based on Quantum Dot Signal and Enzyme-Assisted Target Recycle Amplification. Figure 3 explored the feasibility for detecting CEA based on Mn:ZnCdS@ZnS QD fluorescence probes and polymerase-assisted target recycle amplification. Curve a represents the fluorescence spectrum of Mn:ZnCdS@ZnS QDs, the fluorescence signal is very high. When the QDs were linked to amino-modified DNA P2, the fluorescence signal decreased and a slight redshift of fluorescence emission peak was observed for changed QDs structure (curve b). In the absence of CEA, there is a low background signal due to the adsorption of small amount of QDs signal probe (curve c). When CEA was present and there was no phi29 polymerase, the target could not be recycled, the CdTe QDs labeled P1 was partially bound to SiO2 microspheres, the FL peak intensity improved slightly (curve d). When both CEA and phi29 polymerase were added, more DNA2 were released by recycle of target CEA, which was 4640
DOI: 10.1021/acsanm.9b01003 ACS Appl. Nano Mater. 2019, 2, 4637−4645
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the characteristic absorption peaks of DNA and Mn:ZnCdS@ ZnS QDs at 255 nm and 402 nm (curve f) were observed. Thus, the UV−vis absorption spectra indicated that the sensing of target and assembly of signal probe were successfully performed. Characterization of the Fluorescence Sensing Analysis Methods for Detection of two Targets by Gel Electrophoresis. In Figure 5 (A), lane 1 is a mark, lane 2 is 3
Figure 3. Fluorescence signal responses of the sensing assay: (a) Mn:ZnCdS@ZnS QDs, (b) DNA-Mn:ZnCdS@ZnS QDs, (c) background signal without target CEA, (d) in the presence of target CEA without polymerase, (e) in the presence of target CEA and phi29 polymerase. (excitation wavelength of 410 nm, slit 5.0, photomultiplier voltage 400 V). Figure 5. Electrophoregran of (A) recycle of CEA in the presence of the phi29; (B) Conjugation of DNA S2 with DNA2 and P1 to form DNA trifurcate structure.
bound to DNA S2 to capture more signal probe (P2), thus the signal was significantly enhanced (curve e). These results indicate that enzyme-assisted target cycling could amplify the fluorescence signal of the quantum dot probe, so the amplified fluorescence method based on QDs is feasible for CEA detection. Characterization of the Fluorescence Sensing Analysis Method for Target Detection by UV−vis Absorption Spectra. The fabrication of the fluorescence sensor was verified by UV−vis absorption spectra (Figure 4). Curve a
μL of DNA S1 (10−5 M), lane 3 is the hybridization product of equivalent DNA 2 and S1. Lane 4 showed the specific conjugation of CEA to DNA S1 aptamer, the band position is higher than others due to the larger molecular mass. Lane 5 is the DNA S1/DNA3 double-stranded structure formed by polymerization in the presence of DNA3 and phi29 polymerase, and surplus CEA and DNA S1 were also present, so two bright bands were observed. The results of the bands position in gel electrophoresis indicate that the sensing analysis of target CEA by enzyme-assisted amplification is successful. In Figure 5 (B), lane 1 is marker, lane 2 is 3 μL of 1.0 × 10−5 M DNA S2, lane 3 is equivalent amount of DNA P2. Lane 4 is the formed trifurcated structure signal probe P2 by conjugating DNA S2 to DNA P2 and DNA2, suggesting that the QDs signal probe P2 was successfully formed and applied for target detection. Gel electrophoresis characterization for detection of T4 PNK activity is shown in Figure 6 (A). Lane 1 is mark, lane 2 is
Figure 4. UV−vis absorption spectra characterization for fluorescence assay of CEA. (a) DNA S1; (b) SiO2 microspheres; (c) CEA; (d) Mn:ZnCdS@ZnS QDs; (e) CEA@DNA S1; (f) SiO2@Probe2.
shows an obvious UV absorption peak of DNA at 260 nm, curve b shows that SiO2 microspheres have no UV absorbance peak. CEA exhibits a characteristic absorption peak at 270 nm (curve c). The Mn: ZnCdS@ZnS quantum dots exhibits UV absorption at about 400 nm (curve d). When DNA S1 specifically binds to CEA, it can be seen that two characteristic absorption peaks (curve e) appeared at 256 and 270 nm, respectively, and there is a slight change in peak position, indicating the successful and specific binding of CEA to DNA S1. After the Mn: ZnCdS@ZnS QDs were labeled to probe DNA P2 and then bounded to SiO2 microspheres via DNA S2,
Figure 6. Electrophoregran for (A) conjugation process of signal probe P1; (B) fluorescence assay of target T4 PNK.
3 μL of DNA 6 (10−5 M), and lanes 3, 4, and 5 are the same amount of DNA 7, 8, 9. Lane 6 is an equivalent amount of DNA P1. The higher band position in lane 6 indicated a higher molecular weight of P1, demonstrating that the four DNA strands were successfully hybridized to each other to construct a cross-structured signal probe P1. In Figure 6 (B), lane 1 is the mark, lane 2 is the double-stranded structure of DNA S1/ 4641
DOI: 10.1021/acsanm.9b01003 ACS Appl. Nano Mater. 2019, 2, 4637−4645
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Figure 7. (A) FL signals on the target CEA with various concentrations. (a) 0 g/mL; (b) 0.1 pg/mL; (c) 1.0 pg/mL; (d) 0.01 ng/mL; (e) 0.1 ng/ mL; (f) 1.0 ng/mL; (g) 10 ng/mL; (B) Correlation of the fluorescence change with concentrations of CEA. Inset: Linear correlation of the fluorescence change with logarithmic concentrations of CEA.
and T4-PNK at 1.0 ng/mL and 0.1 U/mL, respectively, the amount of phi29 for fluorescence detection were optimized. The left ordinate in curve a corresponds to the fluorescence signal of CdTe QDs, and the right ordinate in curve b corresponds to the fluorescence signal of Mn:ZnCdS@ZnS QDs. The two fluorescence signals increased with increasing amount of phi29 polymerase, and did not change obviously when the dosage of phi29 polymerase reached 10 U, so 10 U/ 50 μL phi29 was chosen as the optimum value for target detection. Similarly, the concentrations of λExo and ATP also played important role for detection of T4 PNK activity. As shown in SI Figure S2 (A), when T4 PNK is absent, there is no significant increase in the fluorescence signal with increasing ATP concentrations. While the concentration of T4 PNK was at 10 U/mL, the fluorescence signal increased with increasing ATP concentration, and reached a maximum value at 2 mM concentration, so the optimal ATP concentration is 2 mM. Similarly, as shown in SI Figure S2 (B), the effect of λExo dosage on the assay of T4 PNK was explored. When T4 PNK was absent, there is a small change with increasing dosage of λExo due to the nondirectional microshearing. Keeping the amount of T4 PNK at 10 U, the fluorescence signal increased with the increase of λExo, and there is no obvious change of fluorescence signal at 1.0 U of λExo, so 10 U λExo per 50 uL was chosen as the best dosage. The influence of reaction time on the fluorescence signal of T4 PNK sensing system was explored. As shown in SI Figure S3, when T4 PNK is absent, there is no increase in the background fluorescence signal with increasing reaction time, while the fluorescence signal gradually increased with increasing reaction time of T4 PNK, and a maximum value was observed at 120 min. Therefore, 120 min was chosen as the optimum time in the work. Performance of the Fluorescence Strategy for Amplified Assay of CEA. Under the optimized conditions, the fluorescence strategy was used for amplified detection of CEA. As shown inFigure 7A, in the absence of CEA, the fluorescence signal was very low due to adsorption of a small amount of QDs (curve a). When CEA was present, it specifically bound to its aptamer, and a lot of DNA2 was released with the recycle of target CEA, which was bound to DNA S2 and linked more Mn:ZnCdS@ZnS QDs in P2. The
DNA3 by polymerization. In the presence of T4 PNK kinase, ATP and λExo exonuclease, the 5 ’hydroxyl group of DNA3 is phosphorylated by T4 PNK, which is then cleaved by λExo to release single-stranded DNA S1, lane 3 is the product of reaction. Lane 4 is the reaction product of DNA 4 and DNA 5 without DNA Sl, two lower bands in the lane indicated that HCR could not performed. Lane 5 is the prepared signal probe P1. The higher band in lane 6 is the reaction product via HCR of DNA S1 with DNA4 and DNA 5 and then conjugation to signal probe P1. The results suggest that the fluorescence assay for T4 PNK activity was successfully performed. Confocal Fluorescence Imaging Characterization of the Analysis Method for Dectection of two Targets. The feasibility of the fluorescence sensing methods based on the QDs was investigated by confocal fluorescence microscopy imaging. As shown in Supporting Information (SI) Figure S4 (A), when the target CEA specifically binds to its aptamer, the DNA2 is released quantitatively, and then binds to DNA S2 on the surface of the SiO2 microsphere and the Mn:ZnCdS@ZnS QDs signal probe P2. Fluorescence confocal microscopy image displayed bright fluorescence on the surface of SiO2 microspheres, which indicated that the fluorescence sensing method for quantitative detection of target CEA was successfully constructed. As shown in SI Figure S4 (B), when the double-stranded structure DNA S1/DNA3 on the surface of SiO2 microspheres was performed phosphorylation reaction with T4 PNK and ATP and cleaved by λExo, DNA4 and DNA5 were introduced to conduct HCR amplification reaction and then linked with the signal probe P1, bright fluorescence of CdTe QDs on the SiO 2 microspheres was observed, indicating that the fluorescence method for detecting T4 PNK activity was achieved. These fluorescence imaging results demonstrated the fluorescence assay methods based on two signal probes and enzyme-assisted cyclic amplification was successfully fabricated for detection of two targets. Effects of the Enzyme Concentration, Reaction time and ATP Concentration on the Fluorescence Signal for Detecting Targets. According to the design principle, phi 29 polymerase-assisted target recycling plays an important role for detecting CEA and T4 PNK activity. SI Figure S1 explores effects of the phi29 polymerase dosage on fluorescence signal for target detection. Keeping the concentrations of target CEA 4642
DOI: 10.1021/acsanm.9b01003 ACS Appl. Nano Mater. 2019, 2, 4637−4645
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ACS Applied Nano Materials fluorescence signal increased with increase of CEA concentrations (curve b to g), indicating the fluorescence signal can be used for CEA detection. As shown in Figure 7B, the fluorescence signal is linearly related with the logarithm of target CEA in the range of 0.1 pg/mL to 10 ng/mL (R = 0.995) (inset). The detection limit is 0.065 pg/mL (3σ/N, σ is the standard deviation of the blank and N is the slope of the corresponding calibration plot). Five times of the experiments at 1.0 ng/mL CEA using DNA modified Mn:ZnCdS@ZnS QDs in different batches were repeated for estimating the precision of this method, and a relative standard deviation (RSD) of 3.4% were obtained, the results showed that the fluorescence method based on the QDs signal probe and enzyme-assisted target amplification has good reproducibility for CEA detection. Fluorescence Assay of T4 PNK. Based on the formed double-stranded DNA1/DNA3 structure, the activity of T4PNK kinase was further detected by using the cross-structured QDs signal probe. Figure 8 showed the feasibility of the fluorescence sensing assay for T4 PNK. The CdTe quantum dots displayed very
high FL signal (curve a). When the QDs were labeled to DNA to form the fork structure probe P1, the fluorescence signal decreased as the QDs structure changed, and the fluorescence peak slightly shifted (curve b). When the target T4 PNK was absent, there was a low background signal due to the adsorption of a small amount of signal probe (curve c). In the presence of target T4 PNK without λExo, HCR reaction cannot be conducted, the FL signal is also not high (curve d). After T4 PNK and λExo were both present, DNA4 and DNA5 were introduced to conduct HCR amplification reaction, and more signal probes were combined with SiO2 microspheres, so the fluorescence signal is significantly enhanced (curve e), suggesting that the amplified fluorescence analysis of T4 PNK activity is feasible. Figure 9A displayed the fluorescence signals to T4 PNK at various concentrations. Without T4 PNK, the blank signal was low (curve a). When T4 PNK was present, the fluorescence signals were enhanced by HCR amplification, and the signals increased with increase of T4 PNK activity (curve b to g), demonstrating the fluorescence signals have relationship with T4 PNK in the range of 0.001 U/mL to 5 U/mL, which can be used for detection of T4 PNK activity. Figure 9B displayed the relationship of the fluorescence signal with T4 PNK activity, the FL signal was linearly correlated to the logarithmic concentrations of T4 PNK activity in the range of 0.001 U/mL to 10 U/mL (R = 0.992) with a detection limit of 0.001 U/mL (3σ/N, σ is the standard deviation of the blank and N is the slope of the corresponding calibration plot). Five replicates at 0.1 U/mL of T4 PNK were performed to estimate the precision of this assay, the relative standard deviation (RSD) was 4.6%, showing good reproducibility. Selective Analysis of CEA and T4 PNK by the Fluorescence Method. The specificity of the fluorescence method for CEA assay was investigated, and the fluorescence signals of the interference substances were performed under the same experimental conditions. As shown in SI Figure S5 (A), significant fluorescence signal change was observed at 0.1 ng/mL of CEA, while no obvious signal changes were seen for the interferences such as BSA, PSA, and AFP, even at 1.0 ng/ mL, indicating that the method has good selectivity for CEA detection. Similarly, the selectivity investigation for T4 PNK assay was conducted. The concentration of the target T4 PNK was at 0.1
Figure 8. Fluorescence signal responses of the sensing assay: (a) CdTe QDs; (b) P1-CdTe QDs; (c) background signal without target T4-PNK; (d) in the presence of target T4 PNK without HCR reaction, (e) in the presence of the target T4 PNK by HCR reaction. (excitation wavelength: 320 nm, slit 5.0).
Figure 9. (A) FL signals for detection of target T4 PNK with various concentrations (U/mL). (a) 0; (b) 0.001; (c) 0.005; (d) 0.1; (e) 0.5; (f) 1.0; (g) 5.0; (B) Relationship between the fluorescence signal and T4 PNK activity. Inset: Linear correlation of the fluorescence change with logarithmic concentrations of T4 PNK. 4643
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U/mL, and the effects of other proteins on T4 PNK assay in the same conditions were examined. As shown in SI Figure S5 (B), the interfering enzyme such as BSA, ALP, and thrombin even at 1 U/mL did not lead to obvious change to FL signal, while significant signal change was observed at 0.1 U/mL of T4-PNK. Therefore, this method has good selectivity for detection of T4-PNK. Application of the Fluorescence Method for CEA Detection in Real Samples. The potential application of the fluorescence method in real sample analysis was evaluated, the CEA serum samples were obtained from the Affiliated Hospital of Qingdao University, and detected using our developed method. The samples were diluted to 2.28 pg/mL and 35.1 pg/mL, and the detected results using our method were 2.21 pg/mL and 35.8pg/mL, respectively. The consistent results demonstrated that the method can be applied to real biological samples.
CONCLUSIONS In conclusion, a novel fluorescence strategy based on two multiple-branched QDs probes coupled with enzyme-aided target recycle and HCR amplification strategy was designed for versatile detection of CEA and PNK activity. Two new QDs with excellent fluorescence were used to label DNA for fabricating the unique cross-structured signal probes. The amino-modified SiO2 microspheres with good morphology and no interference to fluorescence signal were used to link DNA and QDs signal probes for convenient amplified analysis of two targets. The Mn:ZnCdS@ZnS QDs labeled P2 signal probe combined with polymerase-aided target recycle amplification strategy was first applied to CEA assay. Then the present T4 PNK led to the phosphorylation reaction and HCR to fabricate CdTe QDs P1 for amplified fluorescence detection of PNK. The proposed multichannel detection method is beneficial to the simultaneous assay of different substances in the same system, which possess promising application in biosensing analysis. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.9b01003. Chemicals and materials, DNA sequences, apparatus, effects of the phi29 amount on fluorescence signal for assay of targets, effects of the ATP concentration on the fluorescence signal for detection of T4 PNK, effects of the λExo amount on fluorescence signal for T4 PNK assay, effects of the reaction time on fluorescence signal for T4 PNK detection PDF)
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Article
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Guifen Jie: 0000-0002-6416-6523 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21575072). 4644
DOI: 10.1021/acsanm.9b01003 ACS Appl. Nano Mater. 2019, 2, 4637−4645
Article
ACS Applied Nano Materials
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DOI: 10.1021/acsanm.9b01003 ACS Appl. Nano Mater. 2019, 2, 4637−4645
