DNA Dendrimer–Streptavidin Nanocomplex: an Efficient Signal

May 18, 2017 - We develop a DNA dendrimer–streptavidin (SA) nanocomplex as a novel signal amplifier to create biosensing platforms for disease-relat...
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DNA Dendrimer–Streptavidin Nanocomplex: an Efficient Signal Amplifier for Construction of Biosensing Platforms Yan Zhao, Shichao Hu, Huaming Wang, Kaiwen Yu, Yan Guan, Xiaoyun Liu, Na Li, and Feng Liu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 18, 2017

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DNA Dendrimer–Streptavidin Nanocomplex: an Efficient Signal Amplifier for Construction of Biosensing Platforms Yan Zhao,† Shichao Hu,† Huaming Wang,‡ Kaiwen Yu,† Yan Guan,† Xiaoyun Liu,† Na Li,† and Feng Liu*,† †Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. ‡Hubei Provincial Key Laboratory for Applied Toxicology, Institute of Health Inspection and Testing, Hubei Provincial Center for Disease Control and Prevention, Wuhan 430079, China. KEYWORDS: Nonlinear hybridization chain reaction, DNA dendrimer, streptavidin, biosensing

ABSTRACT: We develop a DNA dendrimer–streptavidin (SA) nanocomplex as a novel signal amplifier to create biosensing platforms for disease-related species. The DNA dendrimer–SA nanocomplex is fabricated by crosslinking the nonlinear hybridization chain reaction based DNA dendrimer with the SA-coupled linker DNA, and possesses multiple sticky ends, a high molecular weight, and a hyperbranched nanostructure with large numbers of DNA duplexes. Taking advantages of the DNA dendrimer–SA nanocomplex and a label-free quartz crystal microbalance (QCM) technology, we first construct a mass-sensitive QCM biosensing platform for nucleic acids, which displays high selectivity and sensitivity with the detection limit of 0.062 nM KRAS gene fragment. Then we present a fluorescent sensing strategy towards HeLa cells by functionalizing the DNA dendrimer–SA nanocomplex using the sgc8 aptamer and the SYBR Green I intercalating dye. The spiked recoveries of targets in physiological media are greater than 90%, demonstrating potential application of created biosensing platforms in clinical diagnosis. This work expands the rule set of designing DNA nanomaterials for development of biosensing strategies, and provides universal platforms for detecting diseaserelated species through simply altering the related capture and reporter DNA sequences.

Developing highly sensitive and selective biosensing strategies for detecting disease-related species is of central importance in disease diagnosis and environmental monitoring.1−3 Recent advances in DNA nanotechnology enable development of a variety of assembled DNA nanostructures as signal amplifiers for effectively improving the performances of biosensing platforms.4−7 Among various DNA nanotechnologies, the hybridization chain reaction (HCR) is one of the well-known amplification strategies.8 Importantly, the linear dsDNA product of HCR with multiple repeated sequences is highly ordered, therefore different kinds of signal indicators can be attached separately on these repeated sequences to generate optical9−11 and electrochemical12−14 output signals. These approaches could further enhance the sensitivity of biosensing platforms. In addition to one-dimensional HCR products, nonlinear HCR systems that enable the formation of branched or dendritic nanostructures with higher dimensions have also drawn attention of researchers in recent years.15−17 In a typical nonlinear HCR system, a trigger DNA sequence initiates an exponential chain-branching growth of assembled DNA dendrimers in a self-sustained way, which leads to an exponential increase in signal intensity and holds great promise for developing biosensing strategies. LaBean’ group presented a nonlinear HCR system for DNA detection, in which a target DNA sequence triggers a HCR between two-loop structured hairpins to form an exponentially growing dendritic nanostructure, but detection sensitivity and selectivity are limited due to the false posi-

tive signal.15 Recently, Hsing’ group designed a hairpin-free nonlinear HCR system in which the quenched double-stranded DNA substrates could successively assemble into the fluorescent DNA dendrimers by the exponential chain-branching growth.16 Basing on that, Hsing’ group developed an immobilization-free and enzyme-free electrochemical sensor for the nucleic acid detection with high sensitivity.17 Despite the great potential of employing nonlinear HCR systems in biosensing, it should be admitted that the related research is still in its early stage. It is still highly desirable to move forward the application of nonlinear HCR systems in developing biosensing strategies. In the current study, we aimed to design nonlinear HCR based signal amplifiers for constructing biosensing platforms. Herein, we develop a DNA dendrimer–streptavidin (SA) nanocomplex as an effective signal amplifier to create two facile and enzyme-free biosensing platforms for diseaserelated species. The DNA dendrimer–SA nanocomplex was fabricated by crosslinking the nonlinear HCR based DNA dendrimer with the SA-coupled linker DNA, and the selfassembly process of the DNA dendrimer–SA nanocomplex was verified by scanning electron microscopy. Taking advantages of the DNA dendrimer–SA nanocomplex and a labelfree quartz crystal microbalance (QCM) technology, we created a facile and mass-sensitive QCM biosensing platform for nucleic acids. Then a new fluorescent probe was prepared based on the functionalization of the DNA dendrimer–SA nanocomplex by the aptamer and the SYBR Green I intercalat-

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ing dye for cancer cell sensing. The performances of two biosensing platforms were explored by using the KRAS gene fragment and the HeLa cell as target models, respectively. This is the first time that the nonlinear HCR based system has been realized to construct the QCM and the fluorescence platforms for nucleic acid and cancer cell sensing, respectively. EXPERIMENTAL SECTION Materials. Tris(2-carboxyethyl)phosphine (TCEP), 6mercapto-1-hexanol (MCH), streptavidin (SA), and poly(styrene-co-maleic acid) (PSMA, Mw = 65000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-(3dimethyl-aminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) was obtained from J&K Scientific Ltd. (Beijing, China). N-hydroxysuccinimide (NHS) was ordered from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). SYBR Green I dye was purchased from Beijing Bridgen Biotechnology Co. Ltd (Beijing, China). RIPA cell lysis solution was obtained from YuanPingHao Bio. (Beijing, China). Fetal bovine serum (FBS) was ordered from Solarbio (Beijing, China). Bovine serum albumin (BSA) was obtained from Beijing Kemeng Purui Biotechnology Co. Ltd (Beijing, China). 50X TAE buffer and all HPLC-purified DNA oligonucleotides (Table S1) were obtained from Sangon Inc. (Shanghai, China). All DNA oligonucleotides and SA were dissolved separately in 1X TAE buffer and stored in the dark at 4 °C, and then filtered by 0.45 µm filter membranes prior to use. All chemicals were of analytical grade, and deionized water was used in all experiments. Preparation of the DNA Dendrimer–SA Nanocomplex. Firstly, the nonlinear HCR based DNA dendrimer was prepared according to the recently reported method.16 The double stranded substrates (Substrate-A and Substrate-B) were fabricated by respectively heating the toehold strands (T1 and T2) with the protective strands (P1 and P2) at 85 °C for 5 min, and then annealed slowly to room temperature. After incubating Substrate-A and Substrate-B with corresponding assistant strands (Assistant-A and Assistant-B) respectively for 30 min, the initiator was introduced to trigger the formation of the nonlinear HCR based DNA dendrimer for 1 h. Next, the SALinker DNA was first obtained by directly incubating the linker DNA with SA for 20 min. The DNA dendrimer–SA nanocomplex was prepared by incubating the DNA dendrimer with the SA-Linker DNA for 20 min. The assembly temperature of the DNA dendrimer–SA nanocomplex was 30 °C and the concentration of the dendrimer–SA nanocomplex as 1X is indicated by the concentrations of the involved reagents in Table S2. Characterization of the DNA Dendrimer–SA Nanocomplex. The silicon wafers were immersed in newly prepared piranha solution (concentrated sulfuric acid and 30% H2O2 in a volume ratio of 3:1) for 5 min and then rinsed with deionized water. 25 µL of the DNA dendrimer and DNA dendrimer–SA nanocomplex (4X) were deposited respectively on the cleaned silicon wafers for 1 h, then gently rinsed the surface of silicon wafers with deionized water and dried in a vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd. Shanghai, China) overnight. The morphologies of the DNA dendrimer and the DNA dendrimer–SA nanocomplex were ob-

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served and recorded using an S-4800 scanning electron microscope (Hitachi, Japan). Caution: the piranha solution is extremely corrosive and potentially explosive, and should be careful to avoid skin contact. QCM Measurements of Nucleic Acids. All on-line QCM measurements were monitored at 20 °C in 1X TAE buffer by a Q-Sense E4 QCM-D instrument with four chambers (Q-Sense AB, Västra Frölunda, Sweden) which can provide real-time response of multiovertone frequencies. The gold-coated crystal chips (5 MHz, AT-cut) (Dongwei Biotechnology Co., Ltd. Hangzhou, China) were immersed in a boiling solution (30% H2O2, 28% ammonia water, and deionized water in a volume ratio of 1:1:5) for 15 min, and then rinsed thoroughly with deionized water and dried by nitrogen gas before loading to measuring cells. The mass probe was obtained by directly incubating the DNA dendrimer–SA nanocomplex (1X) with the DNA reporter (0.2 µM) at 30 °C for 20 min. In a typical experiment, a 0.1 µM capture probe (CP) solution was pretreated by 10 µM TCEP at room temperature for 60 min, then injected into the QCM chambers for modifying the chips for 25 min. Subsequently, a 1 mM MCH solution was injected into the chambers for 10 min to remove the nonspecific CP adsorption and block the chip surface. The running rate of CP and MCH was 20 µL/min set by an Ismatec IPC tubing pump (Glattbrugg, Switzerland). Then the target DNA flowed through the chip surface for 90 min and the mass probe was injected into the QCM chambers for 20 min successively with a running rate of 10 µL/min. The frequency changes were obtained at 9 overtones. Fluorescent Sensing and Imaging of Cancer Cells. The quartz glass slides (1.4 cm × 2.5 cm) were immersed in trimethyl chlorosilane overnight and in PSMA solution (0.67 g PSMA dissolved in 5 mL tetrahydrofuran) for 10 s successively, and then taken out to evaporate the residual solvent at room temperature overnight. The fluorescent probe was obtained by incubating the DNA dendrimer–SA nanocomplex (0.5X) with the sgc8 aptamer reporter (0.1 µM) for 20 min and SYBR Green I dye (2.5 µM) for 20 min successively. The quantum yield of the fluorescent probe was measured according to the procedures of the ESI. In a typical fluorescence cell sensing, the PSMA coated quartz glass slides were first activated with 300 µL mixture of EDC (0.4 mg/mL) and NHS (0.6 mg/mL) for 30 min, and then modified with the sgc8 aptamer capture (0.2 µM, 300 µL) for 2 h. Subsequently, the aptamer capture functionalized quartz glass slides were washed with 1X TAE buffer and blocked by BSA (2.5%, 300 µL) for 10 min. Then the glass slides were incubated with various concentrations of HeLa cells for 30 min, followed by 300 µL fluorescent probe (0.5X) dropped on the glass slides for further signal amplification. After washing with 1X TAE buffer to remove the unbound fluorescence probe, the quartz glass slides were inserted separately in a 1 cm fluorometric cell along the diagonal for fluorescence measurements. The fluorescence spectra were recorded from 500 to 700 nm with the excitation wavelength of 488 nm using an F-7000 fluorescence spectrophotometer (Hitachi, Japan). The fluorescence images were observed by an A1Rsi laser scanning confocal microscopy (Nikon, Japan).

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Figure 1. Schematic representation of the assembly of the DNA dendrimer–SA nanocomplex.

Cell Culture and Treatment. HeLa cells and HEK293T cells were incubated in Dulbecco’s Modified Essential Medium (DMEM, Solarbio, China) containing 10% fetal bovine serum at 37 °C in 5% CO2. Then the cells were harvested by 0.25% trypsin and resuspended in PBS buffer when reaching confluence. The cell density was measured to be 1.0 × 106 cells/mL using a hemocytometer. For the preparation of HeLa cell lysate, the incubated HeLa cells were pelleted at 3,000 rpm for 5 min at 4 °C and then resuspended in RIPA cell lysis solution at a concentration of 5.0 × 106 cells/mL. Then the cells were incubated at −20 °C for 30 min, followed by centrifugation at 12,000 rpm for 30 min at 4 °C. The supernatant was collected and filtered by 0.45 µm filter membrane prior to store at −20 °C. RESULTS AND DISCUSSION Fabrication and Characterization of the DNA Dendrimer–Streptavidin Nanocomplex. The fabrication principle of the DNA dendrimer–streptavidin (SA) nanocomplex is illustrated in Figure 1. First, we describe the fabrication rationale of the DNA dendrimer based on the nonlinear HCR system.16 Two double-stranded DNA substrates (Substrate-A and Substrate-B) and two single-stranded DNA assistants (Assistant-A and Assistant-B) are used as building blocks of nonlinear HCR system. Substrate-A and Substrate-B possess the exposed toehold domain and the loop toehold domain in the middle. After introducing a single-stranded DNA initiator, the self-assembly process begins from the hybridization between the initiator and the exposed toehold domain of Substrate-A through the toehold-mediated strand displacement reaction (SDR), and then the P1 strand is displaced from Substrate-A by hybridizing with Assistant-A, which consecutively open two repeating loops of Substrate-A. The newly exposed two toehold domains of Substrate-A could simultaneously hybridize with the toehold domain of two Substrate-B by the toehold-mediated SDR. Then the P2 strand is displaced from Substrate-B with the help of Assistant-B, leading to the formation of a branched DNA nanostructure with two same single-stranded arms. Given that the new arm sequence is the same as the initiator, a new round of the toehold-mediated SDR begins by using the new arm sequence as the initiator.

This chain-branching growth of the branched DNA nanostructure results in the formation of DNA dendrimer. Subsequently, the preparation of DNA dendrimer–SA nanocomplex is based on the fact that one SA molecule has four binding sites for coupling with the biotinylated single strand DNA (named as linker DNA). The SA-coupled linker DNA (named as SALinker DNA) is prepared by directly incubating the linker DNA with SA, and then introduced into the as-prepared DNA dendrimer solution. Because the designed linker DNA can hybridize with the arm sequences of the DNA dendrimer and simultaneously stop the toehold-mediated SDR, the finally assembled DNA dendrimer–SA nanocomplex is fabricated by crosslinking the DNA dendrimer with SA-Linker DNA as bridges. To demonstrate the self-assembly process of the DNA dendrimer–SA nanocomplex, the morphologies of the DNA dendrimer and the DNA dendrimer–SA nanocomplex were characterized by scanning electron microscopy (SEM). As can be seen in Figure 2A, the DNA dendrimer shows a circular shape and the average diameter calculated from 52 nanoparticles is 140 ± 56 nm, which confirms the triggered chain branching growth of the DNA dendrimer. The DNA dendrimer–SA nanocomplex also has a circular shape and shows an average diameter of 387 ± 113 nm calculated from 36 nanoparticles (Figure 2B). The remarkably growing size of the DNA dendrimer–SA nanocomplex is good visual evidence that supports the occurrence of the SA-Linker DNA mediated crosslinking process as anticipated.

Figure 2. SEM images of (A) DNA dendrimer and (B) DNA dendrimer–SA nanocomplex.

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This novel DNA dendrimer–SA nanocomplex possesses three major advantages: (1) it has multiple sticky ends which make it easy to be functionalized by reporter probes, (2) it has a high molecular weight, and (3) its hyperbranched nanostructure offers large number of DNA duplexes for preparing dyeintercalated fluorescent probes. Thus the proposed DNA dendrimer–SA nanocomplex has great potential as an efficient signal amplifier to create sensing platforms by combining with different instruments for detecting disease-related species, such as nucleic acids and cells. DNA Dendrimer–SA Nanocomplex As a Mass Signal Amplifier for the Nucleic Acid Sensing Platform The quartz crystal microbalance (QCM) is a widely used label-free and mass-sensitive piezoelectric technology that enables detection of biorecognition events in real-time.18,19 Generally, the QCM biosensors show low sensitivity to directly detect nucleic acids by the hybridization reaction without further signal amplification. Hence, to improve the sensitivity of QCM biosensing platforms for nucleic acids, much attention has been focused on developing signal amplification strategies, such as using metal nanoparticles,20,21 liposomes,22 enzymatic reactions,23,24 or proteins25,26 as signal amplifiers. Recently, our group designed self-assembled DNA nanostructures as mass amplifiers to develop the facile and enzyme-free QCM platforms for nucleic acid sensing with good predictability and biocompatibility.27,28 To date, it is still highly desirable to fabricate DNA nanostructure-based mass amplifiers for QCM biosensors. Herein, we utilize the as-prepared DNA dendrimer–SA nanocomplex as a novel mass amplifier to create a QCM platform for highly sensitive and selective nucleic acid sensing.

Figure 3. Schematic representation of the construction and rationale of the amplified QCM biosensing platform for target DNA. (A) Mass probe preparation. (B) Sensing process.

Recognition and Amplified Rationale of the QCM Biosensing Platform. The construction and rationale of the amplified QCM biosensing platform based on the DNA dendrimer–SA nanocomplex as a signal amplifier is illustrated in Figure 3. We choose a 28 nt KRAS gene fragment related to human cancers as a nucleic acid target model (abbreviated as target DNA). As shown in Figure 3A, the mass probe is prepared by incubating a DNA reporter with the DNA dendrimer–SA nanocomplex to specifically recognize the target DNA. The typical sensing process is shown in Figure 3B. Firstly, the crystal chip is functionalized with the hairpin capture probe (CP) and blocked with MCH by the Au–S bond in sequences. Then the target DNA is introduced to the chip surface to open the hairpin CP by a strand displacement reaction.

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Finally, the mass probe is injected to hybridize with the newly exposed sticky end of CP, which produces an amplified frequency shift as the output signal. This novel sensing strategy combines the effective amplification of the high molecular weight DNA dendrimer–SA nanocomplex with the label-free and real-time QCM technique. In addition, the DNA dendrimer–SA nanocomplex as a signal amplifier can be prepared off-line, which would be extremely beneficial for shortening the on-line detection time. Performances of the QCM Biosensing Platform. The signal amplifier is considered as a crucial factor for the improvement of sensing sensitivity, thus we mainly focused on optimizing the assembly parameters of the DNA dendrimer–SA nanocomplex in detail (Figure S1–S7). Sensitivity. To demonstrate the sensitivity of the proposed QCM sensing platform, the real-time frequency responses to different target DNA concentrations were measured under the optimal conditions (Figure 4A). In the absence of target DNA, only a negligible frequency decrease was recorded, indicating no clear adsorption of the mass probe on the chip surface. In contrast, dramatic frequency shifts were monitored in the presence of target DNA with increasing concentration from 0.5 to 30 nM. Figure 4B depicts the linear relationship between the frequency changes and the target DNA concentrations in the range of 0.5–25 nM (r = 0.9978) with a detection limit (LOD) of 0.062 nM calculated by the 3σ/k rule (n = 9), showing about 1.4 and 2.3 orders of magnitude improvement in sensitivity compared to the systems using the nonlinear HCR-based DNA dendrimer as a signal amplifier (LOD = 1.5 nM) and direct detection (LOD = 12 nM), respectively. These results confirm that the rationally designed DNA dendrimer– SA nanocomplex plays a key role in the dramatically enhanced sensitivity of the QCM biosensing platform. The obtained sensitivity is comparable to the best reported QCM nucleic acid sensors based on the target-triggered in situ assembly of the DNA nanostructures as the mass signal amplifiers.27,28 Moreover, the DNA dendrimer–SA nanocomplex can be prepared off-line, thereby efficiently shortening the on-line signal amplification time from 120 min27 and 50 min28 to 20 min for the target DNA detection. Selectivity. To quantitatively evaluate the specificity of the proposed sensing platform, a series of single-base mismatch sequences of KRAS were injected separately to the QCM chamber to obtain the corresponding discrimination factors. The discrimination factor is defined as the ratio of the signal gain obtained with the complementary target DNA to that obtained with the mismatch sequence under the same conditions. As shown in Figure 4C, the discrimination factors are 10.1, 10.9, and 13.7 for A>G, A>T, and A>C substitutions respectively, clearly demonstrating the high selectivity of the fabricated QCM sensing platform. Target DNA Detection in Spiked Samples. To evaluate the robustness of the constructed QCM sensing platform in the physiological medium, a 10-fold diluted HeLa cell lysate was spiked with the target DNA. The spiked recovery of 20 nM target DNA is 90% with a RSD of 4.2% (n = 3) (Figure 4C). Meanwhile, no evident frequency decrease is observed using an unspiked lysate sample, indicating the possibility for biological sample applications.

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Figure 4. (A) Frequency response curves of the DNA dendrimer–SA nanocomplex amplified QCM sensing platform with different target DNA concentrations. (B) Linear relationship between frequency responses and target DNA concentrations. (C) Frequency responses of the DNA dendrimer–SA nanocomplex amplified QCM sensing platform for a series of targets (20 nM): target KRAS, single-base mutant sequences, control sample, and target DNA spiked or unspiked in 10-fold diluted HeLa cell lysates, respectively. The error bars represent the standard deviation of three measurements.

To summarize, we have successfully prepared the DNA dendrimer–SA nanocomplex as an efficient mass signal amplifier to create the QCM sensing platform for highly sensitive and selective detection of nucleic acids. Taking advantages of the DNA dendrimer–SA nanocomplex and a label-free and real-time QCM technology, this biosensing platform achieved remarkable improvement in sensitivity with no need for complex separation process or enzymatic reaction. In addition, the DNA dendrimer–SA nanocomplex can be prepared off-line, which effectively accelerates the on-line detection of the target DNA. The proposed amplification strategy extends the application of nonlinear HCR and QCM technique, and provides a universal platform for detecting other nucleic acid fragments by easily altering the related capture and reporter DNA sequences. DNA Dendrimer–SA Nanocomplex Based Fluorescent Probe for the Cell Sensing Platform. In recent years, aptamer-functionalized nanomaterials based fluorescence signal amplification strategies for cancer cells have attracted considerable attention.29−31 Combining the advantages of aptamers as molecular recognition units and nanomaterials as signal amplifiers such as quantum dots,32,33 silver nanoclusters,34,35 or upconversion nanoparticles,36 the aptamer-functionalized nanomaterials based fluorescence sensing platforms for cancer cells achieve efficient improvement in sensitivity and selectivity, but the toxicity of these nanomaterials and the potential risks for the environment and human health should be evaluated. Recently, the aptamerfunctionalized fluorescent DNA nanostructures such as onedimensional HCR products37,38 and three-dimensional DNA dendrimer39 were applied for constructing the cell sensing platforms, which possesses the advantages of good predictability and biocompatibility. Moreover, to our knowledge, designing the nonlinear HCR based fluorescent DNA nanostructures for cancer cell sensing has not been exploited yet. Herein, we fabricate a new fluorescent probe based on the DNA dendrimer–SA nanocomplex for creating a facile fluorescent sensing platform towards cancer cells. Fabrication and Sensing Rationale of the Fluorescent Platform. Figure 5 shows the construction and rationale of the fluorescent sensing platform for cancer cells based on the

DNA dendrimer–SA nanocomplex. We choose the HeLa cell as a cell model to demonstrate the sensing principle. Based on the high specificity between the sgc8 aptamer and the overexpressed PTK7 membrane protein on HeLa cells, we first fabricate a fluorescent probe by functionalizing the DNA dendrimer–SA nanocomplex with the sgc8 aptamer reporter and the SYBR Green I intercalating dye40 successively (Figure 5A). For the HeLa cell sensing (Figure 5B), the quartz glass slide is first coated with the PSMA dense film, then modified with the sgc8 aptamer capture and blocked with BSA, respectively. By dropping the HeLa cell solution on the modified glass slide, the HeLa cells are captured via the specific binding between the sgc8 aptamer and the PTK7 on HeLa cells. Finally, the sgc8 aptamer functionalized fluorescent probe is introduced to further bind with the captured HeLa cells, which produces an enhanced fluorescence as the output signal. The proposed fluorescent biosensing platform possesses two advantages: (1) The fluorescent signal is effectively amplified because the fluorescent probe contains a large number of SYBR Green I molecules, and (2) the sensing procedures can be conducted facilely, with no requirement for separation process or enzymatic reaction.

Figure 5. Schematic representation of construction and rational of the amplified fluorescence sensing platform for HeLa cells. (A) Fluorescent probe preparation. (B) Sensing process.

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Figure 6. (A) UV-Vis absorption, excitation, and emission spectra of the fluorescence probe. (B) Linear relationship between fluorescence intensity and HeLa cell concentrations. (C) Fluorescence responses of the sensor to 5.0 × 104 cells/mL HeLa cell and HEK293T cell in TAE buffer and 10-fold diluted FBS, respectively. The error bars represent the standard deviation of three measurements.

Performances of the Fluorescent Biosensing Platform. We first evaluated the spectral properties of the prepared fluorescent probe. The absorption, excitation, and emission spectra of fluorescent probe are shown in Figure 6A. Two peaks shown in the absorption spectrum at 260 nm and 488 nm are the characteristic absorption peaks of DNA base pairs and the SYBR Green I respectively. Upon excitation at 488 nm, the maximum emission wavelength of the fluorescent probe is at 525 nm. The quantum yield and the fluorescence lifetime of fluorescent probe are 0.42 and 5.00 ± 0.04 ns, respectively.

Figure 7. Confocal fluorescence microscopy images of the fluorescent sensing platform for HeLa cells and HEK293T cells. The concentrations of each cell were at 1.0 × 105 cells/mL.

Then we investigated the performances of the fluoresence biosensing platform based on our fluorescent probe under the optimal conditions (Figure S8 and S9). As can be seen from Figure 7, the fluorescent probe captured HeLa cells show strong green fluorescence, while only a few non-cancerous HEK293T cells without PTK7 on the cell surface are captured on the glass slide, and no fluorescence is observed. As shown in Figure 6B, the fluorescence intensity of the sensor increases with the concentration of HeLa cells in the range of 0.5 × 104–1.5 × 105 cells/mL (r = 0.9988) with a detection limit (LOD) of 4.4 × 103 cells/mL calculated by the 3σ/k rule (n = 9), showing about 8-fold improvement in sensitivity compared to that of using the nonlinear HCR-based DNA dendrimer as the signal amplifier (LOD = 3.4 × 104 cells/mL). We further investigated the selectivity of the proposed fluoresence sensing platform (Figure 6C). The fluorescence intensity obtained from HeLa cells is 5-fold to that of HEK293T cells in the TE

buffer solution, indicating good specificity towards HeLa cells. The spiked recovery of 5 × 104 cells/mL HeLa cells in 10-fold diluted FBS is 94% with an RSD of 6.1% (n = 3), implying that this biosensing platform exhibits promising potential for practical applications. The results demonstrate the capability of the proposed fluorescence sensing platform in sensitive and selective detection of the HeLa cells. The proposed fluorescent sensing approach also extends the application of nonlinear HCR, and provides a universal platform for other cancer cell sensing by easily altering the related aptamer sequences. CONCLUSIONS In this work, we have successfully fabricated a DNA dendrimer–SA nanocomplex based on the nonlinear HCR system, and used it as an efficient signal amplifier to construct two facile and enzyme-free biosensing platforms. The crucial element of two biosensing platforms is the rationally designed DNA dendrimer–SA nanocomplex that possesses multiple sticky ends, a high molecular weight, and a hyperbranched nanostructure with large numbers of DNA duplexes, and thus can obviously improve the sensing sensitivity and selectivity. The proposed amplification strategy extends the application of nonlinear HCR and the developed biosensing systems can be conducted facilely, with no requirement for separation processes or enzymatic reactions. Moreover, this work provides the universal platforms for detecting disease-related species through simply altering the related capture and reporter DNA sequences, and shows great potential for clinical applications.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.xxxxxxx. DNA oligonucleotide sequences used in this study (Table S1), reagents used in preparing the DNA dendrimer–SA nanocomplex (Table S2), optimization of QCM sensing conditions (Figure S1– S7), fluorescence quantum yield measurement, and optimization of fluorescent sensing conditions (Figure S8 and S9). (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +86 010-62761187. Fax: +86-010-62751708.

ORCID Feng Liu: 0000-0002-3399-9071

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Notes The authors declare no competing financial interest..

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (21675005, 21535006, and 21275013). The authors thank Biolin Scientific AB for offering the Q-Sense E4 instrument.

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